OBJECTIVE:Advanced adrenocortical carcinoma (ACC) is treated with mitotane alone or combined with cytotoxic chemotherapy, yet outcomes remain poor and prognostic models in this setting are lacking. This study aimed to evaluate the prognostic value of clinical parameters in a large cohort of patients with advanced ACC undergoing systemic therapy. METHODS:Multicenter, international cohort study investigating 418 patients with advanced ACC (61.5% = women, median age = 52 years) from 11 centers. Patients received mitotane monotherapy (n = 161), etoposide + doxorubicin + cisplatin ± mitotane (n = 178), or second-line regimens (gemcitabine + capecitabine ± mitotane or temozolomide + mitotane, n = 79). Variables included age, cortisol excess, performance status (ECOG-PS), tumor burden, and neutrophil-to-lymphocyte ratio (NLR) at start of therapy. Outcomes were overall survival (OS), time to progression (TTP), and best objective response. RESULTS:Tumor burden, cortisol excess, ECOG-PS, and NLR ≥5 independently predicted shorter OS (hazard ratio [HR] 1.55-2.68). We developed an integrated ENSAT Risk Score for Advanced ACC combining these variables: tumor burden (0-2), cortisol excess (0/1), ECOG-PS (0-2), and NLR (0/1). A score >2 (poor-risk) was significantly associated with worse OS and TTP across all treatment groups (HRs for OS: 3.05-3.96; TTP: 2.53-3.08). It also predicted poorer response to mitotane (P < .01) and second-line therapies (P = .04). CONCLUSIONS:The ENSAT Risk Score for Advanced ACC is a practical, prognostic tool for patients with advanced ACC receiving systemic therapy. Based on accessible clinical and biochemical markers, it can support treatment decisions and facilitate informed discussions in routine care.

BACKGROUND:Limited availability of corticotropin-releasing hormone (CRH) currently complicates the differentiation of adrenocorticotropin (ACTH)-dependent Cushing's syndrome (CS). The diagnostic value of common screening tests in distinguishing Cushing's disease (CD) from ectopic CS (ECS) remains unclear. OBJECTIVE:To assess the diagnostic performance of screening tests, alone and in combination, in differentiating CD from ECS. METHODS:Retrospective multicenter study enrolling patients with confirmed ACTH-dependent CS and available screening tests at diagnosis. Data are expressed as multiples of upper limit of normal (×ULN). Optimal cut-offs were determined using Youden's Index. Combination with composite score models and machine learning algorithm were performed. RESULTS:A total of 566 patients were included (509 [90%] with CD). The optimal morning ACTH cut-off was 1.8×ULN (sensitivity 74%, specificity 77%, AUC=0.776 [CI-95% .688-.853]). 24h-urinary free cortisol (24h-UFC) showed the best performance (cut-off 5.9×ULN, sensitivity 72%, specificity 83%, AUC=0.854 [.816-.923]), followed by the 1 mg dexamethasone suppression test (12.8×ULN, sensitivity 70%, specificity 86%, AUC=.828 [.740-.906]). Using a composite score, a cut-off of 1.5 yielded 78% sensitivity and 92% specificity (AUC=0.865 [.798-.933]). Combining this score with findings derived from pituitary magnetic resonance imaging (MRI), sensitivity and specificity were 88% and 85% (AUC=0.931 [.887-.974]). Machine learning algorithm (balanced random forest) including screening tests-results yielded a sensitivity of 71% and specificity of 84% (AUC=0.853 [.741-.966]), while including the MRI-findings variant achieved a sensitivity and specificity of 76% and 91% (AUC=0.902 [.805-.999]). CONCLUSION:Combining screening tests in composite scoring and machine learning differentiates subtypes of ACTH-dependent CS and offers a potential diagnostic alternative to the CRH stimulation test.

