Search

IntroductionEndocrine hypertension (EHT) due to pheochromocytoma/paraganglioma (PPGL), Cushing’s syndrome (CS), or primary aldosteronism (PA) is linked to a variety of metabolic alterations and comorbidities. Accordingly, patients with EHT and primary hypertension (PHT) are characterized by distinct metabolic profiles. However, it remains unclear whether the metabolomic differences relate solely to the disease-defining hormonal parameters. Therefore, our objective was to study the association of disease defining hormonal excess and concomitant adrenal steroids with metabolomic alterations in patients with EHT.MethodsRetrospective European multicenter study of 263 patients (mean age 49 years, 50% females; 58 PHT, 69 PPGL, 37 CS, 99 PA) in whom targeted metabolomic and adrenal steroid profiling was available. The association of 13 adrenal steroids with differences in 79 metabolites between PPGL, CS, PA and PHT was examined after correction for age, sex, BMI, and presence of diabetes mellitus.ResultsAfter adjustment for BMI and diabetes mellitus significant association between adrenal steroids and metabolites – 18 in PPGL, 15 in CS, and 23 in PA – were revealed. In PPGL, the majority of metabolite associations were linked to catecholamine excess, whereas in PA, only one metabolite was associated with aldosterone. In contrast, cortisone (16 metabolites), cortisol (6 metabolites), and DHEA (8 metabolites) had the highest number of associated metabolites in PA. In CS, 18-hydroxycortisol significantly influenced 5 metabolites, cortisol affected 4, and cortisone, 11-deoxycortisol, and DHEA each were linked to 3 metabolites.DiscussionsOur study indicates cortisol, cortisone, and catecholamine excess are significantly associated with metabolomic variances in EHT versus PHT patients. Notably, catecholamine excess is key to PPGL’s metabolomic changes, whereas in PA, other non-defining adrenal steroids mainly account for metabolomic differences. In CS, cortisol, alongside other non-defining adrenal hormones, contributes to these differences, suggesting that metabolic disorders and cardiovascular morbidity in these conditions could also be affected by various adrenal steroids.

Summary: 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.

The molecular mechanisms underlying metastasis in pheochromocytoma/paraganglioma (mPPGL) remain to be explored. Here, the authors perform genomic and immunogenomic profiling of mPPGL tumors and suggest potential biomarkers for risk of metastasis and immunotherapy response.

Summary: Background: Pheochromocytomas and paragangliomas have up to a 20% rate of metastatic disease that cannot be reliably predicted. This study prospectively assessed whether the dopamine metabolite, methoxytyramine, might predict metastatic disease, whether predictions might be improved using machine learning models that incorporate other features, and how machine learning-based predictions compare with predictions made by specialists in the field. Methods: In this machine learning modelling study, we used cross-sectional cohort data from the PMT trial, based in Germany, Poland, and the Netherlands, to prospectively examine the utility of methoxytyramine to predict metastatic disease in 267 patients with pheochromocytoma or paraganglioma and positive biochemical test results at initial screening. Another retrospective dataset of 493 patients with these tumors enrolled under clinical protocols at National Institutes of Health (00-CH-0093) and the Netherlands (PRESCRIPT trial) was used to train and validate machine learning models according to selections of additional features. The best performing machine learning models were then externally validated using data for all patients in the PMT trial. For comparison, 12 specialists provided predictions of metastatic disease using data from the training and external validation datasets. Findings: Prospective predictions indicated that plasma methoxytyramine could identify metastatic disease at sensitivities of 52% and specificities of 85%. The best performing machine learning model was based on an ensemble tree classifier algorithm that used nine features: plasma methoxytyramine, metanephrine, normetanephrine, age, sex, previous history of pheochromocytoma or paraganglioma, location and size of primary tumours, and presence of multifocal disease. This model had an area under the receiver operating characteristic curve of 0·942 (95% CI 0·894–0·969) that was larger (p

Abstract Background Maintaining intraoperative haemodynamic stability can reduce cardiovascular complications during surgery for pheochromocytoma and paraganglioma (PPGL). Risk factors such as tumour size and catecholamine levels are reported to predict haemodynamic responses during surgery for PPGL. We hypothesized that additional factors including body composition and genetic information could further improve prediction. Methods Consecutive patients with PPGL confirmed by surgical pathology between June 2010 and June 2019 were retrospectively included. Cross‐sectional computed tomography images at the L3 level were used to assess body composition parameters including skeletal muscle area and visceral fat area. Next‐generation sequencing was performed using a panel containing susceptibility genes of PPGL. Differences in clinical‐genetic characteristics and body composition parameters were analysed and compared in patients with and without intraoperative haemodynamic instability (HDI). Results We included 221 patients with PPGL (median age 47 [38–56] years, and 52% male). Among them, 49.8% had Cluster 2 mutations (related to kinase signalling pathways), 44.8% had sarcopenia, and 52.9% experienced intraoperative HDI. Compared with patients without HDI, more patients with HDI had Cluster 2 mutations (59.8% vs. 38.5%, P = 0.002) and less had sarcopenia (35.9% vs. 54.8%, P = 0.005). Multivariate analysis showed that urine vanillylmandelic acid ≥ 58 μmol/day (adjusted odds ratio [OR] = 1.840, 95% confidence interval [CI] = 1.012–3.347, P = 0.046), tumour size ≥ 4 cm (adjusted OR = 2.278, 95% CI = 1.242–4.180, P = 0.008), and Cluster 2 mutations (adjusted OR = 2.199, 95% CI = 1.128–4.285, P = 0.021) were independent risk factors for intraoperative HDI, while sarcopenia (adjusted OR = 0.475, 95% CI = 0.266–0.846, P = 0.012) decreased the risk. Conclusions Body composition and genotype were associated with intraoperative haemodynamics in patients with PPGL. Our results indicated that inclusion of body composition and genotype in the overall assessment of patients with PPGL helped to predict HDI during surgery, which could assist in implementing preoperative and intraoperative measures to reduce perioperative complications.

Pediatric neural crest-derived adrenal neoplasms include neuroblastoma and pheochromocytoma. Both entities are associated with a high degree of clinical heterogeneity, varying from spontaneous regression to malignant disease with poor outcome. Increased expression and stabilization of HIF2α appears to contribute to a more aggressive and undifferentiated phenotype in both adrenal neoplasms, whereas MYCN amplification is a valuable prognostic marker in neuroblastoma. The present review focuses on HIF- and MYC signaling in both neoplasms and discusses the interaction of associated pathways during neural crest and adrenal development as well as potential consequences on tumorigenesis. Emerging single-cell methods together with epigenetic and transcriptomic analyses provide further insights into the importance of a tight regulation of HIF and MYC signaling pathways during adrenal development and tumorigenesis. In this context, increased attention to HIF-MYC/MAX interactions may also provide new therapeutic options for these pediatric adrenal neoplasms.

