Metabolomics and proteomics in pheochromocytoma and paraganglioma: Translating biochemistry and biology to bedside.

The complexity of omes - the key cellular ensembles (genome and epigenome, transcriptome, proteome, and metabolome) - is becoming increasingly understood in terms of big-data analysis, the omics. Amongst these, proteomics provides a global description of quantitative and qualitative alterations of protein expression (or protein abundance in body fluids) in response to physiologic or pathologic processes while metabolomics offers a functional portrait of the physiological state by quantifying metabolite abundances in biological samples. Here, we summarize how different techniques of proteomic and metabolic analysis can be used to define key biochemical characteristics of pheochromocytomas/paragangliomas (PPGL). The significance of omics in understanding features of PPGL biology that might translate to improved diagnosis and treatment will be highlighted.

Trends in utilization patterns of newer glucose-lowering drugs in US adults with type 2 diabetes and a history of coronary heart disease or heart failure: 2005-2019.

BACKGROUND: The newer glucose-lowering drugs (GLDs), including Glucagon-like peptide-1 receptor agonists (GLP-1RA) and sodium-glucose cotransporter-2 inhibitors (SGLT2i), have demonstrated superior cardio- and renal protective benefits compared to older GLDs in individuals with type 2 diabetes (T2D) at high risk for cardiovascular disease (CVD). OBJECTIVE: This study examined the trends of the newer GLDs use in people with T2D who had a history of coronary heart disease or heart failure in the United States. METHOD: We used 2005-2019 data from the Medical Expenditure Panel Survey (MEPS). Individuals with self-reported diabetes and CVD history were identified. RESULTS: There was a steady increase in the use of GLP-1RA only from 2008 (3 %) to 2019 (21 %) and SGLT2i only from 2014 (5 %) to 2019 (12 %). Individuals with dual use of both newer GLD classes increased from 0.62 % in 2015 to 6 % in 2019. The overall uptake of these two newer drugs in 2019 was less than 40 %. In other words, 60 % of individuals who can substantially benefit from these newer treatments did not use the treatments. CONCLUSION: The use of GLP-1RA and SGLT2i among individuals with T2D and a history of CVD was low and varied by insurance type. Policy-level interventions are needed to improve the use of these newer treatments further. SUMMARY: We examined how newer glucose-lowering drugs are used among individuals with type 2 diabetes and at high risk for coronary heart disease or heart failure in the US. We found that 60 % of individuals who can substantially benefit from these newer treatments did not use the treatments due to the variation of insurance type.

Case report: A novel somatic SDHB variant in a patient with bladder paraganglioma.

Background: Paragangliomas (PGL) are rare neuroendocrine tumors derived from the autonomic nervous system paraganglia. Urinary bladder paragangliomas (UBPGL) originate from the sympathetic neurons of the urinary bladder wall and represent 0.7% of all paragangliomas and <0.05% of all bladder tumors. PGL and UBPGL can be associated with SDHB, SDHD, NF1, and VHL gene variants, with the most common germline alterations found in SDHB and VHL. Case report: We report a case of a 42-year-old woman who presented with menorrhagia/hematuria, uterine leiomyomas, as well as cardiac and bladder masses. The cardiac mass was favored to be a myxoma based on clinical findings, while the bladder mass was diagnosed as UBPGL. A novel SDHB mutation (c.642G>A, p Q214Q), detected in the UBPGL, was proven to be somatic. Although this variant was seemingly synonymous, it was predicted to have a loss of function due to the splice site effect, which was further supported by the immunohistochemical loss of SDHB. Conclusion: This case highlights the challenges of diagnosing an extremely rare entity, bladder paraganglioma, with an emphasis on the multidisciplinary approach to navigate various clinical and imaging findings that may initially be misleading. In addition, a novel loss of function SDHB variant that could have been overlooked as a synonymous variant is herein reported, while also illustrating the importance of both germline and somatic mutation testing.

