Publications by authors named "Silviu Sbiera"

63 Publications

Case Report: Consecutive Adrenal Cushing's Syndrome and Cushing's Disease in a Patient With Somatic , , and Mutations.

Front Endocrinol (Lausanne) 2021 20;12:731579. Epub 2021 Aug 20.

Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital Würzburg, University of Würzburg, Würzburg, Germany.

The occurrence of different subtypes of endogenous Cushing's syndrome (CS) in single individuals is extremely rare. We here present the case of a female patient who was successfully cured from adrenal CS 4 years before being diagnosed with Cushing's disease (CD). The patient was diagnosed at the age of 50 with ACTH-independent CS and a left-sided adrenal adenoma, in January 2015. After adrenalectomy and histopathological confirmation of a cortisol-producing adrenocortical adenoma, biochemical hypercortisolism and clinical symptoms significantly improved. However, starting from 2018, the patient again developed signs and symptoms of recurrent CS. Subsequent biochemical and radiological workup suggested the presence of ACTH-dependent CS along with a pituitary microadenoma. The patient underwent successful transsphenoidal adenomectomy, and both postoperative adrenal insufficiency and histopathological workup confirmed the diagnosis of CD. Exome sequencing excluded a causative germline mutation but showed somatic mutations of the β-catenin protein gene () in the adrenal adenoma, and of both the ubiquitin specific peptidase 8 () and the glucocorticoid receptor () genes in the pituitary adenoma. In conclusion, our case illustrates that both ACTH-independent and ACTH-dependent CS may develop in a single individual even without evidence for a common genetic background.
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http://dx.doi.org/10.3389/fendo.2021.731579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8417750PMC
August 2021

High expression of Sterol-O-Acyl transferase 1 (SOAT1), an enzyme involved in cholesterol metabolism, is associated with earlier biochemical recurrence in high risk prostate cancer.

Prostate Cancer Prostatic Dis 2021 Jul 29. Epub 2021 Jul 29.

University Hospital Würzburg, Department of Internal Medicine I, Division of Endocrinology and Diabetology, Würzburg, Germany.

Background: Prostate cancer (PCa) is the most frequent cancer in men. The prognosis of PCa is heterogeneous with many clinically indolent tumors and rare highly aggressive cases. Reliable tissue markers of prognosis are lacking. Active cholesteryl ester synthesis has been associated with prostate cancer aggressiveness. Sterol-O-Acyl transferases (SOAT) 1 and 2 catalyze cholesterol esterification in humans.

Objective: To investigate the value of SOAT1 and SOAT2 tissue expression as prognostic markers in high risk PCa.

Patients And Methods: Formalin-fixed paraffin-embedded tissue samples from 305 high risk PCa cases treated with radical prostatectomy were analyzed for SOAT1 and SOAT2 protein expression by semi-quantitative immunohistochemistry. The Kaplan-Meier method and Cox proportional hazards modeling were used to compare outcome.

Main Outcome Measure: Biochemical recurrence (BCR) free survival.

Results: SOAT1 expression was high in 73 (25%) and low in 219 (75%; not evaluable: 13) tumors. SOAT2 was highly expressed in 40 (14%) and at low levels in 249 (86%) samples (not evaluable: 16). By Kaplan-Meier analysis, we found significantly shorter median BCR free survival of 93 months (95% confidence interval 23.6-123.1) in patients with high SOAT1 vs. 134 months (112.6-220.2, Log-rank p < 0.001) with low SOAT1. SOAT2 expression was not significantly associated with BCR. After adjustment for age, preoperative PSA, tumor stage, Gleason score, resection status, lymph node involvement and year of surgery, high SOAT1 but not SOAT2 expression was associated with shorter BCR free survival with a hazard ratio of 2.40 (95% CI 1.57-3.68, p < 0.001). Time to clinical recurrence and overall survival were not significantly associated with SOAT1 and SOAT2 expression CONCLUSIONS: SOAT1 expression is strongly associated with BCR free survival alone and after multivariable adjustment in high risk PCa. SOAT1 may serve as a histologic marker of prognosis and holds promise as a future treatment target.
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http://dx.doi.org/10.1038/s41391-021-00431-3DOI Listing
July 2021

Epithelial and Mesenchymal Markers in Adrenocortical Tissues: How Mesenchymal Are Adrenocortical Tissues?

Cancers (Basel) 2021 Apr 6;13(7). Epub 2021 Apr 6.

Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital Würzburg, 97080 Würzburg, Germany.

A clinically relevant proportion of adrenocortical carcinoma (ACC) cases shows a tendency to metastatic spread. The objective was to determine whether the epithelial to mesenchymal transition (EMT), a mechanism associated with metastasizing in several epithelial cancers, might play a crucial role in ACC. 138 ACC, 29 adrenocortical adenomas (ACA), three normal adrenal glands (NAG), and control tissue samples were assessed for the expression of epithelial (E-cadherin and EpCAM) and mesenchymal (N-cadherin, SLUG and SNAIL) markers by immunohistochemistry. Using real-time RT-PCR we quantified the alternative isoform splicing of FGFR 2 and 3, another known indicator of EMT. We also assessed the impact of these markers on clinical outcome. Results show that both normal and neoplastic adrenocortical tissues lacked expression of epithelial markers but strongly expressed mesenchymal markers N-cadherin and SLUG. FGFR isoform splicing confirmed higher similarity of adrenocortical tissues to mesenchymal compared to epithelial tissues. In ACC, higher SLUG expression was associated with clinical markers indicating aggressiveness, while N-cadherin expression inversely associated with these markers. In conclusion, we could not find any indication of EMT as all adrenocortical tissues lacked expression of epithelial markers and exhibited closer similarity to mesenchymal tissues. However, while N-cadherin might play a positive role in tissue structure upkeep, SLUG seems to be associated with a more aggressive phenotype.
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http://dx.doi.org/10.3390/cancers13071736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038668PMC
April 2021

A novel patient-derived cell line of adrenocortical carcinoma shows a pathogenic role of germline MUTYH mutation and high tumour mutational burden.

Eur J Endocrinol 2021 May 4;184(6):823-835. Epub 2021 May 4.

Division of Endocrinology and Diabetology Department of Internal Medicine I, University Hospital, University of Würzburg, Würzburg, Germany.

Background: The response of advanced adrenocortical carcinoma (ACC) to current chemotherapies is unsatisfactory and a limited rate of response to immunotherapy was observed in clinical trials. High tumour mutational burden (TMB) and the presence of a specific DNA signature are characteristic features of tumours with mutations in the gene MUTYH encoding the mutY DNA glycosylase. Both have been shown to potentially predict the response to immunotherapy. High TMB in an ACC cell line model has not been reported yet.

Design And Methods: The JIL-2266 cell line was established from a primary ACC tumour, comprehensively characterised and oxidative damage, caused by a dysfunctional mutY DNA glycosylase, confirmed.

