Cellular landscape of adrenocortical carcinoma at single-nuclei resolution.

Adrenocortical carcinoma (ACC) is a rare yet devastating tumour of the adrenal gland with a molecular pathology that remains incompletely understood. To gain novel insights into the cellular landscape of ACC, we generated single-nuclei RNA sequencing (snRNA-seq) data sets from twelve ACC tumour samples and analysed these alongside snRNA-seq data sets from normal adrenal glands (NAGs). We find the ACC tumour microenvironment to be relatively devoid of immune cells compared to NAG tissues, consistent with known high tumour purity values for ACC as an immunologically "cold" tumour. Our analysis identifies three separate groups of ACC samples that are characterised by different relative compositions of adrenocortical cell types. These include cell populations that are specifically enriched in the most clinically aggressive and hormonally active tumours, displaying hallmarks of reorganised cell mechanobiology and dysregulated steroidogenesis, respectively. We also identified and validated a population of mitotically active adrenocortical cells that strongly overexpress genes POLQ, DIAPH3 and EZH2 to support tumour expansion alongside an LGR4+ progenitor-like or cell-of-origin candidate for adrenocortical carcinogenesis. Trajectory inference suggests the fate adopted by malignant adrenocortical cells upon differentiation is associated with the copy number or allelic balance state of the imprinted DLK1/MEG3 genomic locus, which we verified by assessing bulk tumour DNA methylation status. In conclusion, our results therefore provide new insights into the clinical and cellular heterogeneity of ACC, revealing how genetic perturbations to healthy adrenocortical renewal and zonation provide a molecular basis for disease pathogenesis.

Medical therapy of endogenous Cushing's syndrome with steroidogenesis inhibitors: treatment rationale, available drugs, and therapeutic effects.

Endogenous Cushing's syndrome (CS) is a rare disease characterized by a glucocorticoid excess. If inadequately treated, the latter can lead to increased morbidity and mortality. Surgical removal of the underlying tumor is the first line treatment but is sometimes not feasible or even contraindicated. Additionally, in cases with severe CS, rapid control of hypercortisolism may be required. In these scenarios steroidogenesis inhibitors represent a therapeutic alternative to surgery. Over the last years, the knowledge on the broad therapeutic effects of steroidogenesis inhibitors and the number of available drugs have increased. However, large comparative studies are still lacking. Accordingly, the decision on which drug to be used in a certain patient or clinical setting may be difficult. The aim of this review is to summarize the main characteristics of steroidogenesis inhibitors.

Circulating cell-free DNA-based biomarkers for prognostication and disease monitoring in adrenocortical carcinoma.

OBJECTIVE: Adrenocortical carcinoma (ACC) is a rare aggressive cancer with heterogeneous behaviour. Disease surveillance relies on frequent imaging, which comes with significant radiation exposure. The aim of the study was to investigate the role of circulating cell-free DNA (ccfDNA)-related biomarkers (BMs) for prognostication and monitoring of ACC. DESIGN AND METHODS: We investigated 34 patients with ACC and 23 healthy subjects (HSs) as controls. Circulating cell-free DNA was extracted by commercial kits and ccfDNA concentrations were quantified by fluorimeter (BM1). Targeted sequencing was performed using a customized panel of 27 ACC-specific genes. Leucocyte DNA was used to discriminate somatic variants (BM2), while tumour DNA was sequenced in 22/34 cases for comparison. Serial ccfDNA samples were collected during follow-up in 19 ACC patients (median period 9 months) and analysed in relationship with standard radiological imaging. RESULTS: Circulating cell-free DNA concentrations were higher in ACC than HS (mean ± SD, 1.15 ± 1.56 vs 0.05 ± 0.05 ng/µL, P < .0001), 96% of them being above the cut-off of 0.146 ng/µL (mean HS + 2 SD, positive BM1). At ccfDNA sequencing, 47% of ACC showed at least 1 somatic mutation (positive BM2). A combined ccfDNA-BM score was strongly associated with both progression-free and overall survival (hazard ratio [HR] = 2.63; 95% CI, 1.13-6.13; P = .010, and HR = 5.98; 95% CI, 2.29-15.6; P = .0001, respectively). During disease monitoring, positive BM2 showed the best specificity (100%) and sensitivity (67%) to detect ACC recurrence or progress compared with BM1. CONCLUSION: ccfDNA-related BMs are frequently detected in ACC patients and represent a promising, minimally invasive tool to predict clinical outcome and complement surveillance imaging. Our findings will be validated in a larger cohort of ACCs with long-term follow-up.