Some patients with type 2 diabetes may benefit from intensive glycaemic and blood pressure control: A post-hoc machine learning analysis of ACCORD trial data.

AIM: The action to control cardiovascular risk in diabetes (ACCORD) trial showed a neutral average treatment effect of intensive blood glucose and blood pressure (BP) controls in preventing major adverse cardiovascular events (MACE) in individuals with type 2 diabetes. Yet, treatment effects across patient subgroups have not been well understood. We aimed to identify patient subgroups that might benefit from intensive glucose or BP controls for preventing MACE. MATERIALS AND METHODS: As a post-hoc analysis of the ACCORD trial, we included 10 251 individuals with type 2 diabetes. We applied causal forest and causal tree models to identify participant characteristics that modify the efficacy of intensive glucose or BP controls from 68 candidate variables (demographics, comorbidities, medications and biomarkers) at the baseline. The exposure was (a) intensive versus standard glucose control [glycated haemoglobin (HbA1c) <6.0% vs. 7.0%-7.9%], and (b) intensive versus standard BP control (systolic BP <120 vs. <140 mmHg). The primary outcome was MACE. RESULTS: Compared with standard glucose control, intensive one reduced MACE in those with baseline HbA1c <8.5% [relative risk (RR): 0.79, 95% confidence interval (CI): 0.67-0.93] and those with estimated glomerular filtration rate ≥106 ml/min/1.73 m2 (RR: 0.74, 95% CI: 0.55-0.99). Intensive BP control reduced MACE in those with normal high-density lipoprotein levels (women >55 mg/dl, men >45 mg/dl; RR: 0.51, 95% CI: 0.34-0.74). Risk reductions were not significant in other patient subgroups. CONCLUSIONS: Our findings suggest heterogeneous treatment effects of intensive glucose and BP control and could provide biomarkers for future clinical trials to identify more precise HbA1c and BP treatment goals for individualized medicine.

Disordered-to-ordered transitions in assembly factors allow the complex II catalytic subunit to switch binding partners.

Complex II (CII) activity controls phenomena that require crosstalk between metabolism and signaling, including neurodegeneration, cancer metabolism, immune activation, and ischemia-reperfusion injury. CII activity can be regulated at the level of assembly, a process that leverages metastable assembly intermediates. The nature of these intermediates and how CII subunits transfer between metastable complexes remains unclear. In this work, we identify metastable species containing the SDHA subunit and its assembly factors, and we assign a preferred temporal sequence of appearance of these species during CII assembly. Structures of two species show that the assembly factors undergo disordered-to-ordered transitions without the appearance of significant secondary structure. The findings identify that intrinsically disordered regions are critical in regulating CII assembly, an observation that has implications for the control of assembly in other biomolecular complexes.

Management of phaeochromocytoma and paraganglioma in patients with germline SDHB pathogenic variants: an international expert Consensus statement.

Adult and paediatric patients with pathogenic variants in the gene encoding succinate dehydrogenase (SDH) subunit B (SDHB) often have locally aggressive, recurrent or metastatic phaeochromocytomas and paragangliomas (PPGLs). Furthermore, SDHB PPGLs have the highest rates of disease-specific morbidity and mortality compared with other hereditary PPGLs. PPGLs with SDHB pathogenic variants are often less differentiated and do not produce substantial amounts of catecholamines (in some patients, they produce only dopamine) compared with other hereditary subtypes, which enables these tumours to grow subclinically for a long time. In addition, SDHB pathogenic variants support tumour growth through high levels of the oncometabolite succinate and other mechanisms related to cancer initiation and progression. As a result, pseudohypoxia and upregulation of genes related to the hypoxia signalling pathway occur, promoting the growth, migration, invasiveness and metastasis of cancer cells. These factors, along with a high rate of metastasis, support early surgical intervention and total resection of PPGLs, regardless of the tumour size. The treatment of metastases is challenging and relies on either local or systemic therapies, or sometimes both. This Consensus statement should help guide clinicians in the diagnosis and management of patients with SDHB PPGLs.

