Mitotane Targets Lipid Droplets to Induce Lipolysis in Adrenocortical Carcinoma

INTRODUCTION Adrenocortical carcinoma (ACC) is a rare aggressive cancer with low overall survival. Adjuvant mitotane improves survival but is limited by poor response rates and resistance. Mitotane's efficacy is attributed to the accumulation of toxic free cholesterol, predominantly through cholesterol storage inhibition. However, targeting this pathway has proven unsuccessful. We hypothesize that mitotane-induced free-cholesterol accumulation is also mediated through enhanced breakdown of lipid droplets. METHODOLOGY ATCC-H295R (mitotane-sensitive) and MUC-1 (mitotane-resistant) ACC cells were evaluated for lipid content using specific BODIPY dyes. Protein expression was evaluated by immunoblotting and flow cytometry. Cell viability was measured by quantifying propidium iodide-positive cells following mitotane treatment and pharmacological inhibitors of lipolysis. RESULTS H295R and MUC-1 cells demonstrated similar neutral lipid droplet numbers at baseline. However, evaluation of lipid machinery demonstrated distinct profiles in each model. Analysis of intracellular lipid droplet content showed H295R cells preferentially store cholesteryl esters, whereas MUC-1 cells store triacylglycerol. Decreased lipid droplets were associated with increased lipolysis in H295R and in MUC-1 at toxic mitotane concentrations. Pharmacological inhibition of lipolysis attenuated mitotane-induced toxicity in both models. CONCLUSION We highlight that lipid droplet breakdown and activation of lipolysis represent a putative additional mechanism for mitotane-induced cytotoxicity in ACC. Further understanding of cholesterol and lipids in ACC offers potential novel therapeutic exploitation, especially in mitotane-resistant disease

Liver X receptor inhibition potentiates mitotane induced adrenotoxicity in ACC

Adrenocortical carcinoma (ACC) is a rare aggressive malignancy with a poor outcome largely due to limited treatment options. Here, we propose a novel therapeutic approach through modulating intracellular free cholesterol via the liver X receptor alpha (LXRα) in combination with current first line pharmacotherapy, mitotane. H295R and MUC-1 ACC cell lines were pretreated with LXRα inhibitors in combination with mitotane. In H295R, mitotane (20, 40, 50µM) induced dose-dependent cell death, however, in MUC-1 this only occurred at a supratherapeutic concentration (200µM). LXRα inhibition potentiated mitotane-induced cytotoxicity in both cell lines. This was confirmed through use of the CompuSyn model which showed moderate pharmacological synergism and was indicative of apoptotic cell death via an increase in annexinV and cleaved-caspase 3 expression. Inhibition of LXRα was confirmed through downregulation of cholesterol efflux pumps ABCA1 and ABCG1, however, combination treatment with mitotane attenuated this effect. Intracellular free cholesterol levels were associated with increased cytotoxicity in H295R (r2=0.5210) and MUC-1 (r2=0.9299) cells. While both cell lines exhibited similar levels of free cholesterol at baseline, H295R were cholesterol ester rich whereas MUC-1 were cholesterol ester poor. We highlight the importance of LXRα mediated cholesterol metabolism in the management of ACC, drawing attention to its role in the therapeutics of mitotane sensitive tumours. We also demonstrate significant differences in cholesterol storage between mitotane sensitive and resistant disease.</jats:p

Low DHEAS: A Sensitive and Specific Test for the Detection of Subclinical Hypercortisolism in Adrenal Incidentalomas.

CONTEXT: Subclinical hypercortisolism (SH) occurs in 5% to 30% of adrenal incidentalomas (AIs). Common screening tests for adrenocorticotropin-independent hypercortisolism have substantial false-positive rates, mandating further time and resource-intensive investigations. OBJECTIVE: To determine whether low basal dehydroepiandrosterone sulfate (DHEAS) is a sensitive and specific screening test for SH in AI. SETTING AND PATIENTS: In total, 185 patients with AI were screened for adrenal medullary (plasma metanephrines) and cortical [1 mg overnight dexamethasone suppression test (ONDST), 24-hour urinary free cortisol (UFC), serum DHEAS, plasma renin, and aldosterone] hyperfunction. Positive ONDST [≥1.8 mcg/dL (≥50 nmol/L)] and/or UFC (more than the upper limit of reference range) results were further investigated. We diagnosed SH when at least 2 of the following were met: raised UFC, raised midnight serum cortisol, 48-hour dexamethasone suppression test (DST) cortisol ≥1.8 mcg/dL (≥50 nmol/L). RESULTS: 29 patients (16%) were diagnosed with SH. Adrenocorticotropin was 99%) and specific (91.9%) for the diagnosis of SH. Cortisol following 1 mg ONDST of 1.9 mcg/dL (53 nmol/L) was a sensitive (>99%) screening test for SH but had lower specificity (82.9%). The 24-hour UFC lacked sensitivity (69%) and specificity (72%). CONCLUSION: A single basal measurement of DHEAS offers comparable sensitivity and greater specificity to the existing gold-standard 1 mg DST for the detection of SH in patients with AIs

