First-in-human in-vivo depiction of paraganglioma metabolism by hyperpolarised 13C-magnetic resonance.

Phaeochromocytomas (PCC) and paragangliomas (PGL), cumulatively referred to as PPGLs, are neuroendocrine tumours arising from neural crest-derived cells in the sympathetic and parasympathetic nervous systems. Predicting future tumour behaviour and the likelihood of metastatic disease remains problematic as genotype-phenotype correlations are limited, the disease has variable penetrance and, to date, no reliable molecular, cellular or histological markers have emerged. Tumour metabolism quantification can be considered as a method to delineating tumour aggressiveness by utilising hyperpolarised 13 C-MR (HP-MR). The technique may provide an opportunity to non-invasively characterise disease behaviour. Here, we present the first instance of the analysis of PPGL metabolism via HP-MR in a single case.

Somatic EPAS1 Variants in Pheochromocytoma and Paraganglioma in Patients With Sickle Cell Disease.

CONTEXT: Somatic EPAS1 variants account for 5% to 8% of all pheochromocytoma and paragangliomas (PPGL) but are detected in over 90% of PPGL in patients with congenital cyanotic heart disease, where hypoxemia may select for EPAS1 gain-of-function variants. Sickle cell disease (SCD) is an inherited hemoglobinopathy associated with chronic hypoxia and there are isolated reports of PPGL in patients with SCD, but a genetic link between the conditions has yet to be established. OBJECTIVE: To determine the phenotype and EPAS1 variant status of patients with PPGL and SCD. METHODS: Records of 128 patients with PPGL under follow-up at our center from January 2017 to December 2022 were screened for SCD diagnosis. For identified patients, clinical data and biological specimens were obtained, including tumor, adjacent non-tumor tissue and peripheral blood. Sanger sequencing of exons 9 and 12 of EPAS1, followed by amplicon next-generation sequencing of identified variants was performed on all samples. RESULTS: Four patients with both PPGL and SCD were identified. Median age at PPGL diagnosis was 28 years. Three tumors were abdominal paragangliomas and 1 was a pheochromocytoma. No germline pathogenic variants in PPGL-susceptibility genes were identified in the cohort. Genetic testing of tumor tissue detected unique EPAS1 variants in all 4 patients. Variants were not detected in the germline, and 1 variant was detected in lymph node tissue of a patient with metastatic disease. CONCLUSION: We propose that somatic EPAS1 variants may be acquired through exposure to chronic hypoxia in SCD and drive PPGL development. Future work is needed to further characterize this association.

Diagnostic utility of 11 C-methionine PET/CT in primary hyperparathyroidism in a UK cohort: A single-centre experience and literature review.

OBJECTIVE: Primary hyperparathyroidism is a common endocrine disorder, with 80% of all cases usually caused by one single hyperfunctioning parathyroid adenoma. Conventional imaging modalities for the diagnostic work-up of primary hyperparathyroidism (PHPT) include ultrasound of the neck, 99mTc-sestamibi scintigraphy, and four-dimensional computed tomography (4D-CT). However, the role of other imaging modalities, such as 11C-methionine PET/CT, in the care pathway for PHPT is currently unclear. Here, we report our experience of the diagnostic utility of 11C-methionine PET/CT in a single-center patient cohort (n = 45). DESIGN: Retrospective single-center cohort study. PATIENTS AND MEASUREMENTS: The data of eligible patients that underwent 11C-methionine PET/CT between 2014 and 2022 at Addenbrooke's Hospital (Cambridge, UK) were collected and analyzed. The clinical utility of imaging modalities was determined by comparing the imaging result with histopathological and biochemical outcomes following surgery. RESULTS: In patients with persistent primary hyperparathyroidism following previous surgery, 11C-methionine PET/CT identified a candidate lesion in 6 of 10 patients (60.0%), and histologically confirmed in 5 (50.0%). 11C-methionine PET/CT also correctly identified a parathyroid adenoma in 9 out of 12 patients (75.0%) that failed to be localized on other imaging modalities. 11C-methionine PET/CT had a sensitivity of 70.0% (95% CI 55.8 - 84.2%) for the detection of parathyroid adenomas. CONCLUSIONS: This study highlights a diagnostic role for 11C-methionine PET/CT in patients that have undergone unsuccessful prior surgery or have equivocal or negative prior imaging results, aiding localization and a targeted surgical approach.

Elevated sphingosine-1-phosphate lyase leads to increased metabolism and reduced survival in adrenocortical carcinoma.