IntroductionThe percentage of patients diagnosed with pheochromocytoma and paraganglioma (altogether PPGL) carrying known germline mutations in one of the over fifteen susceptibility genes identified to date has dramatically increased during the last two decades, accounting for up to 35-40% of PPGL patients. Moreover, the application of NGS to the diagnosis of PPGL detects unexpected co-occurrences of pathogenic allelic variants in different susceptibility genes.MethodsHerein we uncover several cases with dual mutations in NF1 and other PPGL genes by targeted sequencing. We studied the molecular characteristics of the tumours with co-occurrent mutations, using omic tools to gain insight into the role of these events in tumour development.ResultsAmongst 23 patients carrying germline NF1 mutations, targeted sequencing revealed additional pathogenic germline variants in DLST (n=1) and MDH2 (n=2), and two somatic mutations in H3-3A and PRKAR1A. Three additional patients, with somatic mutations in NF1 were found carrying germline pathogenic mutations in SDHB or DLST, and a somatic truncating mutation in ATRX. Two of the cases with dual germline mutations showed multiple pheochromocytomas or extra-adrenal paragangliomas - an extremely rare clinical finding in NF1 patients. Transcriptional and methylation profiling and metabolite assessment showed an “intermediate signature” to suggest that both variants had a pathological role in tumour development.DiscussionIn conclusion, mutations affecting genes involved in different pathways (pseudohypoxic and receptor tyrosine kinase signalling) co-occurring in the same patient could provide a selective advantage for the development of PPGL, and explain the variable expressivity and incomplete penetrance observed in some patients.

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors with heterogeneous clinical presentations and potential lethal outcomes. The diagnosis is based on clinical suspicion, biochemical testing, imaging and histopathological confirmation. Increasingly widespread use of imaging studies and surveillance of patients at risk of PPGL due to a hereditary background or a previous tumor is leading to the diagnosis of these tumors at an early stage. This has resulted in an increasing use of the term “silent” PPGL. This term and other variants are now commonly found in the literature without any clear or unified definition. Among the various terms, “clinically silent” is often used to describe the lack of signs and symptoms associated with catecholamine excess. Confusion arises when these and other terms are used to define the tumors according to their ability to synthesize and/or release catecholamines in relation to biochemical test results. In such cases the term “silent” and other variants are often inappropriately and misleadingly used. In the present analysis we provide an overview of the literature and propose standardized terminology in an attempt at harmonization to facilitate scientific communication.

Summary: Background: Arterial hypertension is a major cardiovascular risk factor. Identification of secondary hypertension in its various forms is key to preventing and targeting treatment of cardiovascular complications. Simplified diagnostic tests are urgently required to distinguish primary and secondary hypertension to address the current underdiagnosis of the latter. Methods: This study uses Machine Learning (ML) to classify subtypes of endocrine hypertension (EHT) in a large cohort of hypertensive patients using multidimensional omics analysis of plasma and urine samples. We measured 409 multi-omics (MOmics) features including plasma miRNAs (PmiRNA: 173), plasma catechol O-methylated metabolites (PMetas: 4), plasma steroids (PSteroids: 16), urinary steroid metabolites (USteroids: 27), and plasma small metabolites (PSmallMB: 189) in primary hypertension (PHT) patients, EHT patients with either primary aldosteronism (PA), pheochromocytoma/functional paraganglioma (PPGL) or Cushing syndrome (CS) and normotensive volunteers (NV). Biomarker discovery involved selection of disease combination, outlier handling, feature reduction, 8 ML classifiers, class balancing and consideration of different age- and sex-based scenarios. Classifications were evaluated using balanced accuracy, sensitivity, specificity, AUC, F1, and Kappa score. Findings: Complete clinical and biological datasets were generated from 307 subjects (PA=113, PPGL=88, CS=41 and PHT=112). The random forest classifier provided ∼92% balanced accuracy (∼11% improvement on the best mono-omics classifier), with 96% specificity and 0.95 AUC to distinguish one of the four conditions in multi-class ALL-ALL comparisons (PPGL vs PA vs CS vs PHT) on an unseen test set, using 57 MOmics features. For discrimination of EHT (PA + PPGL + CS) vs PHT, the simple logistic classifier achieved 0.96 AUC with 90% sensitivity, and ∼86% specificity, using 37 MOmics features. One PmiRNA (hsa-miR-15a-5p) and two PSmallMB (C9 and PC ae C38:1) features were found to be most discriminating for all disease combinations. Overall, the MOmics-based classifiers were able to provide better classification performance in comparison to mono-omics classifiers. Interpretation: We have developed a ML pipeline to distinguish different EHT subtypes from PHT using multi-omics data. This innovative approach to stratification is an advancement towards the development of a diagnostic tool for EHT patients, significantly increasing testing throughput and accelerating administration of appropriate treatment. Funding: European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement No. 633983, Clinical Research Priority Program of the University of Zurich for the CRPP HYRENE (to Z.E. and F.B.), and Deutsche Forschungsgemeinschaft (CRC/Transregio 205/1).

Abstract Diagnosis of pheochromocytomas and paragangliomas in patients receiving hemodialysis is troublesome. The aim of the study was to establish optimal conditions for blood sampling for mass spectrometric measurements of normetanephrine, metanephrine and 3-methoxytyramine in patients on hemodialysis and specific reference intervals for plasma metanephrines under the most optimal sampling conditions. Blood was sampled before and near the end of dialysis, including different sampling sites in 170 patients on hemodialysis. Plasma normetanephrine concentrations were lower (P

Catecholamine-producing tumors of childhood include most notably neuroblastoma, but also pheochromocytoma and paraganglioma (PPGL). Diagnosis of the former depends largely on biopsy-dependent histopathology, but this is contraindicated in PPGL where diagnosis depends crucially on biochemical tests of catecholamine excess. Such tests retain some importance in neuroblastoma though continue to largely rely on measurements of homovanillic acid (HVA) and vanillylmandelic acid (VMA), which are no longer recommended for PPGL. For PPGL, urinary or plasma metanephrines are the recommended most accurate tests. Addition of methoxytyramine to the plasma panel is particularly useful to identify dopamine-producing tumors and combined with normetanephrine also shows superior diagnostic performance over HVA and VMA for neuroblastoma. While use of metanephrines and methoxytyramine for diagnosis of PPGL in adults is established, there are numerous pitfalls for use of these tests in children. The establishment of pediatric reference intervals is particularly difficult and complicated by dynamic changes in metabolites during childhood, especially in infants for both plasma and urinary measurements, and extending to adolescence for urinary measurements. Interpretation of test results is further complicated in children by difficulties in following recommended preanalytical precautions. Due to this, the slow growing nature of PPGL and neglected consideration of the tumors in childhood the true pediatric prevalence of PPGL is likely underappreciated. Earlier identification of disease, as facilitated by surveillance programs, may uncover the true prevalence and improve therapeutic outcomes of childhood PPGL. For neuroblastoma there remain considerable obstacles in moving from entrenched to more accurate tests of catecholamine excess.

