The Role of the IGF2 Methylation Score in Diagnosing Adrenocortical Tumors with Unclear Malignant Potential-Feasibility of Formalin-Fixed Paraffin-Embedded Tissue.

The differentiation between benign and malignant adrenocortical tumors based on pathological assessment can be difficult. We present a series of 17 patients with unclear malignant tumors, of whom six had recurrent or metastatic disease. The assessment of the methylation pattern of insulin-like growth factor 2 (IGF2) regulatory regions in fresh frozen material has shown to be valuable in determining the malignancy of adrenocortical tumors, although this has not been elaborately tested in unclear malignant tumors. Since fresh frozen tissue was only available in six of the patients, we determined the feasibility of using formalin-fixed paraffin-embedded (FFPE) tissue for this method. We isolated DNA from FFPE tissue and matched the fresh frozen tissue of three patients with adrenocortical carcinoma. Methylation patterns of IGF2 regulatory regions were determined by pyrosequencing using different amounts of bisulfite-converted DNA (5 ng, 20 ng, 40 ng). Compared to fresh frozen tissue, FFPE tissue had a higher failure rate (fresh frozen 0%; FFPE 18.5%) and poor-to-moderate replicability (fresh frozen rho = 0.89-0.99, median variation 1.6%; FFPE rho = -0.09-0.85, median variation 7.7%). There was only a poor-to-moderate correlation between results from fresh frozen and FFPE tissue (rho = -0.28-0.70, median variation 13.2%). In conclusion, FFPE tissue is not suitable for determining the IGF2 methylation score in patients with an unclear malignant adrenocortical tumor using the currently used method. We, therefore, recommend fresh frozen storage of resection material for diagnostic and biobank purposes.

Fatal Hemophagocytic Lymphohistiocytosis in a Patient with Miliary Tuberculosis: a Case Report.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome induced by cytotoxic T-cells. Mostly, HLH is secondary to infections, malignancies, or autoimmune disorders. HLH triggered by miliary tuberculosis is rare and mortality rates are high. We report a case of a 58-year-old, Caucasian patient admitted to the ICU with respiratory failure. After extensive tests, the diagnosis of HLH was made. Despite aggressive treatment with antibiotics, etoposide, anakinra, and tocilizumab, our patient succumbed to the illness after 18 days in the ICU. Postmortem, a diagnosis of miliary tuberculosis was made, despite negative PCR and culture of mycobacteria during clinical course. Our case demonstrates the challenges of early diagnosis of HLH and the importance of considering miliary tuberculosis as a possible underlying trigger. Supplementary Information: The online version contains supplementary material available at 10.1007/s42399-022-01232-y.

How to Differentiate Benign from Malignant Adrenocortical Tumors?

Adrenocortical carcinoma (ACC) is a rare cancer with a poor prognosis. Adrenal incidentalomas are, however, commonly identified in clinical practice. Discrimination between benign and malignant adrenal tumors is of great importance considering the large differences in clinical behavior requiring different strategies. Diagnosis of ACC starts with a thorough physical examination, biochemical evaluation, and imaging. Computed tomography is the first-level imaging modality in adrenal tumors, with tumor size and Hounsfield units being important features for determining malignancy. New developments include the use of urine metabolomics, also enabling discrimination of ACC from adenomas preoperatively. Postoperatively, the Weiss score is used for diagnosis of ACC, consisting of nine histopathological criteria. Due to known limitations as interobserver variability and lack of accuracy in borderline cases, much effort has been put into new tools to diagnose ACC. Novel developments vary from immunohistochemical markers and pathological scores, to markers at the level of DNA, methylome, chromosome, or microRNA. Molecular studies have provided insights into the most promising and most frequent alterations in ACC. The use of liquid biopsies for diagnosis of ACC is studied, although in a small number of patients, requiring further investigation. In this review, current diagnostic modalities and challenges in ACC will be addressed.

Levoketoconazole, the 2S,4R Enantiomer of Ketoconazole, a New Steroidogenesis Inhibitor for Cushing's Syndrome Treatment.