Chronic Kidney Disease Severity Is Associated With Selective Expansion of a Distinctive Intermediate Monocyte Subpopulation

Chronic kidney disease (CKD) affects 11–13% of the world's population and greatly increases risk of atherosclerotic cardiovascular disease (ASCVD) and death. It is characterized by systemic inflammation and disturbances in the blood leukocytes that remain incompletely understood. In particular, abnormalities in the numbers and relative proportions of the three major monocyte subsets—classical, intermediate, and non-classical—are described in CKD and end-stage renal disease. In this study, we characterized absolute numbers of blood leukocyte subtypes in adults with renal function varying from normal to advanced CKD. The primary aim was to identify monocyte subpopulations that associated most closely with current estimated glomerular filtration rate (eGFR) and subsequent rate of eGFR decline. Leucocyte and monocyte populations were enumerated by multi-color flow cytometry of whole blood and peripheral blood mononuclear cell (PBMC) samples from adults with CKD stage 1–5 (n = 154) and healthy adults (n = 33). Multiple-linear regression analyses were performed to identify associations between numbers of leucocyte and monocyte populations and clinical characteristics including eGFR and rate of eGFR decline with adjustment for age and gender. In whole blood, total monocyte and neutrophil, but not lymphocyte, numbers were higher in adults with CKD 1-5 compared to no CKD and were significantly associated with current eGFR even following correction for age. In PBMC, classical and intermediate monocyte numbers were higher in CKD 1-5 but only intermediate monocyte numbers were significantly associated with current eGFR in an age-corrected analysis. When intermediate monocytes were further sub-divided into those with mid- and high-level expression of class II MHC (HLA-DRmid and HLA-DRhi intermediate monocytes) it was found that only DRhi intermediate monocytes were increased in number in CKD 1-5 compared to no CKD and were significantly associated with eGFR independently of age among the total (No CKD + CKD 1-5) study cohort as well as those with established CKD (CKD 1-5 only). Furthermore, blood number of DRhi intermediate monocytes alone proved to be significantly associated with subsequent rate of renal functional decline. Together, our data confirm neutrophil and monocyte subset dysregulation in CKD and identify a distinct subpopulation of intermediate monocytes that is associated with higher rate of loss of kidney function

Urine steroid metabolomics as a novel diagnostic tool for early detection of recurrence in adrenocortical carcinoma

Introduction: Adrenocortical carcinoma (ACC) is an aggressive malignancy with a high rate of recurrence. Regular post-operative follow-up imaging is necessary, but associated with high radiation exposure and frequent diagnostic ambiguity. Urine steroid metabolomics has recently been introduced as a novel diagnostic tool for the detection of adrenocortical malignancy in patients with adrenal incidentalomas. Here we present the first clinical study assessing the performance of this innovative approach in the context of follow-up after complete (R0) ACC resection. Patients and methods: We included 166 patients from 13 centres registered with the European Network for the Study of Adrenal Tumours (ENSAT). We selected all patients recorded between 2008 and 2015 fulfilling the following criteria: i) recorded on the ENSAT registry as confirmed adrenocortical carcinoma with R0 primary tumour resection and ii) availability of at least two postoperative 24-h urines, one whilst disease-free and the other after recurrence. Twenty-four-hour urines were analysed by gas chromatography–mass spectrometry, with quantification of 38 distinct steroid metabolites. A machine learning-based computational algorithm was employed to detect ACC recurrence. Results: Twenty-one patients developed 22 ACC recurrences during the study period as documented by serial cross-sectional imaging and biopsy where appropriate. Steroid metabolomics predicted disease recurrence at the time of first abnormal imaging with a sensitivity of 84% and specificity of 95%. Adjuvant mitotane in 12/21 patients did not affect accuracy. In the subgroup of patients for whom a diagnostic pre-operative 24-h urine sample was available (n=7), we were able to accurately detect all cases of recurrence (sensitivity and specificity 100%). In seven cases, biochemical evidence of disease recurrence pre-dated the first radiological detection by more than 2 months (range 2–11 months). Conclusion: Our study provides proof-of-principle evidence suggesting a role for urine steroid metabolomics as a potent diagnostic tool in the follow-up monitoring of ACC