"Expression and prognostic relevance of PD-1, PD-L1 and CTLA-4 immune checkpoints in adrenocortical carcinoma".

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with poor prognosis in advanced stages. While therapies targeting the checkpoint molecules programmed cell death 1 (PD-1), its ligand PD-L1 and the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have revolutionized treatment in many cancers, the results in ACC were heterogeneous. Their expression in ACC has not been systematically studied and might explain the variable response to checkpoint inhibitors. The expression of PD-1, PD-L1 and CTLA-4 was examined in 162 tumor samples from 122 ACC patients by immunohistochemistry (threshold of >1%) and correlated with tumoral T lymphocyte infiltration and clinical endpoints. Finally, uni- and multivariate analyses of progression-free and overall survival were performed. PD-1 and PD-L1 were expressed in 26.5% and 24.7% of samples, respectively, with low expression in most tumor samples (median positive cells: 2.1% and 21.7%. In contrast, CTLA-4 expression was observed in 52.5% of ACC with a median of 38.4% positive cells. Positive PD-1 expression was associated with longer progression-free survival (HR: 0.50, 95% CI 0.25-0.98, p=0.04) even after considering prognostic factors. In contrast, PD-L1 and CTLA-4 did not correlate with clinical outcome. Additionally, PD-1 and PD-L1 expression correlated significantly with the amount of CD3+, CD4+, FoxP3+ and CD8+ T cells. The heterogeneous expression of PD1, PD-L1, and CTLA-4 in this large series of well-annotated ACC samples might explain the heterogeneous results of the immunotherapies in advanced ACC. In addition, PD-1 expression is a strong prognostic biomarker that can easily be applied in routine clinical care and histopathological assessment.

The Value of Local Therapies in Advanced Adrenocortical Carcinoma.

International guidelines recommend local therapies (LTs) such as local thermal ablation (LTA; radiofrequency, microwave, cryoablation), transarterial (chemo)embolisation (TA(C)E), and transarterial radioembolisation (TARE) as therapeutic options for advanced adrenocortical carcinoma (ACC). However, the evidence for these recommendations is scarce. We retrospectively analysed patients receiving LTs for advanced ACC. Time to progression of the treated lesion (tTTP) was the primary endpoint. The secondary endpoints were best objective response, overall progression-free survival, overall survival, adverse events, and the establishment of predictive factors by multivariate Cox analyses. A total of 132 tumoural lesions in 66 patients were treated with LTA (n = 84), TA(C)E (n = 40), and TARE (n = 8). Complete response was achieved in 27 lesions (20.5%; all of them achieved by LTA), partial response in 27 (20.5%), and stable disease in 38 (28.8%). For the LTA group, the median tTTP was not reached, whereas it was reached 8.3 months after TA(C)E and 8.2 months after TARE (p < 0.001). The median time interval from primary diagnosis to LT was >47 months. Fewer than four prior therapies and mitotane plasma levels of >14 mg/L positively influenced the tTTP. In summary, this is one of the largest studies on LTs in advanced ACC, and it demonstrates a very high local disease control rate. Thus, it clearly supports the guideline recommendations for LTs in these patients.

Recovery of adrenal function after stopping mitotane in patients with adrenocortical carcinoma.