Purpose Current evidence regarding renal involvement in pheochromocytoma and paraganglioma (PPGL) is scant. More accurate diagnostic methods, such as renal Doppler ultrasound for intrarenal hemodynamic studies, may provide more detailed information on renal function. It might be postulated that renal function in PPGL patients might be altered by high blood pressure and excess secretion of catecholamines. The aim of this prospective study was to assess intrarenal blood flow parameters in PPGL patients included in the prospective monoamine-producing tumour (PMT) study and to evaluate the effects of normalisation of catecholamine production after surgical treatment on long-term renal function. Materials and methods Seventy consecutive patients (aged 46.5 ± 14.0 years) with PPGL were included. Forty-eight patients from the PMT study cohort, matched for age, gender, blood pressure level and presence of hypertension, served as a control group. Renal artery doppler ultrasound spectral analysis included mean resistance index (RRI) and pulsatility index (PI). Forty-seven patients completed 12 months follow-up. Results There were no differences in renal parameters such as RRI, PI and kidney function between PPGL and non-PPGL patients as assessed by renal ultrasound, serum creatinine, eGFR and albumin excretion rate. No correlations between kidney function parameters, intrarenal doppler flow parameters and plasma catecholamines were observed in PPGL patients. At 12 months after surgery, no differences in creatinine level, eGFR, albumin excretion rate, RI and PI were found as compared to baseline results. Conclusions In contrast to patients with other forms of secondary hypertension, our study did not show differences in intrarenal blood flow parameters and renal function between PPGL and non-PPGL subjects. Intrarenal hemodynamics and renal function did not change after normalisation of catecholamine levels by surgical treatment.

Abstract Invasive nonfunctioning (NF) pituitary neuroendocrine tumors (PitNETs) are non‐resectable neoplasms associated with frequent relapses and significant comorbidities. As the current therapies of NF‐PitNETs often fail, new therapeutic targets are needed. The observation that circulating angiopoietin‐2 (ANGPT2) is elevated in patients with NF‐PitNET and correlates with tumor aggressiveness prompted us to investigate the ANGPT2/TIE2 axis in NF‐PitNETs in the GH3 PitNET cell line, primary human NF‐PitNET cells, xenografts in zebrafish and mice, and in MENX rats, the only autochthonous NF‐PitNET model. We show that PitNET cells express a functional TIE2 receptor and secrete bioactive ANGPT2, which promotes, besides angiogenesis, tumor cell growth in an autocrine and paracrine fashion. ANGPT2 stimulation of TIE2 in tumor cells activates downstream cell proliferation signals, as previously demonstrated in endothelial cells (ECs). Tie2 gene deletion blunts PitNETs growth in xenograft models, and pharmacological inhibition of Angpt2/Tie2 signaling antagonizes PitNETs in primary cell cultures, tumor xenografts in mice, and in MENX rats. Thus, the ANGPT2/TIE2 axis provides an exploitable therapeutic target in NF‐PitNETs and possibly in other tumors expressing ANGPT2/TIE2. The ability of tumor cells to coopt angiogenic signals classically viewed as EC‐specific expands our view on the microenvironmental cues that are essential for tumor progression.

Mitotane is the only drug approved for the therapy of adrenocortical carcinoma (ACC). Its clinical use is limited by the occurrence of relapse during therapy. To investigate the underlying mechanisms in vitro, we here generated mitotane-resistant cell lines. After long-term pulsed treatment of HAC-15 human adrenocortical carcinoma cells with 70 μM mitotane, we isolated monoclonal cell populations of treated cells and controls and assessed their respective mitotane sensitivities by MTT assay. We performed exome sequencing and electron microscopy, conducted gene expression microarray analysis and determined intracellular lipid concentrations in the presence and absence of mitotane. Clonal cell lines established after pulsed treatment were resistant to mitotane (IC50 of 102.2 ± 7.3 μM (n = 12) vs 39.4 ± 6.2 μM (n = 6) in controls (biological replicates, mean ± s.d., P = 0.0001)). Unlike nonresistant clones, resistant clones maintained normal mitochondrial and nucleolar morphology during mitotane treatment. Resistant clones largely shared structural and single nucleotide variants, suggesting a common cell of origin. Resistance depended, in part, on extracellular lipoproteins and was associated with alterations in intracellular lipid homeostasis, including levels of free cholesterol, as well as decreased steroid production. By gene expression analysis, resistant cells showed profound alterations in pathways including steroid metabolism and transport, apoptosis, cell growth and Wnt signaling. These studies establish an in vitro model of mitotane resistance in ACC and point to underlying molecular mechanisms. They may enable future studies to overcome resistance in vitro and improve ACC treatment in vivo.

Hypertension is a major global health problem with high prevalence and complex associated health risks. Primary hypertension (PHT) is most common and the reasons behind primary hypertension are largely unknown. Endocrine hypertension (EHT) is another complex form of hypertension with an estimated prevalence varying from 3 to 20% depending on the population studied. It occurs due to underlying conditions associated with hormonal excess mainly related to adrenal tumours and sub-categorised: primary aldosteronism (PA), Cushing’s syndrome (CS), pheochromocytoma or functional paraganglioma (PPGL). Endocrine hypertension is often misdiagnosed as primary hypertension, causing delays in treatment for the underlying condition, reduced quality of life, and costly antihypertensive treatment that is often ineffective. This study systematically used targeted metabolomics and high-throughput machine learning methods to predict the key biomarkers in classifying and distinguishing the various subtypes of endocrine and primary hypertension. The trained models successfully classified CS from PHT and EHT from PHT with 92% specificity on the test set. The most prominent targeted metabolites and metabolite ratios for hypertension identification for different disease comparisons were C18:1, C18:2, and Orn/Arg. Sex was identified as an important feature in CS vs. PHT classification.