Results: Here, we characterise the novel patient-derived ACC cell line JIL-2266, which is deficient in mutY-dependent DNA repair. JIL-2266 cells have a consistent STR marker profile that confirmed congruousness with primary ACC tumour. Cells proliferate with a doubling time of 41 ± 13 h. Immunohistochemistry revealed positivity for steroidogenic factor-1. Mass spectrometry did not demonstrate significant steroid hormone synthesis. JIL-2266 have hemizygous mutations in the tumour suppressor gene TP53 (c.859G>T:p.E287X) and MUTYH (c.316C>T:p.R106W). Exome sequencing showed 683 single nucleotide variants and 4 insertions/deletions. We found increased oxidative DNA damage in the cell line and the corresponding primary tumour caused by impaired mutY DNA glycosylase function and accumulation of 8-oxoguanine.

Conclusion: This model will be valuable as a pre-clinical ACC cell model with high TMB and a tool to study oxidative DNA damage in the adrenal gland.
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http://dx.doi.org/10.1530/EJE-20-1423DOI Listing
May 2021

Circulating microRNA Expression in Cushing's Syndrome.

Front Endocrinol (Lausanne) 2021 22;12:620012. Epub 2021 Feb 22.

Department of Endocrinology, Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians-University, Munich, Germany.

Context: Cushing's syndrome (CS) is a rare disease of endogenous hypercortisolism associated with high morbidity and mortality. Diagnosis and classification of CS is still challenging.

Objective: Circulating microRNAs (miRNAs) are minimally invasive diagnostic markers. Our aim was to characterize the circulating miRNA profiles of CS patients and to identify distinct profiles between the two major CS subtypes.

Methods: We included three groups of patients from the German Cushing's registry: ACTH-independent CS (Cortisol-Producing-Adenoma; CPA), ACTH-dependent pituitary CS (Cushing's Disease; CD), and patients in whom CS had been ruled out (controls). Profiling of miRNAs was performed by next-generation-sequencing (NGS) in serum samples of 15 CS patients (each before and after curative surgery) and 10 controls. Significant miRNAs were first validated by qPCR in the discovery cohort and then in an independent validation cohort of 20 CS patients and 11 controls.

Results: NGS identified 411 circulating miRNAs. Differential expression of 14 miRNAs were found in the pre- and postoperative groups. qPCR in the discovery cohort validated 5 of the significant miRNAs from the preoperative group analyses. Only, miR-182-5p was found to be significantly upregulated in the CD group of the validation cohort. Comparing all CS samples as a group with the controls did not reveal any significant differences in expression.

Outcome: In conclusion, our study identified miR-182-5p as a possible biomarker for CD, which has to be validated in a prospective cohort. Furthermore, our results suggest that presence or absence of ACTH might be at least as relevant for miRNA expression as hypercortisolism itself.
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http://dx.doi.org/10.3389/fendo.2021.620012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937959PMC
February 2021

PKA Cα subunit mutation triggers caspase-dependent RIIβ subunit degradation via Ser phosphorylation.

Sci Adv 2021 Feb 19;7(8). Epub 2021 Feb 19.

Division of Endocrinology and Diabetes, Department of Internal Medicine I, University Hospital, University of Würzburg, 97080 Würzburg, Germany.

Mutations in the gene are the most frequent cause of cortisol-producing adrenocortical adenomas leading to Cushing's syndrome. encodes for the catalytic subunit α of protein kinase A (PKA). We already showed that mutations lead to impairment of regulatory (R) subunit binding. Furthermore, mutations are associated with reduced RIIβ protein levels; however, the mechanisms leading to reduced RIIβ levels are presently unknown. Here, we investigate the effects of the most frequent mutation, L206R, on regulatory subunit stability. We find that Ser phosphorylation of RIIβ is required for its degradation, mediated by caspase 16. Last, we show that the resulting reduction in RIIβ protein levels leads to increased cortisol secretion in adrenocortical cells. These findings reveal the molecular mechanisms and pathophysiological relevance of the R subunit degradation caused by mutations, adding another dimension to the deregulation of PKA signaling caused by mutations in adrenal Cushing's syndrome.
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http://dx.doi.org/10.1126/sciadv.abd4176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895437PMC
February 2021

Single-cell molecular profiling of all three components of the HPA axis reveals adrenal ABCB1 as a regulator of stress adaptation.

Sci Adv 2021 Jan 27;7(5). Epub 2021 Jan 27.

Department of Stress Neurobiology and Neurogenetics, Max Planck Institute of Psychiatry, Munich, Bavaria 80804, Germany.

Chronic activation and dysregulation of the neuroendocrine stress response have severe physiological and psychological consequences, including the development of metabolic and stress-related psychiatric disorders. We provide the first unbiased, cell type-specific, molecular characterization of all three components of the hypothalamic-pituitary-adrenal axis, under baseline and chronic stress conditions. Among others, we identified a previously unreported subpopulation of cells involved in stress adaptation in the adrenal gland. We validated our findings in a mouse stress model, adrenal tissues from patients with Cushing's syndrome, adrenocortical cell lines, and peripheral cortisol and genotyping data from depressed patients. This extensive dataset provides a valuable resource for researchers and clinicians interested in the organism's nervous and endocrine responses to stress and the interplay between these tissues. Our findings raise the possibility that modulating ABCB1 function may be important in the development of treatment strategies for patients suffering from metabolic and stress-related psychiatric disorders.
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http://dx.doi.org/10.1126/sciadv.abe4497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840126PMC
January 2021

Corticotroph tumor progression after bilateral adrenalectomy (Nelson's syndrome): systematic review and expert consensus recommendations.

Eur J Endocrinol 2021 Mar;184(3):P1-P16

Section on Genetics & Endocrinology Eunice Kennedy Shriver National Insitute of Child Health & Human Development (NICHD) National Institute of Health (NIH), NIH Clinical Research Center, Bethesda, Maryland, USA.

Background: Corticotroph tumor progression (CTP) leading to Nelson's syndrome (NS) is a severe and difficult-to-treat complication subsequent to bilateral adrenalectomy (BADX) for Cushing's disease. Its characteristics are not well described, and consensus recommendations for diagnosis and treatment are missing.

Methods: A systematic literature search was performed focusing on clinical studies and case series (≥5 patients). Definition, cumulative incidence, treatment and long-term outcomes of CTP/NS after BADX were analyzed using descriptive statistics. The results were presented and discussed at an interdisciplinary consensus workshop attended by international pituitary experts in Munich on October 28, 2018.

Results: Data covered definition and cumulative incidence (34 studies, 1275 patients), surgical outcome (12 studies, 187 patients), outcome of radiation therapy (21 studies, 273 patients), and medical therapy (15 studies, 72 patients).

Conclusions: We endorse the definition of CTP-BADX/NS as radiological progression or new detection of a pituitary tumor on thin-section MRI. We recommend surveillance by MRI after 3 months and every 12 months for the first 3 years after BADX. Subsequently, we suggest clinical evaluation every 12 months and MRI at increasing intervals every 2-4 years (depending on ACTH and clinical parameters). We recommend pituitary surgery as first-line therapy in patients with CTP-BADX/NS. Surgery should be performed before extrasellar expansion of the tumor to obtain complete and long-term remission. Conventional radiotherapy or stereotactic radiosurgery should be utilized as second-line treatment for remnant tumor tissue showing extrasellar extension.
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http://dx.doi.org/10.1530/EJE-20-1088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060870PMC
March 2021

RNA Sequencing and Somatic Mutation Status of Adrenocortical Tumors: Novel Pathogenetic Insights.

