TY - JOUR A1 - Sbiera, Iuliu A1 - Kircher, Stefan A1 - Altieri, Barbara A1 - Lenz, Kerstin A1 - Hantel, Constanze A1 - Fassnacht, Martin A1 - Sbiera, Silviu A1 - Kroiss, Matthias T1 - Role of FGF Receptors and Their Pathways in Adrenocortical Tumors and Possible Therapeutic Implications JF - Frontiers in Endocrinology N2 - Adrenocortical carcinoma (ACC) is a rare endocrine malignancy and treatment of advanced disease is challenging. Clinical trials with multi-tyrosine kinase inhibitors in the past have yielded disappointing results. Here, we investigated fibroblast growth factor (FGF) receptors and their pathways in adrenocortical tumors as potential treatment targets. We performed real-time RT-PCR of 93 FGF pathway related genes in a cohort of 39 fresh frozen benign and malignant adrenocortical, 9 non-adrenal tissues and 4 cell lines. The expression of FGF receptors was validated in 166 formalin-fixed paraffin embedded (FFPE) tissues using RNA in situ hybridization (RNAscope) and correlated with clinical data. In malignant compared to benign adrenal tumors, we found significant differences in the expression of 16/94 FGF receptor pathway related genes. Genes involved in tissue differentiation and metastatic spread through epithelial to mesechymal transition were most strongly altered. The therapeutically targetable FGF receptors 1 and 4 were upregulated 4.6- and 6-fold, respectively, in malignant compared to benign adrenocortical tumors, which was confirmed by RNAscope in FFPE samples. High expression of FGFR1 and 4 was significantly associated with worse patient prognosis in univariate analysis. After multivariate adjustment for the known prognostic factors Ki-67 and ENSAT tumor stage, FGFR1 remained significantly associated with recurrence-free survival (HR=6.10, 95%CI: 1.78 – 20.86, p=0.004) and FGFR4 with overall survival (HR=3.23, 95%CI: 1.52 – 6.88, p=0.002). Collectively, our study supports a role of FGF pathways in malignant adrenocortical tumors. Quantification of FGF receptors may enable a stratification of ACC for the use of FGFR inhibitors in future clinical trials. KW - normal adrenal glands KW - adrenocortical tumors KW - FGF-pathway KW - FGFR KW - RNA Expression KW - RNAScope KW - unsupervised clustering KW - patient survival Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-251953 SN - 1664-2392 VL - 12 ER - TY - JOUR A1 - Marquardt, André A1 - Landwehr, Laura-Sophie A1 - Ronchi, Cristina L. A1 - di Dalmazi, Guido A1 - Riester, Anna A1 - Kollmannsberger, Philip A1 - Altieri, Barbara A1 - Fassnacht, Martin A1 - Sbiera, Silviu T1 - Identifying New Potential Biomarkers in Adrenocortical Tumors Based on mRNA Expression Data Using Machine Learning JF - Cancers N2 - Simple Summary Using a visual-based clustering method on the TCGA RNA sequencing data of a large adrenocortical carcinoma (ACC) cohort, we were able to classify these tumors in two distinct clusters largely overlapping with previously identified ones. As previously shown, the identified clusters also correlated with patient survival. Applying the visual clustering method to a second dataset also including benign adrenocortical samples additionally revealed that one of the ACC clusters is more closely located to the benign samples, providing a possible explanation for the better survival of this ACC cluster. Furthermore, the subsequent use of machine learning identified new possible biomarker genes with prognostic potential for this rare disease, that are significantly differentially expressed in the different survival clusters and should be further evaluated. Abstract Adrenocortical carcinoma (ACC) is a rare disease, associated with poor survival. Several “multiple-omics” studies characterizing ACC on a molecular level identified two different clusters correlating with patient survival (C1A and C1B). We here used the publicly available transcriptome data from the TCGA-ACC dataset (n = 79), applying machine learning (ML) methods to classify the ACC based on expression pattern in an unbiased manner. UMAP (uniform manifold approximation and projection)-based clustering resulted in two distinct groups, ACC-UMAP1 and ACC-UMAP2, that largely overlap with clusters C1B and C1A, respectively. However, subsequent use of random-forest-based learning revealed a set of new possible marker