Internal Carotid Artery Aneurysm Disguised as Pituitary Macroadenoma.

Hypopituitarism due to an internal carotid artery (ICA) aneurysm is rare. We present a case of hypopituitarism and hyperprolactinemia due to a giant right ICA aneurysm. A 56-year-old woman with a history of primary hypothyroidism presented with fatigue, right-sided headache, and blurred vision. Magnetic resonance (MR) of the brain revealed a sellar mass measuring 3.5 × 2.2 cm involving the right cavernous sinus. Initial neurologic examination was unremarkable, and her biochemical evaluation revealed secondary adrenal insufficiency, central hypogonadism, low serum free thyroxine, and mildly elevated serum prolactin, consistent with stalk effect. Hydrocortisone therapy was started for secondary adrenal insufficiency and her levothyroxine dose was adjusted. The patient was referred to neurosurgery for surgical management of her sellar lesion. Preoperative computed tomography angiography (CTA) of the brain revealed a right ICA aneurysm that contacted the optic chiasm and displaced the pituitary gland. The aneurysm was embolized and diverting stents were placed. Repeat laboratory tests showed resolution of the patient's secondary adrenal insufficiency, normalization of serum prolactin, and an increase in serum gonadotropin concentrations to the postmenopausal range. This case highlights that not all sellar lesions are pituitary adenomas, and CTA should be performed in the evaluation of large cavernous sinus lesions to exclude ICA aneurysm.

Clinical consensus guideline on the management of phaeochromocytoma and paraganglioma in patients harbouring germline SDHD pathogenic variants.

Patients with germline SDHD pathogenic variants (encoding succinate dehydrogenase subunit D; ie, paraganglioma 1 syndrome) are predominantly affected by head and neck paragangliomas, which, in almost 20% of patients, might coexist with paragangliomas arising from other locations (eg, adrenal medulla, para-aortic, cardiac or thoracic, and pelvic). Given the higher risk of tumour multifocality and bilaterality for phaeochromocytomas and paragangliomas (PPGLs) because of SDHD pathogenic variants than for their sporadic and other genotypic counterparts, the management of patients with SDHD PPGLs is clinically complex in terms of imaging, treatment, and management options. Furthermore, locally aggressive disease can be discovered at a young age or late in the disease course, which presents challenges in balancing surgical intervention with various medical and radiotherapeutic approaches. The axiom-first, do no harm-should always be considered and an initial period of observation (ie, watchful waiting) is often appropriate to characterise tumour behaviour in patients with these pathogenic variants. These patients should be referred to specialised high-volume medical centres. This consensus guideline aims to help physicians with the clinical decision-making process when caring for patients with SDHD PPGLs.

Metabolomics in paraganglioma: applications and perspectives from genetics to therapy.

Metabolites represent the highest layer of biological information. Their diverse chemical nature enables networks of chemical reactions that are critical for maintaining life by providing energy and building blocks. Quantification by targeted and untargeted analytical methods using either mass spectrometry or nuclear magnetic resonance spectroscopy has been applied to pheochromocytoma/paraganglioma (PPGL) with the long-term goal to improve diagnosis and therapy. PPGLs have unique features that provide useful biomarkers and clues for targeted treatments. First, high production rates of catecholamines and metanephrines allow for specific and sensitive detection of the disease in plasma or urine. Secondly, PPGLs are associated with heritable pathogenic variants (PVs) in around 40% of cases, many of which occur in genes encoding enzymes, such as succinate dehydrogenase (SDH) and fumarate hydratase (FH). These genetic aberrations lead to the overproduction of oncometabolites succinate or fumarate, respectively, and are detectable in tumors and blood. Such metabolic dysregulation can be exploited diagnostically, with the aim to ensure appropriate interpretation of gene variants, especially those with unknown significance, and facilitate early tumor detection through regular patient follow-up. Furthermore, SDHx and FH PV alter cellular pathways, including DNA hypermethylation, hypoxia signaling, redox homeostasis, DNA repair, calcium signaling, kinase cascades, and central carbon metabolism. Pharmacological interventions targeted toward such features have the potential to uncover treatments against metastatic PPGL, around 50% of which are associated with germline PV in SDHx. With the availability of omics technologies for all layers of biological information, personalized diagnostics and treatment is in close reach.