J Clin Endocrinol Metab 2020 12;105(12)

Division of Endocrinology and Diabetes, Department of Internal Medicine I, University Hospital, University of Würzburg, Würzburg, Germany.

Context: Pathogenesis of autonomous steroid secretion and adrenocortical tumorigenesis remains partially obscure.

Objective: To investigate the relationship between transcriptome profile and genetic background in a large series of adrenocortical tumors and identify new potential pathogenetic mechanisms.

Design: Cross-sectional study.

Setting: University Hospitals of the European Network for the Study of Adrenal Tumors (ENSAT).

Patients: We collected snap-frozen tissue from patients with adrenocortical tumors (n = 59) with known genetic background: 26 adenomas with Cushing syndrome (CS- cortisol-producing adenoma [CPA]), 17 adenomas with mild autonomous cortisol secretion (MACS-CPAs), 9 endocrine-inactive adenomas (EIAs), and 7 adrenocortical carcinomas (ACCs).

Intervention: Ribonucleic acid (RNA) sequencing.

Main Outcome Measures: Gene expression, long noncoding RNA (lncRNA) expression, and gene fusions. Correlation with genetic background defined by targeted Sanger sequencing, targeted panel- or whole-exome sequencing.

Results: Transcriptome analysis identified 2 major clusters for adenomas: Cluster 1 (n = 32) mainly consisting of MACS-CPAs with CTNNB1 or without identified driver mutations (46.9% of cases) and 8/9 EIAs; Cluster 2 (n = 18) that comprised CP-CPAs with or without identified driver mutation in 83.3% of cases (including all CS-CPAs with PRKACA mutation). Two CS-CPAs, 1 with CTNNB1 and 1 with GNAS mutation, clustered separately and relatively close to ACC. lncRNA analysis well differentiate adenomas from ACCs. Novel gene fusions were found, including AKAP13-PDE8A in one CS-CPA sample with no driver mutation.

Conclusions: MACS-CPAs and EIAs showed a similar transcriptome profile, independently of the genetic background, whereas most CS-CPAs clustered together. Still unrevealed molecular alterations in the cAMP/PKA or Wnt/beta catenin pathways might be involved in the pathogenesis of adrenocortical tumors.
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http://dx.doi.org/10.1210/clinem/dgaa616DOI Listing
December 2020

Interplay between glucocorticoids and tumor-infiltrating lymphocytes on the prognosis of adrenocortical carcinoma.

J Immunother Cancer 2020 05;8(1)

Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital Würzburg, Würzburg, Germany

Background: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy. Tumor-related glucocorticoid excess is present in ~60% of patients and associated with particularly poor prognosis. Results of first clinical trials using immune checkpoint inhibitors were heterogeneous. Here we characterize tumor-infiltrating T lymphocytes (TILs) in ACC in association with glucocorticoids as potential explanation for resistance to immunotherapy.

Methods: We performed immunofluorescence analysis to visualize tumor-infiltrating T cells (CD3), T helper cells (CD3CD4), cytotoxic T cells (CD3CD8) and regulatory T cells (Tregs; CD3CD4FoxP3) in 146 ACC tissue specimens (107 primary tumors, 16 local recurrences, 23 metastases). Quantitative data of immune cell infiltration were correlated with clinical data (including glucocorticoid excess).

Results: 86.3% of ACC specimens showed tumor infiltrating T cells (7.7 cells/high power field (HPF)), including T helper (74.0%, 6.7 cells/HPF), cytotoxic T cells (84.3%, 5.7 cells/HPF) and Tregs (49.3%, 0.8 cells/HPF). The number of TILs was associated with better overall survival (HR for death: 0.47, 95% CI 0.25 to 0.87), which was true for CD4- and CD8 subpopulations as well. In localized, non-metastatic ACC, the favorable impact of TILs on overall and recurrence-free survival was manifested even independently of ENSAT (European Network for the Study of Adrenal Tumors) stage, resection status and Ki67 index. T helper cells were negatively correlated with glucocorticoid excess (Phi=-0.290, p=0.009). Patients with glucocorticoid excess and low TILs had a particularly poor overall survival (27 vs. 121 months in patients with TILs without glucocorticoid excess).

Conclusion: Glucocorticoid excess is associated with T cell depletion and unfavorable prognosis. To reactivate the immune system in ACC by checkpoint inhibitors, an inhibition of adrenal steroidogenesis might be pivotal and should be tested in prospective studies.
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http://dx.doi.org/10.1136/jitc-2019-000469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264832PMC
May 2020

Expression of SOAT1 in Adrenocortical Carcinoma and Response to Mitotane Monotherapy: An ENSAT Multicenter Study.

J Clin Endocrinol Metab 2020 08;105(8)

Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital, University of Würzburg, Würzburg, Germany.

Context: Objective response rate to mitotane in advanced adrenocortical carcinoma (ACC) is approximately 20%, and adverse drug effects are frequent. To date, there is no marker established that predicts treatment response. Mitotane has been shown to inhibit sterol-O-acyl transferase 1 (SOAT1), which leads to endoplasmic reticulum stress and cell death in ACC cells.

Objective: To investigate SOAT1 protein expression as a marker of treatment response to mitotane.

Patients: A total of 231 ACC patients treated with single-agent mitotane as adjuvant (n = 158) or advanced disease therapy (n = 73) from 12 ENSAT centers were included. SOAT1 protein expression was determined by immunohistochemistry on formalin-fixed paraffin-embedded specimens.

Setting: Retrospective study at 12 ACC referral centers.

Main Outcome Measure: Recurrence-free survival (RFS), progression-free survival (PFS), and disease-specific survival (DSS).

Results: Sixty-one of 135 patients (45%) with adjuvant mitotane treatment had recurrences and 45/68 patients (66%) with mitotane treatment for advanced disease had progressive disease. After multivariate adjustment for sex, age, hormone secretion, tumor stage, and Ki67 index, RFS (hazard ratio [HR] = 1.07; 95% confidence interval [CI], 0.61-1.85; P = 0.82), and DSS (HR = 1.30; 95% CI, 0.58-2.93; P = 0.53) in adjuvantly treated ACC patients did not differ significantly between tumors with high and low SOAT1 expression. Similarly, in the advanced stage setting, PFS (HR = 1.34; 95% CI, 0.63-2.84; P = 0.45) and DSS (HR = 0.72; 95% CI, 0.31-1.70; P = 0.45) were comparable and response rates not significantly different.

Conclusions: SOAT1 expression was not correlated with clinical endpoints RFS, PFS, and DSS in ACC patients with mitotane monotherapy. Other factors appear to be relevant for mitotane treatment response and ACC patient survival.
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http://dx.doi.org/10.1210/clinem/dgaa293DOI Listing
August 2020

Correction: Steroidogenesis in the NCI-H295 Cell Line Model is Strongly Affected By Culture Conditions and Substrain.

Exp Clin Endocrinol Diabetes 2020 May 12. Epub 2020 May 12.

Department of Internal Medicine I, Division of Endocrinology and Diabetes, University of Würzburg, University Hospital Würzburg, Würzburg, Germany.

