Adrenocortical adenomas are frequent in the general population and can be associated with autonomous cortisol excess, which comes with increased morbidity and mortality. Altered cAMP/PKA signalling is common in sporadic cortisol-producing adenomas, typically due to somatic mutations in the catalytic subunit α of PKA or the G-protein α subunit, Gαs (GNAS), which activate cAMP signalling. We previously identified a novel p.Lys58Gln GNAS somatic variant in a patient with an adenoma and overt Cushing's syndrome that provided enough evidence to warrant further investigation.

We established cel lines with and without the p.Lys58Gln GNAS variant and evaluated adrenocorticotropic hormone (ACTH) receptor signalling using different downstream methods, and assessed cell viability and apoptosis. The Lys58Gln variant showed a significantly higher basal cAMP, pCREB and CRE luciferase reporter concentration and a greater response to ACTH compared to non mutated GNAS. There was also significantly enhanced cell viability and apoptosis in cells with the Lys58Gln variant.

Our study demonstrated that the Lys58Gln variant is associated with constitutive activation of GNAS signalling, similar to other known mutations in adrenocortical adenomas, potentially representing a new pathogenic mechanism in a subset of patients with adrenal Cushing syndrome. 

Somatic alterations are commonly observed in adrenocortical adenomas including cortisol-producing (CPA) [overt Cushing syndrome (CS) or mild autonomous cortisol secretion (MACS)], aldosterone-producing (APA), and non-functioning (NFAT) tumors. 

Aim of the staudy was to test whether somatic variants could be detected in circulating cell-free DNA (ccfDNA) from patients with adenomas and potentially contribute to management strategies.

• We investigated 44 patients and 23 healthy subjects that served as controls. 

• ccfDNA was extracted from blood samples and quantified with fluorimeter. Tumor DNA was isolated from paraffin embedded tissue in 17/44 cases. 

• Matched ccfDNA/T-DNA were sequenced using a customized panel including 32 genes. 

• Leucocyte DNA was used to filter out germline variants.

Patients with adenomas had higher total ccfDNA concentrations than healthy subjects, with CPA-CS showing the highest ccfDNA levels. Within T-DNA, somatic variants were identified in 53% of adenomas: PRKACA in 2/7 CPA-CS, CTNNB1 in 3/5 CPA-MACS and 1/7 CPA-CS, KCNJ5 in 2/5 APA and CACNA1D in 1/5 APA. Somatic mutations were not detected in any of the investigated ccfDNA samples.

In conclusion, ccfDNA concentrations are higher in patients with CPA-CS. Despite the presence of somatic variants in half of tumor samples, we did not detect any at ccfDNA level. Therefore, this approach appears ineffective for pre-operative detection of genetic alterations.

Serum inflammation-based scores can predict clinical outcome in several cancer types, including adrenocortical carcinoma (ACC). It is however unclear whether the extent of inflammation-based scores alterations in ACC reflects malignancy, steroid excess, or both. We investigated a large retrospective cohort of adrenocortical adenomas (ACA, n = 429) and ACC (n = 61) with available baseline full blood count and hormonal evaluation. We examined the relationship between different inflammation-based scores [neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), systemic immune-inflammation index (SII), and prognostic nutrition index (PNI)] and both malignancy and steroid secretion patterns. All inflammation-based scores differed between ACC and ACA: patients with ACC had higher NLR, PLR, SII and lower LMR and PNI levels compared to ACA. NLR showed a positive correlation with cortisol levels after overnight 1 mg-dexamethasone suppression test, both in ACC and ACA. At multivariable analysis, NLR > 2.6 was independently associated with ACC, 1 mg-DST cortisol levels and age, but not with tumour size. Considering the ACC, NLR and SII were higher and PNI was lower in patients with cortisol excess compared to those without cortisol excess. Finally, LMR and NLR differed between inactive-ACC (n = 10) and inactive-ACA (n = 215). 

Inflammation-based scores are related to steroid secretion both in ACC and ACA. ACCs present a higher grade of inflammation regardless of their hormonal secretion, likely as a feature of malignancy itself.

Primary aldosteronism (PA) causes 5–10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup.  

• We measured 34 steroid metabolites in 24-hour urine samples of patients with primary aldosteronism and healthy controls.
  Machine learning applied to the urinary steroid metabolome was highly accurate in identifying primary aldosteronism cases.

• Aldosterone-producing adenomas harbouring mutations of the KCNJ5 gene had specific urine steroid fingerprints.

• Urine steroid metabolome analysis is a non-invasive candidate test for diagnosing and subtyping primary aldosteronism.