OBJECTIVE: Mitotane is the standard therapy of adrenocortical carcinoma (ACC) due to its relative selectivity of its cytotoxic effects toward adrenocortical cells. Therefore, it virtually always leads to adrenal insufficiency. Frequency and characteristics of hypothalamic-pituitary-adrenal axis recovery after discontinuation are ill-defined. METHODS: This was a retrospective study of patients with ACC adjuvantly treated with mitotane for ≥12 months who were disease-free at mitotane stop and had a minimum follow-up ≥1 year. Primary endpoint was adrenal recovery. Cox regression analyses were used to identify predictive factors. Moreover, mitotane plasma elimination rate and hormonal changes after mitotane stop were investigated. RESULTS: Fifty-six patients (36 women) treated with mitotane for a median time of 25 months and an average daily dose of 2.8 g were included. Median time after discontinuation until mitotane levels dropped below 5 and 2 mg/L, and the detection limit was 152 days (interquartile range: 114-202), 280 days (192-370), and 395 days (227-546), respectively. Full adrenal recovery was documented in 32 (57%) patients after a median time of 26 months (95% confidence interval [CI] = 19.6-32.4). In 4 patients (7.1%), adrenal insufficiency persisted >5 years after discontinuation. Mitotane peak ≥ 27 mg/L significantly correlated with longer time to adrenal recovery (hazard ratio [HR] = 0.2, 95% CI = 0.1-0.8, P = .03). Twenty-seven of 38 patients (71%) followed in reference centers achieved adrenal recovery compared with only 5/18 (28%) followed up in non-reference centers (HR = 4.51, 95% CI = 1.71-11.89, P = .002). Other investigated factors were not associated with adrenal function after discontinuation. CONCLUSIONS: Our study demonstrates that adrenal recovery occurs in most patients after stopping mitotane, particularly when followed up in specialized centers, but not in all. Elimination time of mitotane after treatment discontinuation is very long but individually quite variable.

Current Evidence on Local Therapies in Advanced Adrenocortical Carcinoma.

International guidelines emphasise the role of local therapies (LT) for the treatment of advanced adrenocortical carcinoma (ACC). However, large studies are lacking in this field. Therefore, we performed a review of the literature to synthesise current evidence and develop clinical guidance. PubMed database was searched for systematic literature. We identified 119 potentially relevant articles, of which 21 could be included in our final analysis. All were retrospective and reported on 374 patients treated with LT for advanced ACC (12 studies on radiotherapy, 3 on transarterial chemoembolisation and radioembolisation, 4 on image-guided thermal ablation [radiofrequency, microwave ablation, and cryoablation, and two studies reporting treatment with several different LT]). Radiotherapy was frequently performed with palliative intention. However, in most patients, disease control and with higher dosage also partial responses could be achieved. Data for other LT were more limited, but also point towards local disease control in a significant percentage of patients. Very few studies tried to identify factors that are predictive on response. Patients with a disease-free interval after primary surgery of more than 9 months and lesions<5 cm might benefit most. Underreporting of toxicities may be prevalent, but LT appear to be relatively safe overall. Available evidence on LT for ACC is limited. LT appears to be safe and effective in cases with limited disease and should be considered depending on local expertise in a multidisciplinary team discussion.

Morbidity in Patients with Chronic Adrenal Insufficiency - Cardiovascular Risk Factors and Hospitalization Rate Compared to Population Based Controls.

Patients with adrenal insufficiency (AI) have been found to have increased cardiovascular morbidity, partly associated with nonphysiologic glucocorticoid replacement. We included two separate cohorts (cohort 1 n=384 patients, cohort 2 n=180 patients) of patients with chronic primary and secondary AI under standard replacement therapy and compared them to two age- and sex-matched population-based studies (SHIP-TREND/DEGS). Odds ratios with 95% CI for hypertension, hyperlipidemia/HLP, type 2 diabetes/T2DM, obesity, and hospitalization with adjustment for confounders were evaluated by logistic regression. Patient cohort 1 had significantly lower ORs for obesity [0.4 (0.3-0.6), p<0.001] and hypertension [0.5 (0.3-0.6), p<0.001] compared to SHIP-TREND and for obesity [0.7 (0.5-0.9), p=0.01], hypertension [0.4 (0.3-0.5), p<0.001] and HLP [0.4 (0.3-0.6), p<0.001] compared to DEGS. In cohort 2, ORs were significantly lower for HLP compared to both SHIP-TREND [0.4 (0.2-0.7), p=0.001] and DEGS [0.3 (0.2-0.5), p<0.001] and for hypertension [0.7 (0.4-0.9), p=0.04] compared to SHIP-TREND. In patients with SAI from cohort 2, ORs for DM2 [2.5 (1.3-4.9) p=0.009], hypertension [2.5 (1.4-4.5), p=0.002] and obesity [1.9 (1.1-3.1), p=0.02] were significantly higher compared to DEGS, whereas ORs for HLP were significantly lower compared to both SHIP [0.3 (0.1-0.6), p=0.002] and DEGS [0.3 (0.1-0.6), p<0.001]. In most of our AI patients treated with conventional glucocorticoid doses, the risk for T2DM, obesity, hypertension, and HLP was not increased. The number of hospitalizations was significantly higher in AI patients compared to controls, which might reflect increased susceptibility but also a more proactive management of concomitant diseases by physicians and patients.