Despite considerable morbidity and mortality, numerous cases of endocrine hypertension (EHT) forms, including primary aldosteronism (PA), pheochromocytoma and functional paraganglioma (PPGL), and Cushing’s syndrome (CS), remain undetected. We aimed to establish signatures for the different forms of EHT, investigate potentially confounding effects and establish unbiased disease biomarkers. Plasma samples were obtained from 13 biobanks across seven countries and analyzed using untargeted NMR metabolomics. We compared unstratified samples of 106 PHT patients to 231 EHT patients, including 104 PA, 94 PPGL and 33 CS patients. Spectra were subjected to a multivariate statistical comparison of PHT to EHT forms and the associated signatures were obtained. Three approaches were applied to investigate and correct confounding effects. Though we found signatures that could separate PHT from EHT forms, there were also key similarities with the signatures of sample center of origin and sample age. The study design restricted the applicability of the corrections employed. With the samples that were available, no biomarkers for PHT vs. EHT could be identified. The complexity of the confounding effects, evidenced by their robustness to correction approaches, highlighted the need for a consensus on how to deal with variabilities probably attributed to preanalytical factors in retrospective, multicenter metabolomics studies.

Background: Pheochromocytomas and paragangliomas (PPGLs) are rare but potentially harmful tumors that can vary in their clinical presentation. Tumors may be found due to signs and symptoms, as part of a hereditary syndrome or following an imaging procedure. Objective: To investigate potential differences in clinical presentation between PPGLs discovered by imaging (iPPGLs), symptomatic cases (sPPGLs) and those diagnosed during follow-up because of earlier disease/known hereditary mutations (fPPGL). Design: Prospective study protocol, which has enrolled patients from six European centers with confirmed PPGLs. Data were analyzed from 235 patients (37 iPPGLs, 36 sPPGLs, 27% fPPGLs) and compared for tumor volume, biochemical profile, mutation status, presence of metastases and self-reported symptoms. iPPGL patients were diagnosed at a significantly higher age than fPPGLs (P < 0.001), found to have larger tumors (P = 0.003) and higher metanephrine and normetanephrine levels at diagnosis (P = 0.021). Significantly lower than in sPPGL, there was a relevant number of self-reported symptoms in iPPGL (2.9 vs 4.3 symptoms, P < 0.001). In 16.2% of iPPGL, mutations in susceptibility genes were detected, although this proportion was lower than that in fPPGL (60.9%) and sPPGL (21.5%). Patients with PPGLs detected by imaging were older, have higher tumor volume and more excessive hormonal secretion in comparison to those found as part of a surveillance program. Presence of typical symptoms indicates that in a relevant proportion of those patients, the PPGL diagnosis had been delayed. Précis: Pheochromocytoma/paraganglioma discovered by imaging are often symptomatic and carry a significant proportion of germline mutations in susceptibility genes.

Reduced expression of the plasma membrane citrate transporter INDY (acronym I’m Not Dead, Yet) extends life span in lower organisms. Deletion of the mammalian Indy (mIndy) gene in rodents improves metabolism via mechanisms akin to caloric restriction, known to lower blood pressure (BP) by sympathoadrenal inhibition. We hypothesized that mIndy deletion attenuates sympathoadrenal support of BP. Continuous arterial BP and heart rate (HR) were reduced in mINDY-KO mice. Concomitantly, urinary catecholamine content was lower, and the decreases in BP and HR by mIndy deletion were attenuated after autonomic ganglionic blockade. Catecholamine biosynthesis pathways were reduced in mINDY-KO adrenals using unbiased microarray analysis. Citrate, the main mINDY substrate, increased catecholamine content in pheochromocytoma cells, while pharmacological inhibition of citrate uptake blunted the effect. Our data suggest that deletion of mIndy reduces sympathoadrenal support of BP and HR by attenuating catecholamine biosynthesis. Deletion of mIndy recapitulates beneficial cardiovascular and metabolic responses to caloric restriction, making it an attractive therapeutic target.

Continuous activation of hypoxia pathways in pheochromocytomas and paragangliomas (PPGLs) is associated with higher disease aggressiveness, for which effective treatment strategies are still missing. Most of the commonly used in vitro models lack characteristics of these pseudohypoxic tumors, including elevated expression of hypoxia-inducible factor (HIF) 2α. To address this shortcoming, we investigated whether recurrent hypoxia cycles lead to continuous activation of hypoxia pathways under normoxic conditions and whether this pseudohypoxia is associated with increased cellular aggressiveness. Rat pheochromocytoma cells (PC12) were incubated under hypoxia for 24 h every 3–4 days, up to 20 hypoxia–reoxygenation cycles, resulting in PC12 Z20 cells. PC12 Z20 control cells were obtained by synchronous cultivation under normoxia. RNA sequencing revealed upregulation of HIF2α in PC12 Z20 cells and a pseudohypoxic gene signature that overlapped with the gene signature of pseudohypoxic PPGLs. PC12 Z20 cells showed a higher growth rate, and the migration and adhesion capacity were significantly increased compared with control cells. Changes in global methylation, together with the pseudohypoxic conditions, may be responsible for the increased aggressiveness of this new model. The established sub-cell line with characteristics of pseudohypoxic PPGLs represent a complementary model for further investigations, for example, with regard to new therapeutic approaches.