OBJECTIVE: Adrenocortical carcinomas (ACCs) are invasive tumours arising in the adrenal cortex, and steroidogenic tumours are associated with worse prognostic outcomes. Loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1) cause primary adrenal insufficiency and as a key degradative enzyme in the sphingolipid pathway, SGPL1 also influences the balance of pro-proliferative and pro-apoptotic sphingolipids. We, therefore, hypothesized increased SGPL1 may be linked to increased disease severity in ACC. DESIGN: Analyse SGPL1 expression impact on patient survival and adrenal cancer cell phenotype. We analysed two ACC cohorts with survival and corresponding transcriptomic data, focusing on SGPL1 and sphingolipid pathway genes. In vitro, we generated SGPL1-knockout and overexpressing H295R adrenocortical cells to investigate the role of SGPL1 in cell signalling in ACCs. RESULTS: We found increased expression of several sphingolipid pathway receptors and enzymes, most notably SGPL1 correlated with reduced patient survival in both cohorts. Overexpression of SGPL1 in the H295R cell line increased proliferation and migration while reducing apoptosis, while SGPL1 knockout had the opposite effect. RNA-seq revealed a global increase in the expression of genes in the electron transport chain in overexpressing cells, correlating with increased aerobic respiration and glycolysis. Furthermore, the opposite phenotype was seen in cells lacking SGPL1. We subsequently found the increased proliferation is linked to metabolic substrate availability and increased capacity to use different fuel sources, but particularly glucose, in overexpressing cells. CONCLUSIONS: We, therefore, propose that SGPL1-overexpressing ACC tumours reduce patient survival by increasing fuel usage for anabolism and energy production to facilitate growth and invasion.

SDHC phaeochromocytoma and paraganglioma: A UK-wide case series.

OBJECTIVE: Phaeochromocytomas and paragangliomas (PPGL) are rare, but strongly heritable tumours. Variants in succinate dehydrogenase (SDH) subunits are identified in approximately 25% of cases. However, clinical and genetic information of patients with SDHC variants are underreported. DESIGN: This retrospective case series collated data from 18 UK Genetics and Endocrinology departments. PATIENTS: Both asymptomatic and disease-affected patients with confirmed SDHC germline variants are included. MEASUREMENTS: Clinical data including tumour type and location, surveillance outcomes and interventions, SDHC genetic variant assessment, interpretation, and tumour risk calculation. RESULTS: We report 91 SDHC cases, 46 probands and 45 non-probands. Fifty-one cases were disease-affected. Median age at genetic diagnosis was 43 years (range: 11-79). Twenty-four SDHC germline variants were identified including six novel variants. Head and neck paraganglioma (HNPGL, n = 30, 65.2%), extra-adrenal paraganglioma (EAPGL, n = 13, 28.2%) and phaeochromocytomas (PCC) (n = 3, 6.5%) were present. One case had multiple PPGLs. Malignant disease was reported in 19.6% (9/46). Eight cases had non-PPGL SDHC-associated tumours, six gastrointestinal stromal tumours (GIST) and two renal cell cancers (RCC). Cumulative tumour risk (95% CI) at age 60 years was 0.94 (CI: 0.79-0.99) in probands, and 0.16 (CI: 0-0.31) in non-probands, respectively. CONCLUSIONS: This study describes the largest cohort of 91 SDHC patients worldwide. We confirm disease-affected SDHC variant cases develop isolated HNPGL disease in nearly 2/3 of patients, EAPGL and PCC in 1/3, with an increased risk of GIST and RCC. One fifth developed malignant disease, requiring comprehensive lifelong tumour screening and surveillance.

Cabergoline reduces 3-methoxytyramine in a SDHC patient with metastatic paraganglioma and prolactinoma.