PLK1 inhibitors as a new targeted treatment for adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is an aggressive malignancy with limited treatment options. Polo-like kinase 1 (PLK1) is a promising drug target; PLK1 inhibitors (PLK1i) have been investigated in solid cancers and are more effective in TP53-mutated cases. We evaluated PLK1 expression in ACC samples and the efficacy of two PLK1i in ACC cell lines with different genetic backgrounds. PLK1 protein expression was investigated by immunohistochemistry in tissue samples and correlated with clinical data. The efficacy of rigosertib (RGS), targeting RAS/PI3K, CDKs and PLKs, and poloxin (Pol), specifically targeting the PLK1 polo-box domain, was tested in TP53-mutated NCI-H295R, MUC-1, and CU-ACC2 cells and in TP53 wild-type CU-ACC1. Effects on proliferation, apoptosis, and viability were determined. PLK1 immunostaining was stronger in TP53-mutated ACC samples vs wild-type (P = 0.0017). High PLK1 expression together with TP53 mutations correlated with shorter progression-free survival (P= 0.041). NCI-H295R showed a time- and dose-dependent reduction in proliferation with both PLK1i (P< 0.05at 100 nM RGS and 30 µM Pol). In MUC-1, a less pronounced decrease was observed (P< 0.05at 1000 nM RGS and 100 µM Pol). 100 nM RGS increased apoptosis in NCI-H295R (P< 0.001), with no effect on MUC-1. CU-ACC2 apoptosis was induced only at high concentrations (P < 0.05 at 3000 nM RGS and 100 µM Pol), while proliferation decreased at 1000 nM RGS and 30 µM Pol. CU-ACC1 proliferation reduced, and apoptosis increased, only at 100 µM Pol. TP53-mutated ACC cell lines demonstrated better response to PLK1i than wild-type CU-ACC1. These data suggest PLK1i may be a promising targeted treatment of a subset of ACC patients, pre-selected according to tumour genetic signature.

High Filamin a Expression in Adrenocortical Carcinomas Is Associated with a Favourable Tumour Behaviour: A European Multicentric Study.

The insulin-like growth factor 2 (IGF2) promotes cell growth by overactivating the IGF system in an autocrine loop in adrenocortical carcinomas (ACCs). The cytoskeleton protein filamin A (FLNA) acts as a repressor of IGF2 mitogenic signalling in ACC cells. The aims of this study were to test FLNA expression by immunohistochemistry in 119 ACCs and 26 adrenocortical adenomas (ACAs) and to evaluate its relationship with clinicopathological features and outcome in ACCs. We found that 71.4% of ACCs did not express FLNA, whereas FLNA absence was a rare event in ACAs (15.4%, p < 0.001 vs. ACCs). In addition, the expression of FLNA was associated with a less aggressive tumour behaviour in ACCs. Indeed, the subgroup of ACCs with high FLNA showed a lower ENSAT stage, Weiss score, and S-GRAS score compared to ACCs with low FLNA expression (p < 0.05). Moreover, patients with high FLNA had a longer overall survival than those with low FLNA (p < 0.05). In conclusion, our data suggest that FLNA may represent a "protective" factor in ACCs, and the integration of FLNA immunohistochemical expression in ACC tissues along with other clinical and molecular markers could be helpful to improve diagnostic accuracy and prognosis prediction in ACCs.

PPARG dysregulation as a potential molecular target in adrenal Cushing's syndrome.