ABSTRACT: Objective: Both clinical suspicion and diagnosis of pheochromocytoma (PCC) can be challenging in patients where the presentation can be confused with the pharmacophysiological effects of illicit drugs known to activate the sympathetic nervous system. We report on such a patient and outline considerations that can impact diagnostic decision making. Methods: Clinical examination, measurement of plasma metanephrines, followed by magnetic resonance imaging, iodine 123-metaiodobenzylguanidine single-photon emission computed tomography, and histopathology of the resected tumor. Results: A 35-year-old male patient was referred to our center because of a right-sided adrenal mass, incidentally found during an abdominal ultrasound performed due to nausea, vomiting, and lumbar pain. Although he had no history of hypertension, he had complained for over 6 years of severe episodic headache, panic attacks, and profuse sweating. He also had a longer history of methamphetamine abuse. Plasma concentrations of metanephrine (10.7 pmol/L) and normetanephrine (3.83 pmol/L) were 25-fold and 5.6-fold above respective upper limits of reference intervals (0.42 and 0.69 pmol/L). This indicated a PCC, which was confirmed after adrenalectomy. Failure to recognize the patient’s signs and symptoms as attributable to a PCC was unsurprising given that methamphetamine abuse can result in many of the same signs and symptoms as a catecholamine-producing tumor. Conclusion: The abuse of drugs such as methamphetamine can obscure an underlying PCC due to the similarity of several symptoms associated with both conditions. Recognition of a PCC in patients using illicit drugs such as methamphetamine remains challenging. Abbreviations: NE norepinephrine PCC pheochromocytoma

Multiple stressors, including 24-h-shifts characterise the working environment of physicians, influencing well-being, health and performance. We aimed to evaluate the effect of the stressor 24-h-shift on the adrenal medullary and sympathoneural system in physicians with the hypothesis that shift work might have different impacts on both systems. Twenty-two physicians collected two 12-h-urine samples (“daytime” and “nighttime”) during a 24-h shift (“on-duty”) and on a free weekend (“off-duty”), respectively. Urinary excretion rates per m2 body surface area were assessed for the catecholamines epinephrine, norepinephrine and their respective free O-methylated metabolites metanephrine and normetanephrine by LC-MS/MS-analysis. The stressor provoked differential responses of epinephrine and norepinephrine. Epinephrine excretion rates showed significant increases from off to on duty. The largest proportional change (off-duty to on-duty) for epinephrine was observed for nighttime (205%), the increase for daytime was 84%. An increase in norepinephrine from off to on duty was only visible for nighttime collections. For the catecholamine metabolites, normetanephrine paralleled norepinephrine and exhibited an increase in excretion from off to on duty during nighttime collections of 53% whereas there was no change during daytime collections (3%). In conclusion: Whilst the 24-h-shift-work stressor in physicians activates the sympatho-adrenomedullary system, represented by epinephrine, the sympathoneural response through norepinephrine reflects mainly an ambulatory position during working hours.

Pheochromocytoma (PCC) and paraganglioma (PGL) are rare neuroendocrine tumors associated with high cardiovascular morbidity and variable risk of malignancy. The current therapy of choice is surgical resection. Nevertheless, PCCs/PGLs are associated with a lifelong risk of tumor persistence or recurrence. A high rate of germline or somatic mutations in numerous genes has been found in these tumors. For some, the tumorigenic processes are initiated during embryogenesis. Such tumors carry gene mutations leading to pseudohypoxic phenotypes and show more immature characteristics than other chromaffin cell tumors; they are also often multifocal or metastatic and occur at an early age, often during childhood. Cancer stem cells (CSCs) are cells with an inherent ability of self-renewal, de-differentiation, and capacity to initiate and maintain malignant tumor growth. Targeting CSCs to inhibit cancer progression has become an attractive anti-cancer therapeutic strategy. Despite progress for this strategy for solid tumors such as neuroblastoma, brain, breast, and colon cancers, no substantial advance has been made employing similar strategies in PCCs/PGLs. In the current review, we discuss findings related to the identification of normal chromaffin stem cells and CSCs, pathways involved in regulating the development of CSCs, and the importance of the stem cell niche in development and maintenance of CSCs in PCCs/PGLs. Additionally, we examine the development and feasibility of novel CSC-targeted therapeutic strategies aimed at eradicating especially recurrent and metastatic tumors.

Renal cell carcinoma (RCC) is among the 10 most common cancer entities and can be categorised into distinct subtypes by differential expression of Krebs cycle genes. We investigated the predictive value of several targeted metabolites with regards to tumour stages and patient survival in an unselected cohort of 420 RCCs. Unsupervised hierarchical clustering of metabolite ratios identified two main clusters separated by α-ketoglutarate (α-KG) levels and sub-clusters with differential levels of the oncometabolite 2-hydroxyglutarate (2HG). Sub-clusters characterised by high 2HG were enriched in higher tumour stages, suggesting metabolite profiles might be suitable predictors of tumour stage or survival. Bootstrap forest models based on single metabolite signatures showed that lactate, 2HG, citrate, aspartate, asparagine, and glutamine better predicted the cancer-specific survival (CSS) of clear cell RCC patients, whereas succinate and α-ketoglutarate were better CSS predictors for papillary RCC patients. Additionally, this assay identifies rare cases of tumours with SDHx mutations, which are caused predominantly by germline mutations and which predispose to development of different neoplasms. Hence, analysis of selected metabolites should be further evaluated for potential utility in liquid biopsies, which can be obtained using less invasive methods and potentially facilitate disease monitoring for both patients and caregivers.

One of the main problems we face with PPGL is the lack of molecular markers capable of predicting the development of metastases in patients. Telomere-related genes, such as TERT and ATRX, have been recently described in PPGL, supporting the association between the activation of immortalization mechanisms and disease progression. However, the contribution of other genes involving telomere preservation machinery has not been previously investigated. In this work, we aimed to analyze the prognostic value of a comprehensive set of genes involved in telomere maintenance. For this study, we collected 165 PPGL samples (97 non-metastatic/63 metastatic), genetically characterized, in which the expression of 29 genes of interest was studied by NGS. Three of the 29 genes studied, TERT, ATRX and NOP10, showed differential expression between metastatic and non-metastatic cases, and alterations in these genes were associated with a shorter time to progression, independent of SDHB-status. We studied telomere length by Q-FISH in patient samples and in an in vitro model. NOP10 overexpressing tumors displayed an intermediate-length telomere phenotype without ALT, and in vitro results suggest that NOP10 has a role in telomerase-dependent telomere maintenance. We also propose the implementation of NOP10 IHC to better stratify PPGL patients.

Despite all technical progress in modern diagnostic methods and treatment modalities of pheochromocytoma/paraganglioma, early consideration of the presence of these tumors remains the pivotal link towards the best possible outcome for patients. A timely diagnosis and proper treatment can prevent the wide variety of potentially catastrophic cardiovascular complications. Modern biochemical testing should include tests that offer the best available diagnostic performance, measurements of metanephrines and 3-methoxytyramine in plasma or urine. To minimize false-positive test results particular attention should be paid to pre-analytical sampling conditions. In addition to anatomical imaging by computed tomography (CT) or magnetic resonance imaging, new promising functional imaging modalities of photon emission tomography/CT using with somatostatin analogues such as 68Ga-DOTATATE (68Ga-labeled DOTA(0)-Tyr(3)-octreotide) will probably replace 123I-MIBG (iodine-123-metaiodobenzylguanidine) in the near future. As nearly half of all pheochromocytoma patients harbor a mutation in one of the 14 tumor susceptibility genes, genetic testing and counseling should at least be considered in all patients with a proven tumor. Post-surgical annual follow-up of patients by measurements of plasma or urinary metanephrines should last for at least 10 years for timely detection of recurrent or metastatic disease. Patients with a high risk for recurrence or metastatic disease (paraganglioma, young age, multiple or large tumors, genetic background) should be followed up lifelong.