BACKGROUND:Mild autonomous cortisol secretion (MACS), the most common hormonal abnormality in adrenal incidentalomas, is associated with increased cardiometabolic risk. MACS is defined by cortisol concentrations higher than 50 nmol/L after a 1-mg overnight dexamethasone suppression test (1-mg DST). The prognostic value of a single test remains uncertain. We examined longitudinal changes in 1-mg DST results, cumulative cortisol exposure, and their associations with cardiometabolic outcomes and mortality. METHODS:In this retrospective cohort study conducted in 25 adrenal centres that are part of the European Network for the Study of Adrenal Tumours consortium across 14 countries with adults (aged ≥18 years) with benign adrenal incidentalomas diagnosed from Jan 1, 2000, to Dec 1, 2020, two or more 1-mg DSTs, and follow-up of at least 36 months, we used multivariable Cox models to assess associations between longitudinal 1-mg DST results and all-cause mortality and cardiovascular and thrombotic events. Patients with Cushing's syndrome, primary aldosteronism, phaeochromocytoma, or androgen-secreting tumour at baseline; active malignancy within 36 months of incidentaloma diagnosis; or suspicion of unreliable 1-mg DST results were excluded, along with patients taking oral glucocorticoids, strong CYP3A4 modulators, adrenal enzyme inhibitors, oral oestrogen, or selective oestrogen receptor modulators. Cumulative cortisol exposure was estimated from serial 1-mg DSTs. Restricted mean survival time (RMST) quantified differences in event-free time. Data were collected from the electronic health records of all eligible patients at each participating centre. FINDINGS:Among 2525 patients (median follow-up 80 months [IQR 49-122]), 563 (22·3%) had changes in 1-mg DST results leading to a change in diagnosis, most within 3 years of their baseline 1-mg DST. Patients with persistently abnormal 1-mg DST results (ie, MACS-to-MACS patients; n=839) were older and had a greater cardiometabolic burden than those with persistently normal results (ie, non-functioning adrenal tumours [NFAT]-to-NFAT patients; n=1103). MACS-to-MACS patients had a higher rate of worsening hypertension (adjusted hazard ratio 1·34 [95% CI 1·03-1·73]) and a shorter event-free time for worsening hypertension (10-year RMST 60·4 months [56·8-75·5] vs 86·1 months [79·1-93·4]) than NFAT-to-NFAT patients. In crude analyses, patients with higher baseline post-1-mg DST cortisol, patients with greater cumulative cortisol exposure, and MACS-to-MACS patients had shorter survival and event-free time; however, these associations were not independent of age and baseline cardiometabolic risk factors after multivariable adjustment. INTERPRETATION:Longitudinal 1-mg DST changes are common. MACS-to-MACS patients had worsening hypertension, but rates of mortality and cardiovascular or thrombotic events were not significantly associated with 1-mg DST trajectories after adjustment for age and cardiovascular risk factors. These findings identify patients with persistently abnormal 1-mg DST results as a group with high cardiometabolic risk that warrants closer attention to modifiable risk factors. Prospective studies are needed to establish the clinical significance of repeated 1-mg DSTs for risk stratification. FUNDING:National Institute for Health and Care Research Birmingham Biomedical Research Centre, Horizon Europe 2022, and Deutsche Forschungsgemeinschaft.

OBJECTIVE:Distress, fear of progression (FOP), and depression are common in cancer patients and negatively impact patients' health-related quality of life (HRQOL). However, no studies have explored these aspects in adrenocortical carcinoma (ACC), yet. METHODS:We conducted a single-centre observational study in adults (≥18 years) with ACC. We analysed questionnaires addressing FOP, distress (National Comprehensive Cancer Network distress thermometer), depression (patient health questionnaire, PHQ-9), and HRQOL (EORTC-QLQ-C30). Potentially influencing factors were evaluated. Multiple linear regression on HRQOL subscales was performed to identify associated psychosomatic correlations. RESULTS:Within 12 months, 105 patients were included (56.2% female; median age 49 years, 14.3% newly diagnosed, 46.7% tumour-free, 39.0% metastatic). The majority experienced an elevated level of distress (62%) and of FOP (59%). PHQ-9 results suggested major depression in 41%. Of note, these results were neither influenced significantly by tumour burden, hormone excess nor by cancer treatment. Patients reported moderate functioning scores (median 62.2) and moderate symptom burden (median 33.3) in the EORTC-QLQ-C30. Fatigue and insomnia were prominent symptoms. Tumour burden and treatment affected functioning and symptoms, with significant differences in appetite loss, diarrhoea, nausea, vomiting, and financial difficulties. Hormone excess showed no significant impact on these measures. Our questionnaires indicated a need for psychosomatic counselling in 80% of patients. CONCLUSIONS:Patients with ACC show high values for distress, FOP, and depression, which can only partly be explained by clinical characteristics. Our study suggests that psychosomatic support should be offered to all patients with ACC.