Background: We performed a transcriptomic analysis of adrenal signaling pathways in various forms of endogenous Cushing's syndrome (CS) to define areas of dysregulated and druggable targets. Methodology: Next-generation sequencing was performed on adrenal samples of patients with primary bilateral macronodular adrenal hyperplasia (PBMAH, n=10) and control adrenal samples (n=8). The validation groups included cortisol-producing adenoma (CPA, n=9) and samples from patients undergoing bilateral adrenalectomy for Cushing's disease (BADX-CD, n=8). In vivo findings were further characterized using three adrenocortical cell-lines (NCI-H295R, CU-ACC2, MUC1). Results: Pathway mapping based on significant expression patterns identified PPARG (peroxisome proliferator-activated receptor gamma) pathway as the top hit. Quantitative PCR (QPCR) confirmed that PPARG (l2fc<-1.5) and related genes - FABP4 (l2fc<-5.5), PLIN1 (l2fc<-4.1) and ADIPOQ (l2fc<-3.3) - were significantly downregulated (p<0.005) in PBMAH. Significant downregulation of PPARG was also found in BADX-CD (l2fc<-1.9, p<0.0001) and CPA (l2fc<-1.4, p<0.0001). In vitro studies demonstrated that the PPARG activator rosiglitazone resulted in decreased cell viability in MUC1 and NCI-H295R (p<0.0001). There was also a significant reduction in the production of aldosterone, cortisol, and cortisone in NCI-H295R and in Dihydrotestosterone (DHT) in MUC1 (p<0.05), respectively. Outcome: This therapeutic effect was independent of the actions of ACTH, postulating a promising application of PPARG activation in endogenous hypercortisolism.

Performance of DNA-based biomarkers for classification of adrenocortical carcinoma: a prognostic study.

OBJECTIVE: 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. DESIGN AND METHODS: 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). RESULTS: In training cohort, an independent prognostic role was confirmed at multivariate analysis for two DNA-based BMs: alterations in Wnt/ß-catenin and Rb/p53 pathways and hypermethylated PAX5 (both P< .05 for PFS and DFS, hazard ratio [HR] 1.47-2.33). 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 (C index for OS 0.724 and 0.765, PFS 0.717 and 0.670, and DFS 0.699 and 0.644, respectively). CONCLUSIONS: 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.

Outcome of immunotherapy in adrenocortical carcinoma: a retrospective cohort study.

OBJECTIVE: Clinical trials with immune checkpoint inhibitors (ICI) in adrenocortical carcinoma (ACC) have yielded contradictory results. We aimed to evaluate treatment response and safety of ICI in ACC in a real-life setting. DESIGN: Retrospective cohort study of 54 patients with advanced ACC receiving ICI as compassionate use at 6 German reference centres between 2016 and 2022. METHODS: Objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and treatment-related adverse events (TRAE) were assessed. RESULTS: In 52 patients surviving at least 4 weeks after initiation of ICI, ORR was 13.5% (6-26) and DCR was 24% (16-41). PFS was 3.0 months (95% CI, 2.3-3.7). In all patients, median OS was 10.4 months (3.8-17). 17 TRAE occurred in 15 patients, which was associated with a longer PFS of 5.5 (1.9-9.2) vs 2.5 (2.0-3.0) months (HR 0.29, 95% CI, 0.13-0.66, P = 0.001) and OS of 28.2 (9.5-46.8) vs 7.0 (4.1-10.2) months (HR 0.34, 95% CI, 0.12-0.93). Positive tissue staining for programmed cell death ligand 1 (PD-L1) was associated with a longer PFS of 3.2 (2.6-3.8) vs 2.3 (1.6-3.0, P < 0.05) months. Adjusted for concomitant mitotane use, treatment with nivolumab was associated with lower risk of progression (HR 0.36, 0.15-0.90) and death (HR 0.20, 0.06-0.72) compared to pembrolizumab. CONCLUSIONS: In the real-life setting, we observe a response comparable to other second-line therapies and an acceptable safety profile in ACC patients receiving different ICI. The relevance of PD-L1 as a marker of response and the potentially more favourable outcome in nivolumab-treated patients require confirmation.

Bilateral inferior petrosal sinus sampling with human CRH stimulation in ACTH-dependent Cushing's syndrome: results from a retrospective multicenter study.