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http://dx.doi.org/10.1055/a-1169-5951DOI Listing
May 2020

Targeted Gene Expression Profile Reveals CDK4 as Therapeutic Target for Selected Patients With Adrenocortical Carcinoma.

Front Endocrinol (Lausanne) 2020 16;11:219. Epub 2020 Apr 16.

Division of Endocrinology and Diabetology, Department of Internal Medicine, University Hospital of Wuerzburg, Würzburg, Germany.

Adrenocortical carcinomas (ACC) are aggressive tumors with a heterogeneous prognosis and limited therapeutic options for advanced stages. This study aims to identify novel drug targets for a personalized treatment in ACC. RNA was isolated from 40 formalin-fixed paraffin-embedded ACC samples. We evaluated gene expression of 84 known cancer drug targets by reverse transcriptase quantitative real time-PCR and calculated fold change using 5 normal adrenal glands as reference (overexpression by fold change >2.0). The most promising candidate cyclin-dependent kinase 4 (CDK4) was investigated at protein level in 104 ACC samples and tested by experiments in two ACC cell lines (NCI-H295R and MUC1). The most frequently overexpressed genes were (100% of cases, median fold change = 16.5), (95%, fold change = 52.9), (80%, fold change = 6.7) (62%, fold change = 2.6) (60%, fold change = 2.8), and (52%, fold change = 2.3). CDK4 was chosen for functional validation, as it is actionable by approved CDK4/6-inhibitors (e.g., palbociclib). Nuclear immunostaining of CDK4 significantly correlated with mRNA expression (R = 0.52, < 0.005). We exposed both NCI-H295R and MUC1 cell lines to palbociclib and found a concentration- and time-dependent reduction of cell viability, which was more pronounced in the NCI-H295R cells in line with higher CDK4 expression. Furthermore, we tested palbociclib in combination with insulin-like growth factor 1/insulin receptor inhibitor linsitinib showing an additive effect. In conclusion, we demonstrate that RNA profiling is useful to discover potential drug targets and that CDK4/6 inhibitors are promising candidates for treatment of selected patients with ACC.
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http://dx.doi.org/10.3389/fendo.2020.00219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176906PMC
May 2021

Steroidogenesis in the NCI-H295 Cell Line Model is Strongly Affected By Culture Conditions and Substrain.

Exp Clin Endocrinol Diabetes 2020 Oct 29;128(10):672-680. Epub 2020 Apr 29.

Department of Internal Medicine I, Division of Endocrinology and Diabetes, University of Würzburg, University Hospital Würzburg, Würzburg, Germany.

Context: NCI-H295 cells are the most widely used model for adrenal steroidogenesis and adrenocortical carcinoma and have been used for decades in laboratories worldwide. However, reported steroidogenic properties differ considerably.

Objective: To evaluate heterogeneity of steroidogenesis among NCI-H295 cell strains, clarify the influence of culture media and test response to inhibitors of steroidogenesis by using liquid chromatography tandem mass spectrometry (LC-MS/MS).

Methods: NCI-H295 cells were obtained from two cell banks and cultivated in different media. An LC-MS/MS-based panel analysis of thirteen steroids was adapted for cell culture supernatant. Cells were treated with metyrapone, abiraterone and mitotane.

Results: Mineralocorticoid synthesis was strongly affected by passaging as reflected by reduction of aldosterone secretion from 0.158±0.006 to 0.017±0.001 µg/10 cells (p<0.05). Relevant differences were also found for cells from two vendors in terms of aldosterone secretion (0.180±0.001 vs. 0.09±0.002 µg/10 cells, p<0.05). Selection of medium strongly impacted on cortisol secretion with>4-fold difference (40.6±5.5 vs. 182.1±23 µg/10 cells) and reflected differential activation of the glucocorticoid pathway. Exposure to abiraterone, metyrapone and mitotane resulted in characteristic steroidogenic profiles consistent with known mechanism of drug action with considerable differences in metabolites upstream of the blocked enzyme.

Conclusion: We demonstrate that steroid hormone secretion in NCI-H295 cells is strongly affected by the individual strain, passage and growing conditions. These factors should be taken into account in the evaluation of experiments analyzing steroid parameters directly or as surrogate parameters of cell viability.
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http://dx.doi.org/10.1055/a-1105-6332DOI Listing
October 2020

Early Postoperative Circulating miR-483-5p Is a Prognosis Marker for Adrenocortical Cancer.

Cancers (Basel) 2020 Mar 19;12(3). Epub 2020 Mar 19.

Univ. Grenoble Alpes, INSERM, CEA, IRIG, Biology of Cancer and Infection UMR_S 1036, F-38000 Grenoble, France.

We have previously identified serum miR-483-5p as a preoperative diagnosis and prognosis biomarker for adrenocortical cancer (ACC). Here, we aimed to determine whether circulating miR-483-5p levels measured 3 months post-operatively distinguished patients with good prognosis (no recurrence for at least 3 years; NR3yrs) from patients with poor prognosis (recurrence or death within 3 years after surgery; R < 3yrs). We conducted a single-center retrospective analysis using sera from 48 patients with ACC that were initially non-metastatic and treated by surgery. Sera sampled within 3 months after surgery were available in 26 patients. MiR-483-5p absolute circulating levels were measured using quantitative PCR. Thirteen patients showed a recurrence before 3 years (=R < 3yrs). Thirteen patients showed no recurrence within 3 years, including 11 patients with a follow-up longer than 3 years (=NR3yrs). Serum miR-483-5p levels were higher in R < 3yrs than in NR3yrs: 1,541,990 ± 428,377 copies/mL vs. 388,457 ± 62,169 copies/mL ( = 0.002). Receiver operating characteristic analysis showed that a value of 752,898 copies/mL distinguished R < 3yrs from NR3yrs with 61.5% sensitivity (CI 31.6-86.1) and 100% specificity (CI 71.5-100) with an area under the curve of 0.853. Patients with a value below this threshold had a significantly longer recurrence-free and overall survival. In multivariate analysis, miR-483-5p provided the single best prognostic value for recurrence-free survival (RFS) (hazard ratio (HR) for recurrence 5.98, < 0.011) but not for overall survival. Our study suggests that serum miR-483-5p is a potent early post-operative biomarker for ACC prognosis that might be a better predictor of RFS than currently used markers.
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http://dx.doi.org/10.3390/cancers12030724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140036PMC
March 2020

Active steroid hormone synthesis renders adrenocortical cells highly susceptible to type II ferroptosis induction.

Cell Death Dis 2020 03 17;11(3):192. Epub 2020 Mar 17.

Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital, University of Würzburg, Würzburg, Germany.