INTRODUCTION: Racemic ketoconazole (RK) is a steroidogenesis inhibitor used for treatment of Cushing's syndrome. Levoketoconazole (COR-003), the pure 2S,4R enantiomer, is potentially more potent and safe compared to RK. We compared in vitro effects of levoketoconazole and RK on adrenocortical and pituitary adenoma cells. MATERIALS AND METHODS: HAC15 cells and 15 primary human neoplastic adrenocortical cultures (+/- ACTH), and murine (AtT20) and human corticotroph adenoma cultures were incubated with levoketoconazole or RK (0.01-10 µM). Cortisol and ACTH were measured using a chemiluminescence immunoassay system, and steroid profiles by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: In HAC15, levoketoconazole inhibited cortisol at lower concentrations (IC50: 0.300 µM) compared to RK (0.611 µM; P < 0.0001). IC50 values of levoketoconazole for basal cortisol production in primary adrenocortical cultures varied over a 24-fold range (0.00578-0.140 µM), with 2 patients having a higher sensitivity for levoketoconazole vs RK (2.1- and 3.7-fold). LC-MS/MS analysis in selected cases revealed more potent inhibition of cortisol and other steroid profile components by levoketoconazole vs RK. In AtT20, levoketoconazole inhibited cell growth and ACTH secretion (10 µM: -54% and -38%, respectively), and levoketoconazole inhibited cell number in 1 of 2 primary human corticotroph pituitary adenoma cultures (-44%, P < 0.001). CONCLUSION: Levoketoconazole potently inhibits cortisol production in adrenocortical cells, with a variable degree of suppression between specimens. Levoketoconazole inhibits adrenal steroid production more potently compared to RK and might also inhibit ACTH secretion and growth of pituitary adenoma cells. Together with previously reported potential advantages, this indicates that levoketoconazole is a promising novel pharmacotherapy for Cushing's syndrome.

The Efficacy of Mitotane in Human Primary Adrenocortical Carcinoma Cultures.

CONTEXT: Patients with adrenocortical carcinoma (ACC) often fail mitotane treatment and deal with severe toxicity, marking the relevance of predictive parameters for treatment outcome. OBJECTIVE: Determine the effects of mitotane in primary ACC cultures, and correlate sensitivity with patient and tumor characteristics. METHODS: In 32 primary ACC cultures, the effects of mitotane on cell growth and cortisol production were determined. RRM1, SOAT1, and CYP2W1 expression were assessed using reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: The median percentage cell amount inhibition in primary ACC cultures at 50 µM mitotane was 57%. Seven patients were classified as nonresponders, 14 as partial responders, and 11 as responders. The mean median effective concentration (EC50) value of mitotane for inhibition of cell amount in responders was 14.2 µM (95% CI, 11.3-17.9), in partial responders 41.6 µM (95% CI, 33.5-51.8), and could not be calculated in nonresponders. The percentage cortisol-producing ACC was 14%, 43%, and 73% for nonresponders, partial responders, and responders (P = 0.068). Mitotane inhibited cortisol production with a mean EC50 of 1.4 µM (95% CI, 0.9-2.1), which was considerably lower than the EC50 on cell growth. RRM1, SOAT1, and CYP2W1 expression levels were not predictive for mitotane sensitivity in vitro. CONCLUSION: Direct antitumor effects of mitotane on human primary ACC cultures are highly variable between patients, reflecting heterogeneous responses in patients. Cortisol was inhibited at lower concentrations, compared with its effect on cell amount. Cortisol secretion by ACC might be associated with enhanced mitotane sensitivity due to increased direct antitumor effects of mitotane.

Osilodrostat Is a Potential Novel Steroidogenesis Inhibitor for the Treatment of Cushing Syndrome: An In Vitro Study.