OBJECTIVE: Bilateral inferior petrosal sinus sampling (BIPSS) is regarded as gold standard to differentiate between Cushing´s disease (CD) and ectopic Cushing's syndrome (ECS). However, published data e.g. on the diagnostic value of additional prolactin analysis is controversial. Thus, we evaluated the diagnostic performance of BIPSS with and without prolactin in a multicenter study. DESIGN AND METHODS: Retrospective study in 5 European reference centers. Patients with overt adrenocorticotropin (ACTH)-dependent Cushing's syndrome at the time of BIPSS with human corticotropin-releasing hormone stimulation were eligible. Cut-offs for the inferior petrosal sinus (IPS) to peripheral (P) ACTH ratio and the normalized ACTH:prolactin IPS:P ratio were calculated via receiver operator characteristics analyses (reference: CD). RESULTS: 156 patients with BIPSS were identified. Of these, 120 patients (92 (77%) females; 106 (88%) CD, 14 (12%) ECS) had either histopathologically confirmed tumors or biochemical remission and/or adrenal insufficiency after surgery; only this subgroup was analyzed by ROC analysis. The optimal cut-offs for the ACTH IPS:P ratio were ≥1.9 at baseline (sensitivity 82.1% (95%CI 73.2-88.6), specificity 85.7% (95%CI 56.2-97.5), AUC 0.86) and ≥2.1 at 5 minutes post-CRH (sensitivity 91.3% (95%CI 83.6-95.7), specificity 92.9%(95%CI 64.1-99.6), AUC 0.96). A subgroup underwent additional prolactin analysis. An optimal cut-off of ≥1.4 was calculated for the normalized ACTH:prolactin IPS:P ratio (sensitivity 96.0% (95%CI 77.7-99.9), specificity 100% (95%CI 56.1-100), AUC 0.99). CONCLUSION: Our study confirms the high accuracy of BIPSS in the differential diagnosis of ACTH-dependent Cushing's syndrome and suggests that the simultaneous measurement of prolactin might further improve the diagnostic performance of this test.

Clinical course of patients with adrenal incidentalomas and cortisol autonomy: a German retrospective single center cohort study.

Background: Adrenal incidentalomas with cortisol autonomy are associated with increased cardiovascular morbidity and mortality. Specific data on the clinical and biochemical course of affected patients are lacking. Methods: Retrospective study from a tertiary referral centre in Germany. After exclusion of overt hormone excess, malignancy and glucocorticoid medication, patients with adrenal incidentalomas were stratified according to serum cortisol after 1 mg dexamethasone: autonomous cortisol secretion (ACS), >5.0; possible ACS (PACS), 1.9-5.0; non-functioning adenomas (NFA), ≤1.8 µg/dl. Results: A total of 260 patients were enrolled (147 women (56.5%), median follow-up 8.8 (2.0-20.8) years). At initial diagnosis, median age was 59.5 (20-82) years, and median tumour size was 27 (10-116) mm. Bilateral tumours were more prevalent in ACS (30.0%) and PACS (21.9%) than in NFA (8.1%). Over time, 40/124 (32.3%) patients had a shift of their hormonal secretion pattern (NFA to PACS/ACS, n=15/53; PACS to ACS, n=6/47; ACS to PACS, n=11/24; PACS to NFA, n=8/47). However, none of the patients developed overt Cushing's syndrome. Sixty-one patients underwent adrenalectomy (NFA, 17.9%; PACS, 24.0%; ACS, 39.0%). When non-operated patients with NFA were compared to PACS and ACS at last follow-up, arterial hypertension (65.3% vs. 81.9% and 92.0%; p<0.05), diabetes (23.8% vs. 35.6% and 40.0%; p<0.01), and thromboembolic events (PACS: HR 3.43, 95%-CI 0.89-13.29; ACS: HR 5.96, 95%-CI 1.33-26.63; p<0.05) were significantly less frequent, along with a trend towards a higher rate of cardiovascular events in case of cortisol autonomy (PACS: HR 2.23, 95%-CI 0.94-5.32; ACS: HR 2.60, 95%-CI 0.87-7.79; p=0.1). Twenty-five (12.6%) of the non-operated patients died, with higher overall mortality in PACS (HR 2.6, 95%-CI 1.0-4.7; p=0.083) and ACS (HR 4.7, 95%-CI 1.6-13.3; p<0.005) compared to NFA. In operated patients, prevalence of arterial hypertension decreased significantly (77.0% at diagnosis to 61.7% at last follow-up; p<0.05). The prevalence of cardiovascular events and mortality did not differ significantly between operated and non-operated patients, whereas thromboembolic events were significantly less frequent in the surgical treatment group. Conclusion: Our study confirms relevant cardiovascular morbidity in patients with adrenal incidentalomas (especially those with cortisol autonomy). These patients should therefore be monitored carefully, including adequate treatment of typical cardiovascular risk factors. Adrenalectomy was associated with a significantly decreased prevalence of hypertension. However, more than 30% of patients required reclassification according to repeated dexamethasone suppression tests. Thus, cortisol autonomy should ideally be confirmed before making any relevant treatment decision (e.g. adrenalectomy).