SUMMARY: We observed a novel therapeutic response with cabergoline in a male patient with a dopamine-secreting head and neck paraganglioma (HNPGL), macroprolactinoma and germline succinate dehydrogenase C mutation (SDHC). The macroprolactinoma was treated with cabergoline which gave an excellent response. He was found to have raised plasma 3-methoxytyramine of 1014 pmol/L (NR: 0-180 pmol/L); but it was unclear if this was a drug-induced phenomenon from dopamine agonist (DA) therapy. Cabergoline was stopped for 4 weeks and the 3-methoxytyramine level increased significantly to 2185 pmol/L, suggesting a biochemical response of his HNPGL. Subsequently, Gallium-68 Dotatate PET and MRI (Gallium-68 Dotatate PET/MRI) demonstrated a second lesion in the sacrum. Both the HNPGL and metastatic sacral deposit received external beam radiotherapy with a good biochemical and radiological response. CONCLUSION: Our case report highlights the rare potential of germline SDHC mutations causing metastatic paraganglioma and concurrent pituitary tumours. Cabergoline treatment may lower elevated 3-methoxytyramine levels and, therefore, mask the biochemical evidence of metastatic disease but also may have therapeutic relevance in dopamine-secreting pheochromocytomas/paragangliomas (PPGLs). LEARNING POINTS: Several neuroendocrine tumours (NETs) express dopamine D2 and D4 receptors. In this case report, cabergoline significantly reduced plasma 3-methoxytyramine level in a patient with functional HNPGL. Cabergoline might have therapeutic relevance in dopamine-secreting PPGLs. Paragangliomas associated with SDHC mutation classically present with asymptomatic non-functional HNPGL and have rare metastatic potential. The association of pheochromocytoma or paraganglioma and pituitary adenoma is now a well-described rare association (<1%), designated as the three P association. While the three P association is most commonly seen with succinate dehydrogenase B and D mutations, it has also been described in patients with SDHA and SDHC mutations. Cabergoline treatment may lower elevated 3-methoxytyramine levels and mask the biochemical evidence of metastatic disease. Regular functional imaging with Gallium-68 Dotatate PET/MRI provides better evidence of metastatic disease.

Low-grade Cortisol Cosecretion Has Limited Impact on ACTH-stimulated AVS Parameters in Primary Aldosteronism.

CONTEXT: In primary aldosteronism, cosecretion of cortisol may alter cortisol-derived adrenal venous sampling indices. OBJECTIVE: To identify whether cortisol cosecretion in primary aldosteronism alters adrenal venous sampling parameters and interpretation. DESIGN: Retrospective case-control study. SETTING: A tertiary referral center. PATIENTS: 144 adult patients with primary aldosteronism who had undergone both adrenocorticotropic hormone-stimulated adrenal venous sampling and dexamethasone suppression testing between 2004 and 2018. MAIN OUTCOME MEASURES: Adrenal venous sampling indices including adrenal vein aldosterone/cortisol ratios and the selectivity, lateralization, and contralateral suppression indices. RESULTS: 21 (14.6%) patients had evidence of cortisol cosecretion (defined as a failure to suppress cortisol to ≤50 nmol/L post dexamethasone). Patients with evidence of cortisol cosecretion had a higher inferior vena cava cortisol concentration (P = .01) than those without. No difference was observed between the groups in terms of selectivity index, lateralization index, lateralization of aldosterone excess, or adrenal vein cannulation rate. CONCLUSIONS: Cortisol cosecretion alters some parameters in adrenocorticotrophic hormone-stimulated adrenal venous sampling but does not result in alterations in patient management.

The value of 18F-FDG PET-CT and 131I-norcholesterol scintigraphy in the characterization of high-risk adrenal masses.

PURPOSE: To assess the value of F-FDG PET-computed tomography (CT) and I-norcholesterol scintigraphy in noninvasive characterization of high-risk adrenal lesions using surgical pathology as the gold standard. METHODS: We retrospectively reviewed clinical cases referred to the adrenal multidisciplinary team in a tertiary centre over the last 6 years. Inclusion criteria were the presence of indeterminate adrenal lesions and performance of combined imaging with 2-deoxy-2-[fluorine-18] fluoro- D-glucose Positron emission tomography/ computed tomography and I-norcholesterol scans. The accuracy of CT, PET-CT and I-norcholesterol findings was assessed by comparison with the postoperative histopathological outcome. RESULTS: Sixteen patients fulfilled the inclusion criteria. Ten underwent unilateral adrenalectomy, and six had clinical follow-up. The number of cases categorized as concerning on the basis of unenhanced CT, F-FDG PET-CT and I-norcholesterol was 11, 9 and 2, respectively. The mean diameter of adrenal lesions was 4.5 ± 1.9 cm. Average SUVmax of the FDG-avid adrenal lesions was 5.0 ± 2.0 (range 3.5-9.7). Fourteen adrenal masses showed I-norcholesterol uptake. All adrenal masses turned out to be benign lesions. CONCLUSION: Conventional CT and FDG PET parameters are not adequately specific for determination of a benign lesion in this selected cohort of high-risk patients. Use of I-norcholesterol in this patient cohort may provide additional value.