Conditions of impaired adrenal function and tissue destruction, such as in Addison's disease, and treatment resistance of adrenocortical carcinoma (ACC) necessitate improved understanding of the pathophysiology of adrenal cell death. Due to relevant oxidative processes in the adrenal cortex, our study investigated the role of ferroptosis, an iron-dependent cell death mechanism and found high adrenocortical expression of glutathione peroxidase 4 (GPX4) and long-chain-fatty-acid CoA ligase 4 (ACSL4) genes, key factors in the initiation of ferroptosis. By applying MALDI mass spectrometry imaging to normal and neoplastic adrenocortical tissue, we detected high abundance of arachidonic and adrenic acid, two long chain polyunsaturated fatty acids which undergo peroxidation during ferroptosis. In three available adrenal cortex cell models (H295R, CU-ACC1 and CU-ACC-2) a high susceptibility to GPX4 inhibition with RSL3 was documented with EC values of 5.7 × 10, 8.1 × 10 and 2.1 × 10 M, respectively, while all non-steroidogenic cells were significantly less sensitive. Complete block of GPX4 activity by RSL3 led to ferroptosis which was completely reversed in adrenal cortex cells by inhibition of steroidogenesis with ketoconazole but not by blocking the final step of cortisol synthesis with metyrapone. Mitotane, the only approved drug for ACC did not induce ferroptosis, despite strong induction of lipid peroxidation in ACC cells. Together, this report is the first to demonstrate extraordinary sensitivity of adrenal cortex cells to ferroptosis dependent on their active steroid synthetic pathways. Mitotane does not induce this form of cell death in ACC cells.
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http://dx.doi.org/10.1038/s41419-020-2385-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078189PMC
March 2020

Cancer-testis Antigen FATE1 Expression in Adrenocortical Tumors Is Associated with A Pervasive Autoimmune Response and Is A Marker of Malignancy in Adult, but Not Children, ACC.

Cancers (Basel) 2020 Mar 14;12(3). Epub 2020 Mar 14.

NEOGENEX-CANCER CNRS International Associated Laboratory, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France; 1532 Av. Silva Jardim, Curitiba PR 80250-200, Brazil.

The SF-1 transcription factor target gene encodes a cancer-testis antigen that has an important role in regulating apoptosis and response to chemotherapy in adrenocortical carcinoma (ACC) cells. Autoantibodies directed against FATE1 were previously detected in patients with hepatocellular carcinoma. In this study, we investigated the prevalence of circulating anti-FATE1 antibodies in pediatric and adult patients with adrenocortical tumors using three different methods (immunofluorescence, ELISA and Western blot). Our results show that a pervasive anti-FATE1 immune response is present in those patients. Furthermore, expression is a robust prognostic indicator in adult patients with ACC and is associated with increased steroidogenic and decreased immune response gene expression. These data can open perspectives for novel strategies in ACC immunotherapy.
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http://dx.doi.org/10.3390/cancers12030689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140037PMC
March 2020

Effects of Germline CYP2W1*6 and CYP2B6*6 Single Nucleotide Polymorphisms on Mitotane Treatment in Adrenocortical Carcinoma: A Multicenter ENSAT Study.

Cancers (Basel) 2020 Feb 4;12(2). Epub 2020 Feb 4.

Division of Endocrinology and Diabetes, Department of Internal Medicine I, University Hospital, University of Würzburg, 97080 Würzburg, Germany.

Mitotane is the only approved drug for advanced adrenocortical carcinoma (ACC) and no biomarkers are available to predict attainment of therapeutic plasma concentrations and clinical response. Aim of the study was to evaluate the suitability of cytochrome P450(CYP)2W1 and CYP2B6 single nucleotide polymorphisms (SNPs) as biomarkers. A multicenter cohort study including 182 ACC patients (F/M = 121/61) treated with mitotane monotherapy after radical resection (group A, = 103) or in not completely resectable, recurrent or advanced disease (group B, = 79) was performed. , , and were genotyped in germline DNA. Mitotane blood levels were measured regularly. Response to therapy was evaluated as time to progression (TTP) and disease control rate (DCR). Among investigated SNPs, and correlated with mitotane treatment only in group B. Patients with ( = 21) achieved less frequently therapeutic mitotane levels (>14 mg/L) than those with wild type (WT) allele (76.2% vs 51.7%, = 0.051) and experienced shorter TTP (HR = 2.10, = 0.019) and lower DCR (chi-square = 6.948, = 0.008). By contrast, 55% of patients with vs. 28.2% WT ( = 0.016) achieved therapeutic range. Combined, a higher rate of patients with + (60.6%) achieved mitotane therapeutic range ( = 0.034). In not completely resectable, recurrent or advanced ACC, SNP was associated with a reduced probability to reach mitotane therapeutic range and lower response rates, whereas correlated with higher mitotane levels. The association of these SNPs may predict individual response to mitotane.
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http://dx.doi.org/10.3390/cancers12020359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072643PMC
February 2020

The New Genetic Landscape of Cushing's Disease: Deubiquitinases in the Spotlight.

Cancers (Basel) 2019 Nov 8;11(11). Epub 2019 Nov 8.

Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital, University of Würzburg, D-97080 Würzburg, Germany.

Cushing's disease (CD) is a rare condition caused by adrenocorticotropic hormone (ACTH)-producing adenomas of the pituitary, which lead to hypercortisolism that is associated with high morbidity and mortality. Treatment options in case of persistent or recurrent disease are limited, but new insights into the pathogenesis of CD are raising hope for new therapeutic avenues. Here, we have performed a meta-analysis of the available sequencing data in CD to create a comprehensive picture of CD's genetics. Our analyses clearly indicate that somatic mutations in the deubiquitinases are the key drivers in CD, namely USP8 (36.5%) and USP48 (13.3%). While in USP48 only Met415 is affected by mutations, in USP8 there are 26 different mutations described. However, these different mutations are clustering in the same hotspot region (affecting in 94.5% of cases Ser718 and Pro720). In contrast, pathogenic variants classically associated with tumorigenesis in genes like TP53 and BRAF are also present in CD but with low incidence (12.5% and 7%). Importantly, several of these mutations might have therapeutic potential as there are drugs already investigated in preclinical and clinical setting for other diseases. Furthermore, network and pathway analyses of all somatic mutations in CD suggest a rather unified picture hinting towards converging oncogenic pathways.
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http://dx.doi.org/10.3390/cancers11111761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895825PMC
November 2019

Prognostic Relevance of Steroid Sulfation in Adrenocortical Carcinoma Revealed by Molecular Phenotyping Using High-Resolution Mass Spectrometry Imaging.

Clin Chem 2019 10 6;65(10):1276-1286. Epub 2019 Sep 6.

Department of Internal Medicine, Division of Endocrinology and Diabetology, University Hospital Würzburg, Würzburg, Germany;

Background: Adrenocortical carcinoma (ACC) is a rare tumor with variable prognosis even within the same tumor stage. Cancer-related sex hormones and their sulfated metabolites in body fluids can be used as tumor markers. The role of steroid sulfation in ACC has not yet been studied. MALDI mass spectrometry imaging (MALDI-MSI) is a novel tool for tissue-based chemical phenotyping.

Methods: We performed phenotyping of formalin-fixed, paraffin-embedded tissue samples from 72 ACC by MALDI-MSI at a metabolomics level.

Results: Tumoral steroid hormone metabolites-estradiol sulfate [hazard ratio (HR) 0.26; 95% CI, 0.10-0.69; = 0.005] and estrone 3-sulfate (HR 0.22; 95% CI, 0.07-0.63; = 0.003)-were significantly associated with prognosis in Kaplan-Meier analyses and after multivariable adjustment for age, tumor stage, and sex (HR 0.29; 95% CI, 0.11-0.79; = 0.015 and HR 0.30; 95% CI, 0.10-0.91; = 0.033, respectively). Expression of sulfotransferase SULT2A1 was associated with prognosis to a similar extent and was validated to be a prognostic factor in two published data sets. We discovered the presence of estradiol-17β 3,17-disulfate (E2S2) in a subset of tumors with particularly poor overall survival. Electron microscopy revealed novel membrane-delimited organelles in only these tumors. By applying cluster analyses of metabolomic data, 3 sulfation-related phenotypes exhibited specific metabolic features unrelated to steroid metabolism.