Primary aldosteronism (PA) is the leading cause of secondary hypertension. The source of aldosterone hypersecretion is often due to a unilateral aldosterone-producing adenoma, and unilateral laparoscopic adrenalectomy is recommended in such patients. Before surgery, confirmation of unilateral hypersecretion is necessary. This is optimally performed by adrenal venous sampling (AVS). However, AVS is not always successful e.g., due to difficulties in the cannulation of the right adrenal vein. Here we present the case of a 53-year-old female patient with primary aldosteronism, a left-sided adrenal mass and an inconspicuous right adrenal. AVS was performed, but cannulation of the right adrenal vein failed. Therefore, aldosterone hypersecretion also of the right adrenal could not be excluded despite higher aldosterone concentrations in the left renal and adrenal vein. To increase the certainty that the left sided adrenal mass was the source of aldosterone hypersecretion, steroid profiling was performed in a sample from the inferior vena cava. This revealed markedly elevated levels of 18-oxocortisol, 18-hydroxycortisol, 11-deoxycorticosterone, and 11-deoxycortisol, a steroid profile that strongly suggested that the left sided adrenal mass was an aldosterone producing adenoma, most likely due to a somatic KCNJ5 mutation. Following unilateral adrenalectomy, CYP11B2 immunohistochemistry, and genetics analysis of the resected adrenal confirmed a solitary aldosterone-producing adenoma with intense aldosterone synthase expression, which harbored a previously described KCNJ5 Phe154Cys mutation. Biochemical and clinical cure was confirmed 6 months postoperatively.

Recently, activating mutations of the hypoxia-inducible factor 2α gene (HIF2A/EPAS1) have been recognized to predispose to multiple paragangliomas (PGLs) and duodenal somatostatinomas associated with polycythemia, and ocular abnormalities. Previously, mutations in the SDHA/B/C/D, SDHAF2, VHL, FH, PHD1, and PHD2 genes have been associated with HIF activation and the development of pseudohypoxic (cluster-1) PGLs. These tumors overlap in terms of tumor location, syndromic presentation, and noradrenergic phenotype to a certain extent. However, they also differ especially by clinical outcome and by presence of other tumors or abnormalities. In the present study, we aimed to establish additional molecular differences between HIF2A and non-HIF2A pseudohypoxic PGLs. RNA expression patterns of HIF2A PGLs (n = 6) from 2 patients were compared with normal adrenal medullas (n = 8) and other hereditary pseudohypoxic PGLs (VHL: n = 13, SDHB: n = 15, and SDHD: n = 14). Unsupervised hierarchical clustering showed that HIF2A PGLs made up a separate cluster from other pseudohypoxic PGLs. Significance analysis of microarray yielded 875 differentially expressed genes between HIF2A and other pseudohypoxic PGLs after normalization to adrenal medulla (false discovery rate 0.01). Prediction analysis of microarray allowed correct classification of all HIF2A samples based on as little as three genes (TRHDE, LRRC63, IGSF10; error rate: 0.02). Genes with the highest expression difference between normal medulla and HIF2A PGLs were selected for confirmatory quantitative reverse transcriptase polymerase chain reaction. In conclusion, HIF2A PGLs show a characteristic expression signature that separates them from non-HIF2A pseudohypoxic PGLs. Unexpectedly, the most significantly differentially expressed genes have not been previously described as HIF target genes.

Pheochromocytomas and paragangliomas (PCC/PGLs) are rare, mostly catecholamine-producing neuroendocrine tumors of the adrenal gland (PCCs) or the extra-adrenal paraganglia (PGL). They can be separated into three different molecular clusters depending on their underlying gene mutations in any of the at least 20 known susceptibility genes: The pseudohypoxia-associated cluster 1, the kinase signaling-associated cluster 2, and the Wnt signaling-associated cluster 3. In addition to tumor size, location (adrenal vs. extra-adrenal), multiplicity, age of first diagnosis, and presence of metastatic disease (including tumor burden), other decisive factors for best clinical management of PCC/PGL include the underlying germline mutation. The above factors can impact the choice of different biomarkers and imaging modalities for PCC/PGL diagnosis, as well as screening for other neoplasms, staging, follow-up, and therapy options. This review provides a guide for practicing clinicians summarizing current management of PCC/PGL according to tumor size, location, age of first diagnosis, presence of metastases, and especially underlying mutations in the era of precision medicine.

Mutations in the AAAS gene coding for the nuclear pore complex protein ALADIN lead to the autosomal recessive disorder triple A syndrome. Triple A patients present with a characteristic phenotype including alacrima, achalasia and adrenal insufficiency. Patient fibroblasts show increased levels of oxidative stress, and several in vitro studies have demonstrated that the nucleoporin ALADIN is involved in both the cellular oxidative stress response and adrenal steroidogenesis. It is known that ALADIN knock-out mice lack a phenotype resembling human triple A syndrome. The objective of this study was to determine whether the application of chronic oxidative stress by ingestion of paraquat would generate a triple A-like phenotype in ALADIN null mice. Adult male mice were fed either a paraquat (0.25 g/kg diet) or control diet for 11 days. After application of chronic oxidative stress, ALADIN knock-out mice presented with an unexpected compensated glutathione metabolism, but lacked a phenotype resembling human triple A syndrome. We did not observe increased levels of oxidative stress and alterations in adrenal steroidogenesis in mice depleted for ALADIN. This study stresses the species-specific role of the nucleoporin ALADIN, which in mice involves a novel compensatory mechanism for regulating the cellular glutathione redox response.