BACKGROUND:Treatment for patients with bilateral adrenal tumours and cortisol excess is not standardised and poses a therapeutic dilemma. Untreated cortisol excess is associated with cardiometabolic morbidity and mortality, but bilateral adrenalectomy causes adrenal insufficiency and possibly life-threatening adrenal crises. Data on cardiovascular outcomes by treatment modality are scarce. In this study we aimed to evaluate mid-term and long-term clinical and biochemical outcomes in patients with bilateral adrenal tumours and cortisol excess by treatment strategy and diagnosis. METHODS:This retrospective, international cohort study (in 30 centres across 10 countries in Europe plus Singapore and the USA) included patients with bilateral adrenal tumours of 10 mm or larger, post-dexamethasone serum cortisol concentration of 50 nmol/L or higher, and at least 36 months of follow-up, with data collection beween Feb 2, 2024, and Jan 31, 2025. Patients were excluded if they had adrenocorticotropin hormone (ACTH)-dependent cortisol excess, ACTH-dependent nodular adrenal hyperplasia, partial glucocorticoid resistance syndrome, a diagnosis inconsistent with benign adrenocortical lesions, or received systemic oral or intravenous glucocorticoids other than replacement therapy following adrenalectomy. Primary endpoints were all-cause mortality and clinical and biochemical remission rates. Secondary endpoints were the incidence of cardiovascular events, prevalence of vascular and metabolic comorbidities, and incidence of adrenal crises. FINDINGS:Of 629 patients who were diagnosed between Jan 1, 2000, and Jan 31, 2022, 105 (17%) had Cushing's syndrome and 524 (83%) had mild autonomous cortisol secretion (MACS), median age was 62 years (IQR 54·0-68·0), and 426 (68%) were female. 85 (81%) of 105 patients with Cushing's syndrome underwent surgery, and 384 (73%) of 524 patients with MACS received non-specific symptomatic treatment (ie, never underwent adrenalectomy or received steroidogenesis inhibitors). Over a median follow-up of 6·8 years, biochemical remission was achieved in 46 (45%) of 102 patients with Cushing's syndrome and in 67 (13%) of 517 patients with MACS. In both groups, 7% of patients died (Cushing's syndrome: seven of 105; MACS: 38 of 524) and 12% (13 of 105) of patients with Cushing's syndrome and 16% (82 of 524) of those with MACS had at least one cardiovascular event, without substantial differences across treatments. Smoking emerged as key modifiable mortality and cardiovascular risk factor in all patients, and in patients with MACS who only received non-specific symptomatic therapy, post-dexamethasone cortisol was also associated with increased mortality. Bilateral adrenalectomy led to full biochemical remission, few non-fatal adrenal crises, and improved arterial hypertension. Unilateral adrenalectomy and steroidogenesis inhibitors yielded heterogeneous biochemical outcomes and no substantial comorbidity improvement. Non-specific symptomatic treatment in MACS was associated with worsening of all investigated comorbidities. INTERPRETATION:Although mortality and cardiovascular event rates were similar across treatments, surgery led to better biochemical control and more favourable comorbidity outcomes. FUNDING:None.

CONTEXT:Methylation of FKBP5, a glucocorticoid (GC)-receptor co-chaperone, negatively correlates with cortisol levels in both healthy individuals and Cushing patients, potentially serving as an indicator of GC exposure. OBJECTIVE:We explored whether GC replacement correlates with FKBP5 methylation in patients with adrenal insufficiency (AI), aiming to assess the adequacy of therapy. METHODS:In an explorative cross-sectional analysis, we analyzed FKBP5 gene methylation at 54 CpG sites using bisulfite pyrosequencing in 120 patients with chronic primary (n = 72) and secondary (n = 48) AI on hydrocortisone replacement. Results were correlated with GC replacement, salivary cortisol, 24-hour urinary cortisol, FKBP5 polymorphisms, physician-guided therapy adjustments, and a predefined clinical score for assessment of GC exposure. Methylation levels were further compared with patients with cortisol-producing adrenal adenomas (CPA, n = 64). RESULTS:Significant negative correlations were found between methylation levels and GC dose, clinical GC replacement score, and salivary and urinary cortisol levels. Patients advised to increase their GC dose showed higher methylation levels than those recommended to reduce or maintain their dose. AI patients exhibited similar or lower methylation levels compared to those with CPA. CONCLUSION:The correlation between FKBP5 methylation status, GC replacement dose, and clinical evaluation of therapy suggests a dose-dependent effect of GC replacement on FKBP5 methylation. This encourages further research into the added value of FKBP5 methylation analysis in assessing the quality of replacement therapy. The lower methylation levels in AI compared to CPA suggest a potential impact of the cortisol peaks arising under conventional GC treatment on FKBP5-methylation.