Metabolic Characterization and Glyceraldehyde-3-Phosphate Dehydrogenase-Dependent Regulation of Epithelial Sodium Channels in hPheo1 Wild-type and SDHB Knockdown Cells.

Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the protein expression and activity, respectively, of these ion channels. Our western blot experiments show both ENaC alpha and ASIC1/2 are expressed in both hPheo1 WT and SDHB KD cells, with lower levels of a cleaved 60 kDa form of ENaC in SDHB KD cells. Single-channel patch clamp studies corroborate these results and further indicate channel activity is decreased in SDHB KD cells. Additional experiments showed a more significant decreased membrane potential in SDHB KD cells, which were sensitive to amiloride compared to WT cells. We provide evidence for the differential expression and activity of ENaC and ASIC hybrid channels in hPheo1 WT and SDHB KD cells, providing an important area of investigation in understanding SDHB-related disease.

Case report: Two sisters with a germline CHEK2 variant and distinct endocrine neoplasias.

Genetic testing has become the standard of care for many disease states. As a result, physicians treating patients who have tumors often rely on germline genetic testing results for making clinical decisions. Cases of two sisters carrying a germline CHEK2 variant are highlighted whereby possible other genetic drivers were discovered on tumor analysis. CHEK2 (also referred to as CHK2) loss of function has been firmly associated with breast cancer development. In this case report, two siblings with a germline CHEK2 mutation also had distinct endocrine tumors. Pituitary adenoma and pancreatic neuroendocrine tumor (PNET) was found in the first sibling and pheochromocytoma (PCC) discovered in the second sibling. Although pituitary adenomas, PNETs, and PCC have been associated with NF1 gene mutations, the second sister with a PCC did have proven germline CHEK2 with a pathogenic somatic NF1 mutation. We highlight the clinical point that unless the tumor is sequenced, the real driver mutation that is causing the patient's tumor may remain unknown.

Genetic impairment of succinate metabolism disrupts bioenergetic sensing in adrenal neuroendocrine cancer.

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of the mitochondrial tricarboxylic acid (TCA) cycle enzyme subunit succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect, triggers dysregulation of [Ca2+]i, and aberrantly activates calpain and protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a phospho-signaling cascade where GSK3 inhibition inactivates energy sensing by AMP kinase through dephosphorylation of the AMP kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC. A potent Cdk5 inhibitor, MRT3-007, reverses this phospho-cascade, invoking a senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis.

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.

Germline SUCLG2 Variants in Patients With Pheochromocytoma and Paraganglioma.

BACKGROUND: Pheochromocytoma and paraganglioma (PPGL) are neuroendocrine tumors with frequent mutations in genes linked to the tricarboxylic acid cycle. However, no pathogenic variant has been found to date in succinyl-CoA ligase (SUCL), an enzyme that provides substrate for succinate dehydrogenase (SDH; mitochondrial complex II [CII]), a known tumor suppressor in PPGL. METHODS: A cohort of 352 patients with apparently sporadic PPGL underwent genetic testing using a panel of 54 genes developed at the National Institutes of Health, including the SUCLG2 subunit of SUCL. Gene deletion, succinate levels, and protein levels were assessed in tumors where possible. To confirm the possible mechanism, we used a progenitor cell line, hPheo1, derived from a human pheochromocytoma, and ablated and re-expressed SUCLG2. RESULTS: We describe 8 germline variants in the guanosine triphosphate-binding domain of SUCLG2 in 15 patients (15 of 352, 4.3%) with apparently sporadic PPGL. Analysis of SUCLG2-mutated tumors and SUCLG2-deficient hPheo1 cells revealed absence of SUCLG2 protein, decrease in the level of the SDHB subunit of SDH, and faulty assembly of the complex II, resulting in aberrant respiration and elevated succinate accumulation. CONCLUSIONS: Our study suggests SUCLG2 as a novel candidate gene in the genetic landscape of PPGL. Large-scale sequencing may uncover additional cases harboring SUCLG2 variants and provide more detailed information about their prevalence and penetrance.