The adrenal medulla is composed predominantly of chromaffin cells producing and secreting the catecholamines dopamine, norepinephrine, and epinephrine. Catecholamine biosynthesis and secretion is a complex and tightly controlled physiologic process. The pathways involved have been extensively studied, and various elements of the underlying molecular machinery have been identified. In this review, we provide a detailed description of the route from stimulus to secretion of catecholamines by the normal adrenal chromaffin cell compared to chromaffin tumor cells in pheochromocytomas. Pheochromocytomas are adrenomedullary tumors that are characterized by uncontrolled synthesis and secretion of catecholamines. This uncontrolled secretion can be partly explained by perturbations of the molecular catecholamine secretory machinery in pheochromocytoma cells. Chromaffin cell tumors also include sympathetic paragangliomas originating in sympathetic ganglia. Pheochromocytomas and paragangliomas are usually locally confined tumors, but about 15% do metastasize to distant locations. Histopathological examination currently poorly predicts future biologic behavior, thus long term postoperative follow-up is required. Therefore, there is an unmet need for prognostic biomarkers. Clearer understanding of the cellular mechanisms involved in the secretory characteristics of pheochromocytomas and sympathetic paragangliomas may offer one approach for the discovery of novel prognostic biomarkers for improved therapeutic targeting and monitoring of treatment or disease progression.

Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors with a strong hereditary background and a large genetic heterogeneity. Identification of the underlying genetic cause is crucial for the management of patients and their families as it aids differentiation between hereditary and sporadic cases. To improve diagnostics and clinical management we tailored an enrichment based comprehensive multi-gene next generation sequencing panel applicable to both analyses of tumor tissue and blood samples. We applied this panel to tumor samples and compared its performance to our current routine diagnostic approach. Routine diagnostic sequencing of 11 PPGL susceptibility genes was applied to blood samples of 65 unselected PPGL patients at a single center in Dresden, Germany. Predisposing germline mutations were identified in 19 (29.2%) patients. Analyses of 28 PPGL tumor tissues using the dedicated PPGL panel revealed pathogenic or likely pathogenic variants in known PPGL susceptibility genes in 21 (75%) cases, including mutations in IDH2, ATRX and HRAS. These mutations suggest sporadic tumor development. Our results imply a diagnostic benefit from extended molecular tumor testing of PPGLs and consequent improvement of patient management. The approach is promising for determination of prognostic biomarkers that support therapeutic decision-making.

Function-integrative textiles bear the potential for a variety of applications in the medical field. Recent clinical investigations suggest that the application of a function-integrative fabric could have a positive impact on currently applied diagnostic procedures of a specific type of tumour. In this context, the fabric should enable local warming of a patient’s upper extremity as well as blood flow measurement. Existing solutions comprise a warming system but lack a measuring apparatus for blood flow determination. With regard to the quality of results of current diagnostic procedures, the local warming of the patients’ upper extremity and the simultaneous determination of the blood flow plateau are crucial. In the present paper, the development process of a function-integrative sleeve is introduced. Besides the development of an adaptable sleeve-design, the manufacturing process of an integrated warming system was also addressed. Furthermore, the identification of crucial physiological effects, using a Laser Doppler Perfusion Monitor, is introduced. During testing of the function-integrative sleeve, modulation of the desired physiological effects was observed. The results support the initial assumptions and dictate further investigations on increasing user-friendliness and cost-efficiency during adjusting and determining the physiological effects in the course of tumour diagnosis.

We previously identified a novel syndrome in patients characterized by paraganglioma, somatostatinoma, and polycythemia. In these patients, polycythemia occurs long before any tumor develops, and tumor removal only partially corrects polycythemia, with recurrence occurring shortly after surgery. Genetic mosaicism of gain-of-function mutations of the EPAS1 gene (encoding HIF2α) located in the oxygen degradation domain (ODD), typically p.530–532, was shown as the etiology of this syndrome. The aim of the present investigation was to demonstrate that these mutations are necessary and sufficient for the development of the symptoms. We developed transgenic mice with a gain-of-function Epas1A529V mutation (corresponding to human EPAS1A530V), which demonstrated elevated levels of erythropoietin and polycythemia, a decreased urinary metanephrine-to-normetanephrine ratio, and increased expression of somatostatin in the ampullary region of duodenum. Further, inhibition of HIF2α with its specific inhibitor PT2385 significantly reduced erythropoietin levels in the mutant mice. However, polycythemia persisted after PT2385 treatment, suggesting an alternative erythropoietin-independent mechanism of polycythemia. These findings demonstrate the vital roles of EPAS1 mutations in the syndrome development and the great potential of the Epas1A529V animal model for further pathogenesis and therapeutics studies.

Cyclooxygenase 2 (COX-2) is a key enzyme of the tumorigenesis-inflammation interface and can be induced by hypoxia. A pseudohypoxic transcriptional signature characterizes pheochromocytomas and paragangliomas (PPGLs) of the cluster I, mainly represented by tumors with mutations in von Hippel−Lindau (VHL), endothelial PAS domain-containing protein 1 (EPAS1), or succinate dehydrogenase (SDH) subunit genes. The aim of this study was to investigate a possible association between underlying tumor driver mutations and COX-2 in PPGLs. COX-2 gene expression and immunoreactivity were examined in clinical specimens with documented mutations, as well as in spheroids and allografts derived from mouse pheochromocytoma (MPC) cells. COX-2 in vivo imaging was performed in allograft mice. We observed significantly higher COX-2 expression in cluster I, especially in VHL-mutant PPGLs, however, no specific association between COX-2 mRNA levels and a hypoxia-related transcriptional signature was found. COX-2 immunoreactivity was present in about 60% of clinical specimens as well as in MPC spheroids and allografts. A selective COX-2 tracer specifically accumulated in MPC allografts. This study demonstrates that, although pseudohypoxia is not the major determinant for high COX-2 levels in PPGLs, COX-2 is a relevant molecular target. This potentially allows for employing selective COX-2 inhibitors as targeted chemotherapeutic agents and radiosensitizers. Moreover, available models are suitable for preclinical testing of these treatments.

Pheochromocytomas and paragangliomas (PPGLs) with activated pseudohypoxic pathways are associated with an immature catecholamine phenotype and carry a higher risk for metastasis. For improved understanding of the underlying mechanisms we investigated the impact of hypoxia and pseudohypoxia on catecholamine biosynthesis in pheochromocytoma cells naturally lacking Hif2α (MPC and MTT) or expressing both Hif1α and Hif2α (PC12). Cultivation under extrinsic hypoxia or in spheroid culture (intrinsic hypoxia) increased cellular dopamine and norepinephrine contents in all cell lines. To distinguish further between Hif1α- and Hif2α-driven effects we expressed Hif2α in MTT and MPC-mCherry cells (naturally lacking Hif2α). Presence of Hif2α resulted in similarly increased cellular dopamine and norepinephrine under hypoxia as in the control cells. Furthermore, hypoxia resulted in enhanced phosphorylation of tyrosine hydroxylase (TH). A specific knockdown of Hif1α in PC12 diminished these effects. Pseudohypoxic conditions, simulated by expression of Hif2α under normoxia resulted in increased TH phosphorylation, further stimulated by extrinsic hypoxia. Correlations with PPGL tissue data led us to conclude that catecholamine biosynthesis under hypoxia is mainly mediated through increased phosphorylation of TH, regulated as a short-term response (24−48 h) by HIF1α. Continuous activation of hypoxia-related genes under pseudohypoxia leads to a HIF2α-mediated phosphorylation of TH (permanent status).