Pheochromocytomas are increasingly diagnosed in incidentally detected adrenal masses. However, the characteristics of incidental pheochromocytomas are unclear. Therefore, we aimed to assess the proportion and clinical, biochemical, radiological, genetic, histopathological, and follow-up characteristics of incidental pheochromocytomas.

A retrospective review was conducted of patients with pheochromocytoma seen between January 2010 and October 2022 at our tertiary care center. The diagnosis was confirmed histologically or by the combined presence of increased plasma and/or urinary metanephrines (MN), indeterminate adrenal mass on cross-sectional imaging, and metaiodobenzylguanidine avidity.

We identified 167 patients with pheochromocytoma; 144 of whom underwent adrenalectomy. Among them, 28% of patients presented with adrenergic symptoms and/or uncontrolled hypertension, while 69% patients presented with an incidentally detected adrenal mass. Incidentally detected patients were olderthan those detected due to clinical suspicion or after genetic screening. Incidentally detected pheochromocytomas were smaller than tumors detected due to adrenergic symptoms/uncontrolled hypertension, but larger than tumors identified by genetic screening. Increased MN excretion showed a similar pattern (symptomatic/uncontrolled hypertension > incidental > genetic screening). 

In conclusion, the majority of pheochromocytomas are diagnosed incidentally and have distinct clinical, radiological, biochemical, and genetic features. Their detection at older age but smaller size may point to a different underlying tumor biology.

Benign adrenocortical tumours are diagnosed in ∼5% of adults and are associated with cortisol excess in 30%-50% of cases. Adrenal Cushing's syndrome (CS) is rare and leads to multiple haematological alterations. However, little is known about the effects of the much more frequent mild autonomous cortisol secretion (MACS) on immune function. The aim of this study was to evaluate the haematological alterations in benign adrenocortical tumours with different degrees of cortisol excess.

We investigated 375 patients: 215 with non-functioning adrenal tumours (NFAT), 138 with MACS, and 22 with CS. We evaluated the relationship between the degree of cortisol excess and full blood count as well as multiple inflammation-based scores, including the neutrophil-to-lymphocyte ratio (NLR), the lymphocyte-to-monocyte ratio (LMR), and the systemic immune-inflammation index (SII).

We observed a gradual and significant increase of leucocytes, neutrophils, and monocytes across the spectrum of cortisol excess, from NFAT over MACS to CS. Neutrophil-to-lymphocyte ratio and SII were significantly higher in both MACS and CS when compared to NFAT. Conversely, LMR was lower in MACS and CS than in NFAT but also significantly lower in CS compared to MACS

In conclusionn, Neutrophil-to-lymphocyte ratio, SII, and LMR correlated with the degree of cortisol excess in benign adrenocortical tumours and were altered in patients with CS and MACS. 

These findings suggest that, similar to clinically overt CS, MACS also affects the immune function, potentially contributing to the MACS-associated comorbidities.

Adrenocortical carcinoma (ACC) is a rare aggressive malignancy with heterogeneous clinical outcomes. Recent studies proposed a combination of clinical/histopathological parameters (S-GRAS score) or molecular biomarkers (BMs) to improve prognostication. We performed a comparative analysis of DNA-based BMs by evaluating their added prognostic value to the S-GRAS score.

A total of 194 formalin-fixed, paraffin-embedded (FFPE) ACC samples were analysed, including a retrospective training cohort (n = 107) and a prospective validation cohort (n = 87). Targeted DNA sequencing and pyrosequencing were used to detect somatic single-nucleotide variations in ACC-specific genes and methylation in the promoter region of paired box 5 (PAX5). The European Network for the Study of Adrenocortical Tumors (ENSAT) tumour stage, age, symptoms at presentation, resection status, and Ki-67 were combined to calculate S-GRAS. Endpoints were overall (OS), progression-free (PFS), and disease-free survival (DFS). Prognostic role was evaluated by multivariable survival analysis and their performance compared by Harrell's concordance index (C index).

In training cohort, an independent prognostic role was confirmed at multivariate analysis for two DNA-based BMs: alterations in Wnt/β-catenin and Rb/p53 pathways and hypermethylated PAX5. These were combined to S-GRAS to obtain a combined (COMBI) score. At comparative analysis, the best discriminative prognostic model was COMBI score in both cohorts for all endpoints, followed by S-GRAS score .

In conclusion, targeted DNA-based BM evaluated on routinely available FFPE samples improves prognostication of ACC beyond routinely available clinical and histopathological parameters. 

This approach may help to better individualise patient's management.