Subacute Thyroiditis and Heart Failure in a Patient Presenting With COVID-19.

A 67-year-old male was admitted with shortness of breath and diarrhea. His COVID-19 polymerase chain reaction test was positive, and he was found to be in acute heart failure. Troponin levels were elevated, echocardiogram showed ejection fraction of 24%, and his electrocardiogram was normal. Inflammatory markers were elevated. Further testing revealed suppressed thyroid-stimulating hormone and elevated free thyroxine (T4). Differential diagnosis at this point included possible myocarditis from the viral illness, exacerbation of heart failure from the viral infection or from thyrotoxicosis was considered. Patient's heart failure improved with initiation of heart failure therapies; however, biochemically, his thyroid function tests (TFTs) did not improve, despite empiric methimazole. Thyroid antibody tests were unremarkable. Thyroid ultrasound showed mildly enlarged thyroid gland with no increased vascularity and 5-mm bilateral cysts. Thyroid dysfunction was attributed to subacute thyroiditis from COVID-19, methimazole was tapered, and prednisone was initiated. The patient's TFTs improved. With the ongoing COVID-19 pandemic, it is imperative that clinicians keep a broad differential in individuals presenting with heart failure, and obtaining baseline TFTs may be reasonable. Rapid treatment of the underlying thyroiditis is important in these patients to improve the cardiovascular outcomes. In our experience, steroid therapy showed a rapid improvement in the TFTs.

Vanishing Adrenal Mass in Pregnancy.

OBJECTIVES: The evaluation of an adrenal mass is challenging. We present the case of a 33-year-old pregnant woman who was found to have an adrenal incidentaloma. Four months after the initial imaging, the mass vanished. METHODS: We described the case of a pregnant woman with hypertension and an incidentally found right adrenal mass. RESULTS: A magnetic resonance imaging scan showed a right adrenal mass measuring 7.9 × 3.9 × 3.0 cm with a multilobulated appearance. Initial biochemical testing was concerning for a pheochromocytoma with positive metanephrines during hospitalization while being treated for an infection. Repeat outpatient adrenal hormone results, including metanephrines, were negative. Four months after her initial magnetic resonance imaging scan, the right adrenal mass was no longer present. CONCLUSION: A 33-year-old pregnant woman was found to have a right adrenal mass that later vanished as a result of the resolution of a unilateral adrenal hemorrhage. Predisposing factors to adrenal hemorrhage in the presented case include pregnancy, infection, and hypertension.

Succinate Mediates Tumorigenic Effects via Succinate Receptor 1: Potential for New Targeted Treatment Strategies in Succinate Dehydrogenase Deficient Paragangliomas.