CONTEXT: Metyrapone and ketoconazole, frequently used steroidogenesis inhibitors for treatment of Cushing syndrome, can be associated with side effects and limited efficacy. Osilodrostat is a CYP11B1 and CYP11B2 inhibitor, with unknown effects on other steroidogenic enzymes. OBJECTIVE: To compare the effects of osilodrostat, metyrapone, and ketoconazole on adrenal steroidogenesis, and pituitary adenoma cells in vitro. METHODS: HAC15 cells, 17 primary human adrenocortical cell cultures, and pituitary adenoma cells were incubated with osilodrostat, metyrapone, or ketoconazole (0.01 to 10 µM). Cortisol and ACTH were measured using chemiluminescence immunoassays, and steroid profiles by liquid chromatography-mass spectrometry. RESULTS: In HAC15 cells, osilodrostat inhibited cortisol production more potently (IC50: 0.035 µM) than metyrapone (0.068 µM; P < 0.0001), and ketoconazole (0.621 µM; P < 0.0001). IC50 values of osilodrostat and metyrapone for basal cortisol production varied with a 25- and 18-fold difference, respectively, with comparable potency. Aldosterone production was inhibited more potently by osilodrostat vs metyrapone and ketoconazole. Osilodrostat and metyrapone treatment resulted in strong inhibition of corticosterone and cortisol, 11-deoxycortisol accumulation, and modest effects on adrenal androgens. No pituitary-directed effects of osilodrostat were observed. CONCLUSIONS: Under our study conditions, osilodrostat is a potent cortisol production inhibitor in human adrenocortical cells, comparable with metyrapone. All steroidogenesis inhibitors showed large variability in sensitivity between primary adrenocortical cultures. Osilodrostat might inhibit CYP11B1 and CYP11B2, in some conditions to a lesser extent CYP17A1 activity, and a proximal step in the steroidogenesis. Osilodrostat is a promising treatment option for Cushing syndrome, and in vivo differences with metyrapone are potentially driven by pharmacokinetic differences.

Identification of Mutations in Cell-Free Circulating Tumor DNA in Adrenocortical Carcinoma: A Case Series.

Context: The disease course of adrenocortical carcinoma (ACC) patients is heterogeneous. A marker for prognosis and treatment response would facilitate choices for diagnosis and therapy. In other cancer types, circulating cell-free tumor DNA predicted tumor dynamics. Case Descriptions: The present pilot study included six patients. Next-generation sequencing (NGS) showed mutations in three ACC cases. From these patients, blood was drawn before (1 to 2 weeks) and after surgery and cell-free circulating DNA (cfDNA) was isolated. Tumor-specific mutations were found in the cfDNA of one of the three patients, with metastasized ACC at diagnosis. NGS of the tumor showed an NRAS mutation (c.182A>G:p.Q61R) in 78%, a TP53 mutation (c.856G>A:p.E286K) in 60%, and a TERT gene mutation (1295250C>T) in 28% of the reads. The preoperative cfDNA showed the same mutations at a frequency of 64%, 32%, and 2%, respectively. The postoperative cfDNA showed the same mutations but at lower frequencies (52%, 16%, and 3%, respectively). The patient was postoperatively treated with mitotane and chemotherapy. No mutations were detected in the corresponding leukocyte DNA or in the cfDNA from the two other patients. Conclusions: To the best of our knowledge, we report for the first time mutations occurring at high levels in cfDNA collected before and after surgery from one of three patients, after previous identification in the tumor. However, in the cfDNA from two patients with known mutations, we were unable to reliably detect mutations in the cfDNA. Our results indicate that mutation detection in cfDNA can vary among ACC patients, and other approaches might be required to detect the tumor response and monitor progressive disease.

Cushing's syndrome: an update on current pharmacotherapy and future directions.

INTRODUCTION: Endogenous Cushing's syndrome (CS) is characterized by chronic overproduction of cortisol and is associated with increased mortality and morbidity. It can be caused by a pituitary adenoma, ectopic adrenocorticotropic hormone (ACTH) production or primary adrenal disease. Successful tumor-directed surgery is the keystone treatment. When surgery is unsuccessful, contraindicated or in case of acute disease, pharmacotherapy is indicated to treat hypercortisolism. AREAS COVERED: In this review, pharmacotherapeutic options for CS will be covered discussing the different possible targets, that is: i) inhibition of ACTH secretion; ii) suppression of steroidogenesis; and iii) blockade of cortisol effects at tissue level. Preclinical and clinical studies will be discussed considering mono- and combination therapy, taking into account efficacy, toxicity and mechanism of action. Per CS entity, future directions of pharmacotherapies will be addressed. EXPERT OPINION: The number of medical treatment options for CS has increased in the past years. In contrast to decades ago, prospective trials are now being performed focusing on pituitary-directed drugs like pasireotide, the glucocorticoid receptor blocker mifepristone and 'new generation' steroid synthesis inhibitors. Future studies will focus on tumor-shrinking effects of neuromodulatory drugs, the optimal order and combination of pharmacotherapy, long-term efficacy and safety and new targets for medical treatment of CS.