Conclusions: MALDI-MSI provides novel insights into the pathophysiology of ACC. Steroid hormone sulfation may be used for prognostication and treatment stratification. Sulfation-related metabolic reprogramming may be of relevance also in conditions beyond the rare ACC and can be directly investigated by the use of MALDI-MSI.
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http://dx.doi.org/10.1373/clinchem.2019.306043DOI Listing
October 2019

Value of Molecular Classification for Prognostic Assessment of Adrenocortical Carcinoma.

JAMA Oncol 2019 Oct;5(10):1440-1447

Institut Cochin, INSERM U1016, CNRS UMR8104, Paris Descartes University, Paris, France.

Importance: The risk stratification of adrenocortical carcinoma (ACC) based on tumor proliferation index and stage is limited. Adjuvant therapy after surgery is recommended for most patients. Pan-genomic studies have identified distinct molecular groups closely associated with outcome.

Objective: To compare the molecular classification for prognostic assessment of ACC with other known prognostic factors.

Design, Setting, And Participants: In this retrospective biomarker analysis, ACC tumor samples from 368 patients who had undergone surgical tumor removal were collected from March 1, 2005, to September 30, 2015 (144 in the training cohort and 224 in the validation cohort) at 21 referral centers with a median follow-up of 35 months (interquartile range, 18-74 months). Data were analyzed from March 2016 to March 2018.

Exposures: Meta-analysis of pan-genomic studies (transcriptome, methylome, chromosome alteration, and mutational profiles) was performed on the training cohort. Targeted biomarker analysis, including targeted gene expression (BUB1B and PINK1), targeted methylation (PAX5, GSTP1, PYCARD, and PAX6), and targeted next-generation sequencing, was performed on the training and validation cohorts.

Main Outcomes And Measures: Disease-free survival. Cox proportional hazards regression and C indexes were used to assess the prognostic value of each model.

Results: Of the 368 patients (mean [SD] age, 49 [16] years), 144 were in the training cohort (100 [69.4%] female) and 224 were in the validation cohort (142 [63.4%] female). In the training cohort, pan-genomic measures classified ACC into 3 molecular groups (A1, A2, and A3-B), with 5-year survival of 9% for group A1, 45% for group A2, and 82% for group A3-B (log-rank P < .001). Molecular class was an independent prognostic factor of recurrence in stage I to III ACC after complete surgery (hazard ratio, 55.91; 95% CI, 8.55-365.40; P < .001). The combination of European Network for the Study of Adrenal Tumors (ENSAT) stage, tumor proliferation index, and molecular class provided the most discriminant prognostic model (C index, 0.88). In the validation cohort, the molecular classification, determined by targeted biomarker measures, was confirmed as an independent prognostic factor of recurrence (hazard ratio, 5.96 [95% CI, 1.81-19.58], P = .003 for the targeted classifier combining expression, methylation, and chromosome alterations; and 2.61 [95% CI, 1.31-5.19], P = .006 for the targeted classifier combining methylation, chromosome alterations, and mutational profile). The prognostic value of the molecular markers was limited for patients with stage IV ACC.

Conclusions And Relevance: The findings suggest that in localized ACC, targeted classifiers may be used as independent markers of recurrence. The determination of molecular class may improve individual prognostic assessment and thus may spare unnecessary adjuvant treatment.
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http://dx.doi.org/10.1001/jamaoncol.2019.1558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624825PMC
October 2019

Driver mutations in USP8 wild-type Cushing's disease.

Neuro Oncol 2019 10;21(10):1273-1283

Department of Medicine I, Division of Endocrinology and Diabetes, University Hospital Würzburg (UKW), Würzburg, Germany.

Background: Medical treatment in Cushing's disease (CD) is limited due to poor understanding of its pathogenesis. Pathogenic variants of ubiquitin specific peptidase 8 (USP8) have been confirmed as causative in around half of corticotroph tumors. We aimed to further characterize the molecular landscape of those CD tumors lacking USP8 mutations in a large cohort of patients.

Methods: Exome sequencing was performed on 18 paired tumor-blood samples with wild-type USP8 status. Candidate gene variants were screened by Sanger sequencing in 175 additional samples. The most frequent variant was characterized by further functional in vitro assays.

Results: Recurrent somatic hotspot mutations in another deubiquitinase, USP48, were found in 10.3% of analyzed samples. Several possibly damaging variants were found in TP53 in 6 of 18 samples. USP48 variants were associated with smaller tumors and trended toward higher frequency in female patients. They also changed the structural conformation of USP48 and increased its catalytic activity toward its physiological substrates histone 2A and zinc finger protein Gli1, as well as enhanced the stimulatory effect of corticotropin releasing hormone (CRH) on pro-opiomelanocortin production and adrenocorticotropic hormone secretion.

Conclusions: USP48 pathogenic variants are relatively frequent in USP8 wild-type tumors and enhance CRH-induced hormone production in a manner coherent with sonic hedgehog activation. In addition, TP53 pathogenic variants may be more frequent in larger CD tumors than previously reported.
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http://dx.doi.org/10.1093/neuonc/noz109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784271PMC
October 2019

Impact of USP8 Gene Mutations on Protein Deregulation in Cushing Disease.

J Clin Endocrinol Metab 2019 07;104(7):2535-2546

Division of Endocrinology and Diabetes, Department of Internal Medicine I, University Hospital, University of Wuerzburg, Wuerzburg, Germany.

Context: Cushing disease (CD) is a rare disorder with severe sequels and incompletely understood pathogenesis. The underlying corticotroph adenomas harbor frequently somatic mutations in the ubiquitin-specific peptidase 8 (USP8) gene. These mutations render USP8 hyperactive and prevent client proteins from degradation.

Objective: To investigate the impact of USP8 mutations on proteins deregulated in CD.

Design: One hundred eight pituitary adenomas (75 corticotroph [58 USP8 wild type (WT) and 17 USP8 mutated], 14 somatotroph, and 19 nonfunctioning) were investigated by immunohistochemistry. All evaluated proteins [USP8, arginine vasopressin receptor 1b and 2, corticotropin-releasing hormone receptor, cAMP response element-binding protein (CREB), p27/kip1, cyclin E, heat shock protein 90 (HSP90), orphan nuclear receptor 4, epidermal growth factor receptor, histone deacetylase 2, glucocorticoid receptor, cyclin-dependent kinase 5 and Abelson murine leukemia viral oncogene homolog 1 enzyme substrate 1] were known to be deregulated in CD. Furthermore, AtT20 cells were transfected with USP8 to investigate the expression of possible downstream proteins by immunoblot.