Testicular germ cell tumors are the most common solid malignancy in young adult males with non-seminomatous representing a clinically aggressive subtype. Although cisplatin (CP)-based chemotherapy is highly effective, a subset of patients develop resistance. This study explored a potential new treatment by targeting WEE1, a key cell-cycle regulator, using the drug adavosertib in non-seminoma cell models, aiming to overcome CP resistance. Two non-seminoma cell lines, NCCIT and NT2/D1, and their CP-resistant subclones (NCCIT-R and NT2/D1-R) were used. The effects of adavosertib and CP, alone or in combination, on cell viability, cell-cycle progression, apoptosis, and DNA damage markers was assessed. WEE1 was expressed in non-seminoma cell models. Adavosertib reduced cell viability in a dose-dependent manner across all cell models in both 2D and 3D cultures. IC50 values were in low micromolar range (NCCIT: 0.550 µM; NCCIT-R: 0.630 µM; NT2/D1: 0.415 µM; NT2/D1-R: 0.630 µM). Adavosertib altered cell-cycle distribution, increasing S-phase population. Western blot analysis revealed that inhibiting WEE1 increases CDK1 activity and mitotic marker pH3, indicating disrupted cell cycle control. This leads to replication stress and forces cells with DNA damage into early mitosis, causing mitotic catastrophe. The treatment also triggered higher levels of DNA damage and cell death, as shown by increased caspase activity and apoptosis markers (cleaved-PARP and cleaved-Caspase 3). In combination treatments, adavosertib enhanced CP efficacy across all models, including resistant lines. Overall, our results provide compelling preclinical evidence supporting the combination of WEE1 inhibition with standard chemotherapy as a promising strategy to overcome CP-resistance in non-seminoma testicular cancer.

CONTEXT:Most of the patients included in the clinical trials and real-world studies with osilodrostat only include patients with adrenocorticotropic hormone dependent Cushing syndrome (CS), while data on the efficacy and safety of osilodrostat in patients with adrenal CS is scarce. OBJECTIVE:To assess the efficacy and safety of osilodrostat in adrenal CS. METHODS:International study of patients with adrenal CS: patients treated with osilodrostat at any time were enrolled in the safety evaluation and those treated for longer than 4 weeks, in the efficacy evaluation. Patients were classified as responders if they experienced a reduction in urinary free cortisol (UFC) >50% (complete responders when UFC levels were below the upper limit of normal [ULN] and partial responders if there was a reduction >50% but not normalization). RESULTS:Twenty-eight patients with adrenal CS were enrolled: 16 with adrenocortical carcinoma and 12 with benign disease. Osilodrostat was used in monotherapy in 22 patients and in combination with metyrapone in 6 cases. In those patients treated for longer than 4 weeks (n = 21), 66.7% were classified as responders (28.6% with complete response and 38.1% with partial response), and for those treated for longer than 12 weeks, the rate of response increased to 87.5% The use of osilodrostat as a nonfirst-line therapy (odds ratio 15.0, P = .010) was a predictor of response. Osilodrostat led to a significant decrease in systolic blood pressure and body weight (P < .05). Nine patients developed one or more adverse events and in 56% (n = 5) led to osilodrostat discontinuation. CONCLUSION:Osilodrostat controls hypercortisolism in 66.7% of patients with adrenal CS treated for longer than 4 weeks and in 87.5% of cases treated for longer than 12 weeks, with a positive impact on blood pressure and body weight. Patients who received osilodrostat after other previous steroidogenesis inhibitors have a higher probability of response.