DLK1/PREF1 marks a novel cell population in the human adrenal cortex.

The adrenal cortex governs fundamental metabolic processes though synthesis of glucocorticoid, mineralocorticoids and androgens. Studies in rodents have demonstrated that the cortex undergoes a self-renewal process and that capsular/subcapsular stem/progenitor cell pools differentiate towards functional steroidogenic cells supporting the dynamic centripetal streaming of adrenocortical cells throughout life. We previously demonstrated that the Notch atypical ligand Delta-like homologue 1 (DLK1)/preadipocyte factor 1 (PREF1) is expressed in subcapsular Sf1 and Shh-positive, CYP11B1-negative and CYP11B2-partially positive cortical progenitor cells in rat adrenals, and that secreted DLK1 can modulate GLI1 expression in H295R cells. Here we show that the human adrenal cortex remodels with age to generate clusters of relatively undifferentiated cells expressing DLK1. These clusters (named DLK1-expressing cell clusters or DCCs) increased with age in size and were found to be different entities to aldosterone-producing cell clusters, another well-characterized and age-dependent cluster structure. DLK1 was markedly overexpressed in adrenocortical carcinomas but not in aldosterone-producing adenomas. Thus, this data identifies a novel cell population in the human adrenal cortex and might suggest a yet-to be identified role of DLK1 in the pathogenesis of adrenocortical carcinoma in humans.

Adrenal cancer in neurofibromatosis type 1: case report and DNA analysis.

UNLABELLED: Adrenal cortical carcinoma (ACC) has previously only been reported in eight patients with type 1 neurofibromatosis (NF1). There has not been any clear evidence of a causal association between NF1 gene mutations and adrenocortical malignancy development. We report the case of a 49-year-old female, with no family history of endocrinopathy, who was diagnosed with ACC on the background of NF1, due to a novel germline frame shift mutation (c.5452_5453delAT) in exon 37 of the NF1 gene. A left adrenal mass was detected by ultrasound and characterised by contrast computerised tomography (CT) scan. Biochemical tests showed mild hypercortisolism and androgen excess. A 24-h urinary steroid profile and (18)flouro deoxy glucose PET suggested ACC. An open adrenalectomy was performed and histology confirmed ACC. This is the first reported case with DNA analysis, which demonstrated the loss of heterozygosity (LOH) at the NF1 locus in the adrenal cancer, supporting the hypothesis of an involvement of the NF1 gene in the pathogenesis of ACC. LOH analysis of the tumour suggests that the loss of neurofibromin in the adrenal cells may lead to tumour formation. LEARNING POINTS: ACC is rare but should be considered in a patient with NF1 and adrenal mass when plasma metanephrines are normal.Urinary steroid metabolites and PET/CT are helpful in supporting evidence for ACC.The LOH at the NF1 region of the adrenal tumour supports the role of loss of neurofibromin in the development of ACC.

Bioluminescence resonance energy transfer reveals the adrenocorticotropin (ACTH)-induced conformational change of the activated ACTH receptor complex in living cells.

The melanocortin 2 receptor (MC2R) accessory protein (MRAP) is a small single-transmembrane domain protein that plays a pivotal role in the function of the MC2R. The pituitary hormone, ACTH, acts via this receptor complex to stimulate adrenal steroidogenesis. Using both coimmunoprecipitation and bioluminescence resonance energy transfer (BRET), we show that the MC2R is constitutively homodimerized in cells. Furthermore, consistent with previous data, we also show that MRAP exists as an antiparallel homodimer. ACTH enhanced the BRET signal between MC2R homodimers as well as MC2R-MRAP heterodimers. However, ACTH did not enhance the physical interaction between these dimers as determined by coimmunoprecipitation. Real-time BRET analysis of the MRAP-MC2R interaction revealed two distinct phases of the ACTH-dependent BRET increase, an initial complex series of changes occurring over the first 2 min and a later persistent increase in BRET signal. The slower ACTH-dependent phase was inhibited by the protein kinase A inhibitor KT5720, suggesting that signal transduction was a prerequisite for this later conformational change. The MRAP-MC2R BRET approach provides a unique tool with which to analyze the activation of this receptor.

Phenotypic characteristics of familial glucocorticoid deficiency (FGD) type 1 and 2.