Paragangliomas and pheochromocytomas (PPGLs) are chromaffin tumors associated with severe catecholamine-induced morbidities. Surgical removal is often curative. However, complete resection may not be an option for patients with succinate dehydrogenase subunit A-D (SDHx) mutations. SDHx mutations are associated with a high risk for multiple recurrent, and metastatic PPGLs. Treatment options in these cases are limited and prognosis is dismal once metastases are present. Identification of new therapeutic targets and candidate drugs is thus urgently needed. Previously, we showed elevated expression of succinate receptor 1 (SUCNR1) in SDHB PPGLs and SDHD head and neck paragangliomas. Its ligand succinate has been reported to accumulate due to SDHx mutations. We thus hypothesize that autocrine stimulation of SUCNR1 plays a role in the pathogenesis of SDHx mutation-derived PPGLs. We confirmed elevated SUCNR1 expression in SDHx PPGLs and after SDHB knockout in progenitor cells derived from a human pheochromocytoma (hPheo1). Succinate significantly increased viability of SUCNR1-transfected PC12 and ERK pathway signaling compared to control cells. Candidate SUCNR1 inhibitors successfully reversed proliferative effects of succinate. Our data reveal an unrecognized oncometabolic function of succinate in SDHx PPGLs, providing a growth advantage via SUCNR1.

Real-World Experience With Automated Insulin Pump Technology in Veterans With Type 1 Diabetes.

BACKGROUND: Advancements in diabetes technology now allow insulin pump and continuous glucose monitor (CGM) technology to be a part of usual US Department Veterans Affairs (VA) clinical care. The automated insulin pump (AIP) delivers insulin automatically based on CGM readings. In randomized clinical trials the closed-loop system has shown to improve glycemic control in children and younger adults with type 1 diabetes mellitus (T1DM) while preventing hypoglycemia. However, its safety and efficacy is less well known in older veterans with T1DM. In this VA pilot study, we aimed to assess AIP technology in the real world of an older population of veterans with T1DM followed in the outpatient setting. METHODS: Thirty-seven patients with T1DM new to AIP seen at the Malcom Randall VA Medical Center in Gainesville, Florida, were evaluated between March and December of 2018 on an Medtronic Minimed 670G Insulin Pump System. We collected demographic as well as clinical data before and after the initiation of AIP, including standard insulin pump/CGM information (sensor wear, time in target glucose range, time in automated mode, other). RESULTS: At the time of the initiation of AIP, the mean (SD) age of patients was 59.1 (14.4) years; 35 identified as male and 2 as female. The mean (SD) duration of T1DM was 25.3 (12.0) years. Patients transitioned from either insulin injections or other non-AIP pump to AIP safely-there was no increase in hypoglycemia, and the mean (SD) hemoglobin A1c decreased from 7.6% (0.8) to 7.3% (0.8) by the second follow-up visit. CONCLUSION: In this real-world study, AIP use was both safe and viable as a tool for T1DM management with older veterans. This technology further engaged veterans in monitoring their blood sugars and achieving more optimal glycemic control. Future long-term, larger studies are much needed in this setting.

Activation of RAS Signalling is Associated with Altered Cell Adhesion in Phaeochromocytoma.

Phaeochromocytomas and paragangliomas (PPGLs) are neuroendocrine catecholamine-producing tumours that may progress into inoperable metastatic disease. Treatment options for metastatic disease are limited, indicating a need for functional studies to identify pharmacologically targetable pathophysiological mechanisms, which require biologically relevant experimental models. Recently, a human progenitor phaeochromocytoma cell line named "hPheo1" was established, but its genotype has not been characterised. Performing exome sequencing analysis, we identified a KIF1B T827I mutation, and the oncogenic NRAS Q61K mutation. While KIF1B mutations are recurring somatic events in PPGLs, NRAS mutations have hitherto not been detected in PPGLs. Therefore, we aimed to assess its implications for the hPheo1 cell line, and possible relevance for the pathophysiology of PPGLs. We found that transient downregulation of NRAS in hPheo1 led to elevated expression of genes associated with cell adhesion, and enhanced adhesion to hPheo1 cells' extracellular matrix. Analyses of previously published mRNA data from two independent PPGL patient cohorts (212 tissue samples) revealed a subcluster of PPGLs featuring hyperactivated RAS pathway-signalling and under-expression of cell adhesion-related gene expression programs. Thus, we conclude that NRAS activity in hPheo1 decreases adhesion to their own extracellular matrix and mirrors a transcriptomic RAS-signalling-related phenomenon in PPGLs.