Clinical study on 6 cases of urosepsis associated with septic shock

6例のseptic shockを伴うurosepsis症例について全例で糖尿病, 脳血管障害, 癌のいずれかを合併していた.基礎疾患として, 尿路閉塞の他糖尿病に合併する腎乳頭壊死を伴う腎膿瘍, 気腫性腎盂腎炎が重要であった.尿路閉塞のあるseptic shockでは, 抗shock療法や, DICに対する抗凝固療法の効果は疑問であるAt Asama General Hospital, we experienced six cases of urosepsis with septic shock during a period of five years between 1989 and 1993. All six patients, whose average age was 74 years old, recovered. In four patients, the condition was caused by obstructive uropathy. The remaining two cases were caused by renal inflammatory disease, which was complicated by diabetes mellitus. One of them was renal abscess with renal papillary necrosis, and the other was emphysematous pyelonephritis. The patients, who exhibited symptoms such as gram-negative bacteremia, severe hypotension, tachycardia, decrease of urine volume and mental disturbance, were diagnosed with urosepsis with septic shock. In all cases, symptoms such as a high fever of over 39 degrees C, hypoxemia and thrombocytopenia were observed. Renal dysfunction was found in 67%, and both liver dysfunction and disseminated intravascular coagulation (DIC) were found in 50% of the cases. Since no patients suffered from adult respiratory distress syndrome, a high survival rate was apparent. Anti-shock therapy and anti-coagulation therapy were ineffective for the patients who had septic shock due to urinary tract obstruction. Urinary tract drainage was required to treat the latter patients. Nephrectomy could not be avoided in renal parenchymatous inflammatory disease. In the future, what might be essential in therapeutics against urosepsis with septic shock, particularly to avoid nephrectomy, are the treatments such as immunotherapy against endotoxins and their mediators, and hemoperfusion for the removal of endotoxins

Image-based computer modeling assessment of microwave ablation for treatment of adrenal tumors

To assess the feasibility of delivering microwave ablation for targeted treatment of aldosterone producing adenomas using image-based computational models. We curated an anonymized dataset of diagnostic 11C-metomidate PET/CT images of 14 patients with aldosterone producing adenomas (APA). A semi-automated approach was developed to segment the APA, adrenal gland, and adjacent organs within 2 cm of the APA boundary. The segmented volumes were used to implement patient-specific 3D electromagnetic-bioheat transfer models of microwave ablation with a 2.45 GHz directional microwave ablation applicator. Ablation profiles were quantitatively assessed based on the extent of the APA target encompassed by an ablative thermal dose, while limiting thermal damage to the adjacent normal adrenal tissue and sensitive critical structures. Across the 14 patients, adrenal tumor volumes ranged between 393 mm3 and 2,395 mm3. On average, 70% of the adrenal tumor volumes received an ablative thermal dose of 240CEM43, while limiting thermal damage to non-target structures, and thermally sparing 83.5–96.4% of normal adrenal gland. Average ablation duration was 293 s (range: 60–600 s). Simulations indicated coverage of the APA with an ablative dose was limited when the axis of the ablation applicator was not well aligned with the major axis of the targeted APA. Image-based computational models demonstrate the potential for delivering microwave ablation to APA targets within the adrenal gland, while limiting thermal damage to surrounding non-target structures.</p

Cardiometabolic Disease Burden and Steroid Excretion in Benign Adrenal Tumors A Cross-Sectional Multicenter Study

Background: Benign adrenal tumors are commonly discovered on cross-sectional imaging. Mild autonomous cortisol secretion (MACS) is regularly diagnosed, but its effect on cardiometabolic disease in affected persons is ill defined. Objective: To determine cardiometabolic disease burden and steroid excretion in persons with benign adrenal tumors with and without MACS. Design: Cross-sectional study. Setting: 14 endocrine secondary and tertiary care centers (recruitment from 2011 to 2016). Participants: 1305 prospectively recruited persons with benign adrenal tumors. Measurements: Cortisol excess was defined by clinical assessment and the 1-mg overnight dexamethasone-suppression test (serum cortisol: 138 nmol/L and absence of typical clinical Cushing syndrome [CS] features, definitive MACS [MACS-2]). Net steroid production was assessed by multisteroid profiling of 24-hour urine by tandem mass spectrometry. Results: Of the 1305 participants, 49.7% had NFAT (n = 649; 64.1% women), 34.6% had MACS-1 (n = 451; 67.2% women), 10.7% had MACS-2 (n = 140; 73.6% women), and 5.0% had CS (n = 65; 86.2% women). Prevalence and severity of hypertension were higher in MACS-2 and CS than NFAT (adjusted prevalence ratios [aPRs] for hypertension: MACS-2, 1.15 [95% CI, 1.04 to 1.27], and CS, 1.37 [CI, 1.16 to 1.62]; aPRs for use of ≥3 antihypertensives: MACS-2, 1.31 [CI, 1.02 to 1.68], and CS, 2.22 [CI, 1.62 to 3.05]). Type 2 diabetes was more prevalent in CS than NFAT (aPR, 1.62 [CI, 1.08 to 2.42]) and more likely to require insulin therapy for MACS-2 (aPR, 1.89 [CI, 1.01 to 3.52]) and CS (aPR, 3.06 [CI, 1.60 to 5.85]). Urinary multisteroid profiling revealed an increase in glucocorticoid excretion from NFAT over MACS-1 and MACS-2 to CS, whereas androgen excretion decreased. Limitations: Cross-sectional design; possible selection bias. Conclusion: A cardiometabolic risk condition, MACS predominantly affects women and warrants regular assessment for hypertension and type 2 diabetes