Renewal of the catecholamine-secreting chromaffin cell population of the adrenal medulla is necessary for physiological homeostasis throughout life. Definitive evidence for the presence or absence of an adrenomedullary stem cell has been enigmatic. In this work, we demonstrate that a subset of sustentacular cells endowed with a support role, are in fact adrenomedullary stem cells. Through genetic tracing and comprehensive transcriptomic data of the mouse adrenal medulla, we show that cells expressing Sox2/SOX2 specialise as a unique postnatal population from embryonic Schwann Cell Precursors and are also present in the normal adult human adrenal medulla. Postnatal SOX2+ cells give rise to chromaffin cells of both the adrenaline and noradrenaline lineages in vivo and in vitro. We reveal that SOX2+ stem cells have a second, paracrine role in maintaining adrenal chromaffin cell homeostasis, where they promote proliferation through paracrine secretion of WNT6. This work identifies SOX2+ cells as a true stem cell for catecholamine-secreting chromaffin cells.

The insulin-like growth factor 2 (IGF2) is overexpressed in 90% of adrenocortical carcinomas (ACC) and promotes cell proliferation via IGF1R and isoform A of insulin receptor (IRA). However, IGF2 role in ACC tumourigenesis has not been completely understood yet, and the contribution of IGF1R and IRA in mediating ACC cell growth has been poorly explored. This study aimed to investigate IGF1R and IR expression and localisation, including the expression of IR isoforms, in ACC and adrenocortical adenomas (ACA), and their role in IGF2-driven proliferation. Immunohistochemistry staining of IGF1R and IR was performed on 118 ACC and 22 ACA to evaluate their expression and cellular localisation and statistical analyses were carried out to assess correlations with clinicopathological data. The expression of IRA and IRB in ACC and ACA tissues, ACC cell lines and ACC and ACA primary cultures was determined by RT-qPCR. To appraise the specific role of IGF1R and IR in mediating IGF2 mitogenic pathway, single and double silencing of receptors and their inhibition in 2 ACC cell lines derived from primary tumours (H295R and JIL-2266) and 2 derived from metastatic tumours (MUC-1 and TVBF-7) as well as in ACC and ACA primary cultures were performed. We found a higher IGF1R plasma membrane localisation in ACC compared to ACA. In ACC this localisation was associated with higher Ki67 and Weiss score. IR was expressed in about half of ACC and in all ACA but, in ACC, it was associated with higher Ki67 and Weiss score. RT-qPCR revealed that the prevalent isoform of IR was IRA in ACC and ACA, but not in normal adrenals. In ACC cell lines, double IGF1R + IR silencing reduced cell proliferation in JIL-2266, MUC-1 and TVBF-7 but not in H295R. In ACC, but not ACA, primary cultures, cell proliferation was reduced after IR but not IGF1R knockdown. Overall, these data suggest that IGF1R localisation and IR expression represent new biomarkers predicting tumour aggressiveness, as well as possible molecular markers useful to patients' stratification for more individualized IGF1R-IR targeted therapies or for novel pharmacological approaches specifically targeting IRA isoform.

CONTEXT:Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy with difficult to predict clinical outcomes. The S-GRAS score combines clinical and histopathological variables (tumor stage, grade, resection status, age, and symptoms) and showed good prognostic performance for patients with ACC. OBJECTIVE:To improve ACC prognostic classification by applying robust machine learning (ML) models. METHOD:We developed ML models to enhance outcome prediction using the published S-GRAS dataset (n = 942) as the training cohort and an independent dataset (n = 152) for validation. Sixteen ML models were constructed based on individual clinical variables. The best-performing models were used to develop a web-based tool for individualized risk prediction. RESULTS:Quadratic Discriminant Analysis, Light Gradient Boosting Machine, and AdaBoost Classifier models exhibited the highest performance, predicting 5-year overall mortality (OM), and 1-year and 3-year disease progression (DP) with F1 scores of 0.79, 0.63, and 0.83 in the training cohort, and 0.72, 0.60, and 0.83 in the validation cohort. Sensitivity and specificity for 5-year OM were at 77% and 77% in the training cohort, and 65% and 81% in the validation cohort, respectively. A web-based tool (https://acc-survival.streamlit.app) was developed for easily applicable and individualized risk prediction of mortality and disease progression. CONCLUSION:S-GRAS parameters can efficiently predict outcome in patients with ACC, even using a robust ML model approach. Our web app instantly estimates the mortality and disease progression for patients with ACC, representing an accessible tool to drive personalized management decisions in clinical practice.