New Findings on Presentation and Outcome of Patients With Adrenocortical Cancer: Results From a National Cohort Study.

CONTEXT: Because of the rarity of adrenocortical cancer (ACC), only a few population-based studies are available, and they reported limited details in the characterization of patients and their treatment. OBJECTIVE: To describe in a nationwide cohort the presentation of patients with ACC, treatment strategies, and potential prognostic factors. METHODS: Retrospective analysis of 512 patients with ACC, diagnosed in 12 referral centers in Italy from January 1990 to June 2018. RESULTS: ACC diagnosed as incidentalomas accounted for overall 38.1% of cases, with a frequency that increases with age and with less aggressive pathological features than symptomatic tumors. Women (60.2%) were younger than men and had smaller tumors, which more frequently secreted hormones. Surgery was mainly done with an open approach (72%), and after surgical resection, 62.7% of patients started adjuvant mitotane therapy. Recurrence after tumor resection occurred in 56.2% of patients. In patients with localized disease, cortisol secretion, ENSAT stage III, Ki67%, and Weiss score were associated with an increased risk of recurrence, whereas margin-free resection, open surgery, and adjuvant mitotane treatment were associated with reduced risk. Death occurred in 38.1% of patients and recurrence-free survival (RFS) predicted overall survival (OS). In localized disease, age, cortisol secretion, Ki67%, ENSAT stage III, and recurrence were associated with increased risk of mortality. ACCs presenting as adrenal incidentalomas showed prolonged RFS and OS. CONCLUSION: Our study shows that ACC is a sex-related disease and demonstrates that an incidental presentation is associated with a better outcome. Given the correlation between RFS and OS, RFS may be used as a surrogate endpoint in clinical studies.

Recurrence-Free Survival in Early and Locally Advanced Large Cell Neuroendocrine Carcinoma of the Lung after Complete Tumor Resection.

BACKGROUND: Large Cell Neuroendocrine Carcinoma (LCNEC) is a rare subtype of lung cancer with poor clinical outcomes. Data on recurrence-free survival (RFS) in early and locally advanced pure LCNEC after complete resection (R0) are lacking. This study aims to evaluate clinical outcomes in this subgroup of patients and to identify potential prognostic markers. METHODS: Retrospective multicenter study including patients with pure LCNEC stage I-III and R0 resection. Clinicopathological characteristics, RFS, and disease-specific survival (DSS) were evaluated. Univariate and multivariate analyses were performed. RESULTS: 39 patients (M:F = 26:13), with a median age of 64 years (44-83), were included. Lobectomy (69.2%), bilobectomy (5.1%), pneumonectomy (18%), and wedge resection (7.7%) were performed mostly associated with lymphadenectomy. Adjuvant therapy included platinum-based chemotherapy and/or radiotherapy in 58.9% of cases. After a median follow-up of 44 (4-169) months, the median RFS was 39 months with 1-, 2- and 5-year RFS rates of 60.0%, 54.6%, and 44.9%, respectively. Median DSS was 72 months with a 1-, 2- and 5-year rate of 86.8, 75.9, and 57.4%, respectively. At multivariate analysis, age (cut-off 65 years old) and pN status were independent prognostic factors for both RFS (HR = 4.19, 95%CI = 1.46-12.07, p = 0.008 and HR = 13.56, 95%CI 2.45-74.89, p = 0.003, respectively) and DSS (HR = 9.30, 95%CI 2.23-38.83, p = 0.002 and HR = 11.88, 95%CI 2.28-61.84, p = 0.003, respectively). CONCLUSION: After R0 resection of LCNEC, half of the patients recurred mostly within the first two years of follow-up. Age and lymph node metastasis could help to stratify patients for adjuvant therapy.