Results: Whereas most of the investigated proteins were not differentially expressed, the cell-cycle inhibitor p27 was significantly reduced in USP8 mutated corticotroph adenoma (H-score 2.0 ± 1.0 vs 1.1 ± 1.1 in WT adenomas; P = 0.004). In contrast, the chaperone HSP90 was expressed higher (0.5 ± 0.4 vs 0.2 ± 0.4; P = 0.29), and the phosphorylation of the transcription factor CREB was increased in USP8 mutated adenomas (1.30.5 ± 0.40.9 vs 0.70.5 ± 0.40.7; P = 0.014). Accordingly, AtT20 cells transfected with the USP8 P720R mutant had higher phosphorylated CREB (pCREB) levels than WT transfected cells (1.3 ± 0.14 vs 1 ± 0.23; P = 0.13).

Conclusions: We could demonstrate that USP8 mutations are associated with deregulation of p27/kip1, HSP90, and pCREB. These findings suggest that these proteins are direct or indirect clients of USP8 and could therefore be potential targets for therapeutic approaches in patients with CD.
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http://dx.doi.org/10.1210/jc.2018-02564DOI Listing
July 2019

Alterations in Protein Kinase A Substrate Specificity as a Potential Cause of Cushing Syndrome.

Endocrinology 2019 02;160(2):447-459

Institute of Pharmacology and Toxicology and Bio-Imaging Center, University of Würzburg, Würzburg, Germany.

Cushing syndrome is a severe endocrine disorder of cortisol excess associated with major metabolic and cardiovascular sequelae. We recently identified somatic mutations in PRKACA, the gene encoding the catalytic (C) α subunit of protein kinase A (PKA), as being responsible for cortisol-producing adrenocortical adenomas (CPAs), which are a major cause of Cushing syndrome. In spite of previous studies on the two initially identified mutations (L206R, 199_200insW), the mechanisms of action of the clinically highly relevant PRKACA mutations remain poorly understood. Here, by investigating a large panel of PRKACA mutations, including all those identified so far in Cushing syndrome, we unexpectedly found that not all mutations interfere with the binding of regulatory (R) subunits as previously hypothesized. Because several mutations lie in a region of PKA Cα involved in substrate recognition, we investigated their consequences on substrate specificity by quantitative phosphoproteomics. We found that all three mutations analyzed (L206R, 200_201insV, and d244-248+E249Q) cause major changes in the preference of PKA for its targets, leading to hyperphosphorylation of several PKA substrates, most notably including histone H1.4 at Ser36, which is required for and promotes mitosis. This is reflected by a ninefold hyperphosphorylation of H1.4 in CPAs carrying the L206R mutation. Thus, our findings suggest that in addition to hampering binding to R subunits, PRKACA mutations act by altering PKA substrate specificity. These findings shed light on the molecular events leading to Cushing syndrome and illustrate how mutations altering substrate specificity of a protein kinase may cause human disease.
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http://dx.doi.org/10.1210/en.2018-00775DOI Listing
February 2019

Hsp90 inhibition in adrenocortical carcinoma: Limited drug synergism with mitotane.

Mol Cell Endocrinol 2019 01 10;480:36-41. Epub 2018 Oct 10.

University Hospital Würzburg, Department of Internal Medicine I, Division of Endocrinology and Diabetes, Würzburg, Germany. Electronic address:

90 kDa heat shock proteins (Hsp90) act as protein chaperones and play a role in modulating endoplasmic reticulum (ER) stress. Hsp90 inhibitors are under clinical investigation as cancer treatment. Mitotane therapy of adrenocortical carcinoma (ACC) has been shown to act through lipid-induced ER-stress. To explore the potential of Hsp90 inhibitors in ACC as a single agent and in combination with mitotane, we analyzed two independent gene expression data sets of adrenal tumors in silico and treated the ACC cell line model NCI-H295 with Hsp90 inhibitors BIIB021 (B) and CCT18159 (C) alone and in combination with mitotane. ER-stress markers were monitored by immunoblotting. Drug synergism was quantified using the median effect model with cell viability as read-out. Cytosolic Hsp90 isoforms AA1 and AB1 were significantly overexpressed in ACC. Viability of H295 cells was impaired by B and C as single agents with an EC of 5.7 × 10M and 12.1 × 10M. B but not C dose-dependently increased XBP1 splicing and CHOP expression indicative of ER-stress activation. ER-stress marker expression was enhanced by co-incubation of B with 10  μM but not 5  μM mitotane. Maximal CHOP expression was induced by 25 μM mitotane alone with no additional effect of B. Combination indices (CI) of B and C with mitotane ranged from 0.64 to 1.38 and 0.68 to 1.30, respectively where CI values < 0.5 support clinically-relevant drug synergism. In conclusion, Hsp90 paralogs are differentially expressed in ACC and B but not C activates ER-stress in ACC cells. No meaningful drug synergism of Hsp90 inhibitors with mitotane was observed.
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http://dx.doi.org/10.1016/j.mce.2018.10.009DOI Listing
January 2019

Targeted Molecular Analysis in Adrenocortical Carcinomas: A Strategy Toward Improved Personalized Prognostication.

J Clin Endocrinol Metab 2018 12;103(12):4511-4523

Department of Medicine I, Division of Endocrinology and Diabetes, University Hospital, University of Würzburg, Würzburg, Germany.

Context: Adrenocortical carcinoma (ACC) has a heterogeneous prognosis, and current medical therapies have limited efficacy in its advanced stages. Genome-wide multiomics studies identified molecular patterns associated with clinical outcome.

Objective: Here, we aimed at identifying a molecular signature useful for both personalized prognostic stratification and druggable targets, using methods applicable in clinical routine.

Design: In total, 117 tumor samples from 107 patients with ACC were analyzed. Targeted next-generation sequencing of 160 genes and pyrosequencing of 4 genes were applied to formalin-fixed, paraffin-embedded (FFPE) specimens to detect point mutations, copy number alterations, and promoter region methylation. Molecular results were combined with clinical/histopathological parameters (tumor stage, age, symptoms, resection status, and Ki-67) to predict progression-free survival (PFS).

Results: In addition to known driver mutations, we detected recurrent alterations in genes not previously associated with ACC (e.g., NOTCH1, CIC, KDM6A, BRCA1, BRCA2). Best prediction of PFS was obtained integrating molecular results (more than one somatic mutation, alterations in Wnt/β-catenin and p53 pathways, high methylation pattern) and clinical/histopathological parameters into a combined score (P < 0.0001, χ2 = 68.6). Accuracy of prediction for early disease progress was 83.3% (area under the receiver operating characteristic curve: 0.872, 95% confidence interval 0.80 to 0.94). Furthermore, 17 potentially targetable alterations were found in 64 patients (e.g., in CDK4, NOTCH1, NF1, MDM2, and EGFR and in DNA repair system).

Conclusions: This study demonstrates that molecular profiling of FFPE tumor samples improves prognostication of ACC beyond clinical/histopathological parameters and identifies new potential drug targets. These findings pave the way to precision medicine in this rare disease.
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http://dx.doi.org/10.1210/jc.2018-01348DOI Listing
December 2018

High-Resolution Tissue Mass Spectrometry Imaging Reveals a Refined Functional Anatomy of the Human Adult Adrenal Gland.

Endocrinology 2018 03;159(3):1511-1524

Department of Internal Medicine, Division of Endocrinology and Diabetology, University Hospital Würzburg, University of Würzburg, Würzburg, Germany.