CONTEXT: Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder as a result of mutation in genes encoding either the ACTH receptor [melanocortin 2 receptor (MC2R)] or its accessory protein [melanocortin 2 receptor accessory protein (MRAP)]. The disorder is known as FGD type 1 and 2, respectively. OBJECTIVE: The aim of the study was to compare the phenotype/genotype relationships between FGD 1 and 2. DESIGN AND PATIENTS: Forty patients with missense MC2R mutations and 22 patients with MRAP mutations were included. Forty-four of these patients had been referred for genetic screening and 18 were patients published by other authors. RESULTS: The median age at presentation for FGD type 1 was variable at 2.0 years; range 0.02-16 years, and this was associated with unusually tall stature, mean height SDS + 1.75 +/- 1.53 (mean +/- SD). In contrast, FGD type 2 presented at a much earlier median age (0.08 years; range at birth to 1.6 years) (P < 0.01) and patients were of normal height SDS + 0.12 +/- 1.35 (P < 0.001). No differences in baseline cortisol or ACTH levels were seen between FGD types 1 and 2. CONCLUSION: FGD type 2 appears to present earlier. This may reflect the functional significance of the underlying mutations in that all MRAP mutations are nonsense or splice site mutations that result in abolition of a functional protein, whereas most of the MC2R mutations are missense mutations and give rise to proteins with some residual function. Tall stature is associated with mutations in MC2R but not in MRAP. There were no other significant clinical distinctions between the two.

The genetics of familial glucocorticoid deficiency.

Familial glucocorticoid deficiency is an autosomal recessive disorder resulting from defects in the action of adrenocorticotropic hormone (ACTH) to stimulate glucocorticoid synthesis in the adrenal. Production of mineralocorticoids by the adrenal is normal. Patients present in early life with low or undetectable cortisol and--because of the failure of the negative feedback loop to the pituitary and hypothalamus--grossly elevated ACTH levels. About half of all cases result from mutations in the ACTH receptor (melanocortin 2 receptor) or from mutations in the melanocortin 2 receptor accessory protein (MRAP), but other genetic causes of this potentially lethal disorder remain to be discovered.

MRAP and MRAP2 are bidirectional regulators of the melanocortin receptor family.

The melanocortin receptor (MCR) family consists of 5 G protein-coupled receptors (MC1R-MC5R) with diverse physiologic roles. MC2R is a critical component of the hypothalamic-pituitary-adrenal axis, whereas MC3R and MC4R have an essential role in energy homeostasis. Mutations in MC4R are the single most common cause of monogenic obesity. Investigating the way in which these receptors signal and traffic to the cell membrane is vital in understanding disease processes related to MCR dysfunction. MRAP is an MC2R accessory protein, responsible for adrenal MC2R trafficking and function. Here we identify MRAP2 as a unique homologue of MRAP, expressed in brain and the adrenal gland. We report that MRAP and MRAP2 can interact with all 5 MCRs. This interaction results in MC2R surface expression and signaling. In contrast, MRAP and MRAP2 can reduce MC1R, MC3R, MC4R, and MC5R responsiveness to [Nle4,D-Phe7]alpha-melanocyte-stimulating hormone (NDP-MSH). Collectively, our data identify MRAP and MRAP2 as unique bidirectional regulators of the MCR family.

Functional consequence of a novel Y129C mutation in a patient with two contradictory melanocortin-2-receptor mutations.

CONTEXT: Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disease, characterised by isolated glucocorticoid deficiency in the absence of mineralocorticoid deficiency. Inactivating mutations in the ACTH receptor (melanocortin-2-receptor, MC2R) are well described and account for approximately 25% of cases. By contrast, activating MC2R mutations are extremely rare. PATIENT: We report a child of Saudi Arabian origin who was diagnosed with FGD following hypoglycaemic episodes that resulted in spastic quadriplegia. METHODS AND RESULTS: MC2R gene analysis revealed an unusual combination of two homozygous missense mutations, consisting of the novel mutation Y129C and the previously described F278C activating mutation. Parents were heterozygous at both of these sites. In vitro analysis of the Y129C mutation using a fluorescent cell surface assay showed that this mutant was unable to reach the cell surface in CHO cells stably transfected with MC2R accessory protein (MRAP), despite the demonstration of an interaction with MRAP by co-immunoprecipitation. The double mutant Y129C-F278C also failed to traffic to the cell surface. CONCLUSION: The tyrosine residue at position 129 in the second intracellular loop is critical in MC2R folding and/or trafficking to the cell surface. Furthermore, the absence of cell surface expression of MC2R would account for the lack of activation of the receptor due to the F278C mutation located at the C-terminal tail. We provide a novel molecular explanation for a child with two opposing mutations causing severe FGD.