Vitamin D and Bone Metabolism in Adult Patients with Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is a genetic multisystemic autosomal dominant disorder determining reduced life expectancy due to higher risk of developing benign and malignant tumors. Low levels of vitamin D and reduced bone mineral density (BMD) have been reported in young patients with NF1. However, correlation between vitamin D and NF1 phenotype needs to be elucidated. Aim of this study was to assess vitamin D levels and bone metabolism in NF1 patients, analyzing potential correlations with clinical phenotype. A cross-sectional study was carried out in a monocentric series of NF1 patients, evaluating genotype, clinical phenotype, BMD, biochemical evaluation with focus on serum 25OH-vitamin D, parathyroid hormone (PTH), calcium and phosphate levels. Correlations between clinical manifestations, neurofibromas, and vitamin D status have been studied in comparison with healthy controls. 31 NF1 adult patients were matched for sex, age and body mass index with 31 healthy controls. A significantly difference in vitamin D level emerged in NF1 patients compared to controls. Interestingly low vitamin D levels correlated with a more aggressive phenotype and with a bigger size of neurofibromas. These data underline that vitamin D deficiency/insufficiency may play a role in clinical severity of neurofibromas in patients with NF1, suggesting the need to check bone status and replace vitamin D in these patients.

Coincidence of primary adrenocortical carcinoma and melanoma: three CASE reports.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a heterogeneous prognosis, while adrenal metastasis from other primary cancers, including melanoma, may occur more frequently. ACC may rarely occur as part of familial cancer syndromes, but even in sporadic cases, a significant proportion of patients had other malignancies before or after diagnosis of ACC. Herein we present three cases where sporadic ACC was identified in patients with coexistent or previous history of melanoma. CASE DESCRIPTION: Patient 1 - A 37-yr-old man with a superficial spreading BRAF-positive melanoma was found to harbour a progressively growing left adrenal mass. Initially, he was suspected of having adrenal metastasis, but the histology after adrenalectomy confirmed ACC. Patient 2 - A 68-year-old man with a history of recurrent BRAF-positive melanoma was diagnosed with disseminated metastatic melanoma recurrence, including a rapidly enlarging left adrenal mass. Consequently, he underwent left adrenalectomy, and histology again confirmed ACC. Patient 3 - A 50-yr-old man was referred with histological diagnosis of metastatic ACC. He had a background history of pT1 melanoma. We undertook targeted sequencing of ACC tissue samples in all cases. Somatic variants were observed in the known driver genes CTNNB1 (Patient 1), APC and KMT2D (Patient 2), and APC and TP53 (Patient 3). Germline TP53 variants (Li-Fraumeni syndrome) were excluded in all cases. Retrospective review of our patient cohort in the last 21 years revealed a frequency of 0.5% of histologically diagnosed melanoma metastasis among patients referred for adrenal masses. On the other hand, 1.6% of patients with histologically confirmed ACC had a previous history of melanoma. CONCLUSION: Sporadic ACC can occur in the background of melanoma, even if adrenal metastasis might appear to be the most likely diagnosis. Coexistent primary adrenal malignancy should be considered and investigated for in all patients with a history of melanoma with suspicious adrenal lesions.

Lack of sensitivity of diagnostic Cushing-scores in Germany: a multicenter validation.

OBJECTIVE: Endogenous Cushing's syndrome (CS) is a severe condition, often diagnosed at a late stage. To reduce mortality, early diagnosis plays an important role. Two screening tools for early identification of patients with CS have been developed in multicentric cohorts, but have not yet been validated in cohorts with different geographic backgrounds. DESIGN: We validated the Spanish score published by Leon-Justel et al. in 2016 and the Italian score by Parasiliti-Caprino et al. published in 2021 in our cohort. METHODS: In the multicentric German Cushing registry, patients with confirmed and expected but ruled out Cushing's syndrome are prospectively diagnosed and followed up. We validated both scores in a cohort of 458 subjects: 176 patients with confirmed CS and 282 patients with suspected, but finally excluded CS. RESULTS: Using the Spanish score, 17.5% of our patients with proven CS biochemical screening would not have been recommended. This concerned patients with pituitary CS (22%) and with adrenal CS (10%). On the contrary, only 14% of patients without CS would have received a recommendation for biochemical screening. Using the Italian score, 29% of patients with proven CS were classified into the low-risk classes not recommended for biochemical screening. This mostly affected patients with adrenal (31%) and pituitary CS (30%). About 12% of subjects without CS would have received a biochemical screening recommendation. CONCLUSIONS: Both scores had limited sensitivity and high specificity in a German validation cohort. Further research is necessary to develop a screening score, which is effective in different healthcare systems and ethnicities.