In the adrenal gland, neuroendocrine cells that synthesize catecholamines and epithelial cells that produce steroid hormones are united beneath a common organ capsule to function as a single stress-responsive organ. The functional anatomy of the steroid hormone-producing adrenal cortex and the catecholamine-producing medulla is ill defined at the level of small molecules. Here, we report a comprehensive high-resolution mass spectrometry imaging (MSI) map of the normal human adrenal gland. A large variety of biomolecules was accessible by matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance MSI, including nucleoside phosphates indicative of oxidative phosphorylation, sterol and steroid metabolites, intermediates of glycolysis and the tricarboxylic acid cycle, lipids, and fatty acids. Statistical clustering analyses yielded a molecularly defined adrenal anatomy of 10 distinct molecular zones including a highly structured corticomedullary interface. By incorporating pathway information, activities of carbohydrate, amino acid, and lipid metabolism as well as endocrine bioactivity were revealed to be highly spatially organized, which could be visualized as different molecularly defined zones. Together, these findings provide a molecular definition of human adult adrenal gland structure beyond classical histological anatomy.
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http://dx.doi.org/10.1210/en.2018-00064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839739PMC
March 2018

ERCC1 as predictive biomarker to platinum-based chemotherapy in adrenocortical carcinomas.

Eur J Endocrinol 2018 Feb 29;178(2):181-188. Epub 2017 Nov 29.

Division of Endocrinology and Diabetes, Department of Internal Medicine I, University Hospital, University of Wuerzburg, Wuerzburg, Germany.

Objective: Platinum-based chemotherapy (PBC) is the most effective cytotoxic treatment for advanced adrenocortical carcinoma (ACC). Excision repair cross complementing group 1 (ERCC1) plays a critical role in the repair of platinum-induced DNA damage. Two studies investigating the role of ERCC1 immunostaining as a predictive marker for the response to PBC in ACC had reported conflicting results. Both studies used the ERCC1-antibody clone 8F1 that later turned out to be not specific. The aim of this study was to evaluate the predictive role of ERCC1 with a new specific antibody in a larger series of ACC.

Design And Methods: 146 ACC patients with available FFPE slides were investigated. All patients underwent PBC (median cycles = 6), including cisplatin ( = 131) or carboplatin ( = 15), in most cases combined with etoposide ( = 144), doxorubicin ( = 131) and mitotane ( = 131). Immunostaining was performed with the novel ERCC1-antibody clone 4F9. The relationship between ERCC1 expression and clinicopathological parameters, as well as best objective response to therapy and progression-free survival (PFS) during PBC was evaluated.

Results: High ERCC1 expression was observed in 66% of ACC samples. During PBC, 43 patients experienced objective response (29.5%), 49 stable disease (33.6%), 8 mixed response (5.5%) and 46 progressive disease (31.5%) without any relationship with the ERCC1 immunostaining. No significant correlation was also found between ERCC1 expression and progression-free survival (median 6.5 vs 6 months,     , HR = 1.23, 95% CI = 0.82-2.0).

Conclusion: ERCC1 expression is not directly associated with sensitivity to PBC in ACC. Thus, other predictive biomarkers are required to support treatment decisions in patients with ACC.
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http://dx.doi.org/10.1530/EJE-17-0788DOI Listing
February 2018

Gemcitabine-Based Chemotherapy in Adrenocortical Carcinoma: A Multicenter Study of Efficacy and Predictive Factors.

J Clin Endocrinol Metab 2017 11;102(11):4323-4332

Division of Endocrinology and Diabetes, Department of Internal Medicine I, University Hospital, University of Wuerzburg, Wuerzburg 97070, Germany.

Context: Adrenocortical carcinoma (ACC) is rare and confers an unfavorable prognosis in advanced stages. Other than combination chemotherapy with cisplatin, etoposide, doxorubicin, and mitotane, the second- and third-line regimens are not well-established. Gemcitabine (GEM)-based chemotherapy was suggested in a phase 2 clinical trial with 28 patients. In other solid tumors, human equilibrative nucleoside transporter type 1 (hENT1) and/or ribonucleotide reductase catalytic subunit M1 (RRM1) expression have been associated with resistance to GEM.

Objective: To assess the efficacy of GEM-based chemotherapy in ACC in a real-world setting and the predictive role of molecular parameters.

Design: Retrospective multicenter study.

Setting: Referral centers of university hospitals.

Patients And Materials: A total of 145 patients with advanced ACC were treated with GEM-based chemotherapy (132 with concomitant capecitabine). Formalin-fixed paraffin-embedded tumor material was available for 70 patients for immunohistochemistry.

Outcome Measures: The main outcome measures were progression-free survival (PFS) and an objective response to GEM-based chemotherapy. The secondary objective was the predictive role of hENT1 and RRM1.

Results: The median PFS for the patient population was 12 weeks (range, 1 to 94). A partial response or stable disease was achieved in 4.9% and 25.0% of cases, with a median duration of 26.8 weeks. Treatment was generally well tolerated, with adverse events of grade 3 or 4 occurring in 11.0% of cases. No substantial effect of hENT1 and/or RRM1 expression was observed in response to GEM-based chemotherapy.

Conclusions: GEM-based chemotherapy is a well-tolerated, but modestly active, regimen against advanced ACC. No reliable molecular predictive factors could be identified. Owing to the scarce alternative therapeutic options, GEM-based chemotherapy remains an important option for salvage treatment for advanced ACC.
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http://dx.doi.org/10.1210/jc.2017-01624DOI Listing
November 2017

Assessment of VAV2 Expression Refines Prognostic Prediction in Adrenocortical Carcinoma.

J Clin Endocrinol Metab 2017 09;102(9):3491-3498

Université Côte d'Azur, Sophia Antipolis, 06560 Valbonne, France.

Context: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with overall poor prognosis. The Ki67 labeling index (LI) has a major prognostic role in localized ACC after complete resection, but its estimates may suffer from considerable intra- and interobserver variability. VAV2 overexpression induced by increased Steroidogenic Factor-1 dosage is an essential factor driving ACC tumor cell invasion.

Objective: To assess the prognostic role of VAV2 expression in ACC by investigation of a large cohort of patients.

Design, Setting, And Participants: A total of 171 ACC cases (157 primary tumors, six local recurrences, eight metastases) from seven European Network for the Study of Adrenal Tumors centers were studied.

Outcome Measurements: H-scores were generated to quantify VAV2 expression. VAV2 expression was divided into two categories: low (H-score, <2) and high (H-score, ≥2). The Ki67 LI retrieved from patients' pathology records was also categorized into low (<20%) and high (≥20%). Clinical and immunohistochemical markers were correlated with progression-free survival (PFS) and overall survival (OS).

Results: VAV2 expression and Ki67 LI were significantly correlated with each other and with PFS and OS. Heterogeneity of VAV2 expression inside the same tumor was very low. Combined assessment of VAV2 expression and Ki67 LI improved patient stratification to low-risk and high-risk groups.

Conclusion: Combined assessment of Ki67 LI and VAV2 expression improves prognostic prediction in ACC.
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http://dx.doi.org/10.1210/jc.2017-00984DOI Listing
September 2017
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