ABSTRACT: Objective: Surgical management of critical ischemic heart disease in a patient with a pheochromocytoma poses a significant challenge for endocrinologists, surgeons, and anesthesiologists in terms of the sequence and optimal interval between operations for each condition. This case report exemplifies the practical problem of how to manage two major surgical procedures with their interrelated effects on outcome.Methods: We report on a patient with pheochromocytoma who presented with symptoms of severe multivessel coronary artery disease during medical preparation for pheochromocytoma surgery, requiring coronary artery bypass grafting (CABG).Results: A 74-year-old woman was diagnosed with a symptomatic pheochromocytoma of the right adrenal gland. After 3 days of presurgical preparation with the alpha-adrenoceptor blocker phenoxybenzamine, she presented with symptoms of multivessel ischemic heart disease, for which CABG was required. A successful two-stage procedure was carried out, first performing CABG under protection of phenoxybenzamine, followed by successful laparoscopic adrenalectomy a few weeks later. Both procedures had an uneventful course without major blood pressure fluctuations.Conclusion: Although it is important that the surgical approach is individualized in patients with the concurrent presence of a pheochromocytoma and critical ischemic heart disease, cardiac revascularization under alpha-adrenoceptor blockade prior to pheochromocytoma removal is generally the preferred sequence as this carries the lowest risk of complications.Abbreviations: CABG = coronary artery bypass grafting CAD = coronary artery disease

Epigenetic changes provide a frequent mechanism for transcriptional silencing of genes in cancer cells. We previously established that epigenetic mechanisms are important for control of group IIA phospholipase A2 (PLA2G2A) gene transcription in human DU-145 prostate cells. In this study, we analyzed the involvement of such mechanisms in the regulation of five sPLA2 isozymes and the M-type receptor of sPLA2 (sPLA2-R) in human leukemic Jurkat cells. These cells constitutively expressed sPLA2-IB, sPLA2-III, sPLA2-X, and sPLA2-R but not sPLA2-IIA and sPLA2-V. Transcription of sPLA2-IIA and sPLA2-V was, however, detected after exposure of cells to the DNA demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC). Expression of sPLA2-IIA was further enhanced by additional exposure to interferon-γ and blocked by inhibitors of specificity protein 1, nuclear factor κB, and Janus kinase/signal transducer and activator of transcription-dependent pathways. Sequence analysis and methylation-specific polymerase chain reaction of bisulfite-modified genomic DNA revealed two 5′-CpG sites (-111 and -82) in the sPLA2-IIA proximal promoter that were demethylated after 5-aza-dC treatment. These sites may be involved in the DNA binding of specificity protein 1 and other transcription factors. Similar findings after treatment of human U937 leukemia cells with 5-aza-dC indicate that this mechanism of PLA2G2A gene silencing is not restricted to Jurkat and DU-145 cells. These data establish that regulation of sPLA2-IIA and sPLA2-V in Jurkat and other cells involves epigenetic silencing by DNA hypermethylation.

The differentiation of dopamine-producing neurons from chromaffin progenitors might represent a new valuable source for replacement therapies in Parkinson's disease. However, characterization of their differentiation potential is an important prerequisite for efficient engraftment. Based on our previous studies on isolation and characterization of chromaffin progenitors from adult adrenals, this study investigates their potential to produce dopaminergic neurons and means to enhance their dopaminergic differentiation. Chromaffin progenitors grown in sphere culture showed an increased expression of nestin and Mash1, indicating an increase of the progenitor subset. Proneurogenic culture conditions induced the differentiation into neurons positive for neural markers β-III-tubulin, MAP2, and TH accompanied by a decrease of Mash1 and nestin. Furthermore, Notch2 expression decreased concomitantly with a downregulation of downstream effectors Hes1 and Hes5 responsible for self-renewal and proliferation maintenance of progenitor cells. Chromaffin progenitor-derived neurons secreted dopamine upon stimulation by potassium. Strikingly, treatment of differentiating cells with retinoic and ascorbic acid resulted in a twofold increase of dopamine secretion while norepinephrine and epinephrine were decreased. Initiation of dopamine synthesis and neural maturation is controlled by Pitx3 and Nurr1. Both Pitx3 and Nurr1 were identified in differentiating chromaffin progenitors. Along with the gained dopaminergic function, electrophysiology revealed features of mature neurons, such as sodium channels and the capability to fire multiple action potentials. In summary, this study elucidates the capacity of chromaffin progenitor cells to generate functional dopaminergic neurons, indicating their potential use in cell replacement therapies.

Pheochromocytomas are rare endocrine tumors that can present insidiously and remain undiagnosed until death or onset of clear manifestations of catecholamine excess. They are often referred to as one of the ‘great mimics’ in medicine. These tumors can no longer be regarded as a uniform disease entity, but rather as a highly heterogeneous group of chromaffin cell neoplasms with different ages of onset, secretory profiles, locations, and potential for malignancy according to underlying genetic mutations. These aspects all have to be considered when the tumor is encountered, thereby enabling optimal management for the patient. Referral to a center of specialized expertise for the disease should be considered wherever possible. This is not only important for surgical management of patients, but also for post-surgical follow up and screening of disease in patients with a hereditary predisposition to the tumor. While preoperative management has changed little over the last 20 years, surgical procedures have evolved so that laparoscopic resection is the standard of care and partial adrenalectomy should be considered in all patients with a hereditary condition. Follow-up testing is essential and should be recommended and ensured on a yearly basis. Managing such patients must now also take into account possible underlying mutations and the appropriate selection of genes for testing according to disease presentation. Patients and family members with identified mutations then require an individualized approach to management. This includes consideration of distinct patterns of biochemical test results during screening and the appropriate choice of imaging studies for tumor localization according to the mutation and associated differences in predisposition to adrenal, extra-adrenal and metastatic disease.