genes showing significant differential expression in the described clusters (e.g., SOAT1, EIF2A1). For validation purposes, we used a secondary dataset based on a previous study from our group, consisting of 4 normal adrenal glands and 52 benign and 7 malignant tumor samples. The results largely confirmed those obtained for the TCGA-ACC cohort. In addition, the ENSAT dataset showed a correlation between benign adrenocortical tumors and the good prognosis ACC cluster ACC-UMAP1/C1B. In conclusion, the use of ML approaches re-identified and redefined known prognostic ACC subgroups. On the other hand, the subsequent use of random-forest-based learning identified new possible prognostic marker genes for ACC. KW - adrenocortical carcinoma KW - in silico analysis KW - machine learning KW - bioinformatic clustering KW - biomarker prediction Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246245 SN - 2072-6694 VL - 13 IS - 18 ER - TY - JOUR A1 - Sbiera, Iuliu A1 - Kircher, Stefan A1 - Altieri, Barbara A1 - Fassnacht, Martin A1 - Kroiss, Matthias A1 - Sbiera, Silviu T1 - Epithelial and Mesenchymal Markers in Adrenocortical Tissues: How Mesenchymal Are Adrenocortical Tissues? JF - Cancers N2 - 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. KW - adrenocortical tissues KW - EMT KW - epithelial markers KW - mesenchymal markers KW - recurrence-free survival Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236486 SN - 2072-6694 VL - 13 IS - 7 ER - TY - JOUR A1 - Detomas, Mario A1 - Altieri, Barbara A1 - Schlötelburg, Wiebke A1 - Appenzeller, Silke A1 - Schlaffer, Sven A1 - Coras, Roland A1 - Schirbel, Andreas A1 - Wild, Vanessa A1 - Kroiss, Matthias A1 - Sbiera, Silviu A1 - Fassnacht, Martin A1 - Deutschbein, Timo T1 - Case Report: Consecutive Adrenal Cushing’s Syndrome and Cushing’s Disease in a Patient With Somatic CTNNB1, USP8, and NR3C1 Mutations JF - Frontiers in Endocrinology N2 - 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 (CTNNB1) in the adrenal adenoma, and of both the ubiquitin specific peptidase 8 (USP8) and the glucocorticoid receptor (NR3C1) 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. KW - Cushing’s syndrome KW - Cushing’s disease KW - hypercortisolism KW - glucocorticoid excess KW - USP8 KW - CTNNB1 KW - NR3C1 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-244596 SN - 1664-2392 VL - 12 ER - TY - JOUR A1 - Chifu, Irina A1 - Heinze, Britta A1 - Fuss, Carmina T. A1 - Lang, Katharina A1 - Kroiss, Matthias A1 - Kircher, Stefan A1 - Ronchi, Cristina L. A1 - Altieri, Barbara A1 - Schirbel, Andreas A1 - Fassnacht, Martin A1 - Hahner, Stefanie T1 - Impact of the Chemokine Receptors CXCR4 and CXCR7 on Clinical Outcome in Adrenocortical Carcinoma JF - Frontiers in Endocrinology N2 - Chemokine receptors have a negative impact on tumor progression in several human cancers and have therefore been of interest for molecular imaging and targeted therapy. However, their clinical and prognostic significance in adrenocortical carcinoma (ACC) is unknown. The aim of this study was to evaluate the chemokine receptor profile in ACC and to analyse its association with clinicopathological characteristics and clinical outcome. A chemokine receptor profile was initially evaluated by quantitative PCR in 4 normal adrenals, 18 ACC samples and human ACC cell line NCI-H295. High expression of CXCR4 and CXCR7 in both healthy and malignant adrenal tissue and ACC cells was confirmed. In the next step, we analyzed the expression and cellular localization of CXCR4 and CXCR7 in ACC by immunohistochemistry in 187 and 84 samples, respectively. These results were correlated with clinicopathological parameters and survival outcome. We detected strong membrane expression of CXCR4 and CXCR7 in 50% of ACC samples. Strong cytoplasmic CXCR4 staining was more frequent among samples derived from metastases compared to primaries (p=0.01) and local recurrences (p=0.04). CXCR4 membrane staining positively correlated with proliferation index Ki67 (r=0.17, p=0.028). CXCR7 membrane staining negatively correlated with Ki67 (r=−0.254, p=0.03) but positively with tumor size (r=0.3, p=0.02). No differences in progression-free or overall survival were observed between patients with strong and weak staining intensities for CXCR4 or CXCR7. Taken together, high expression of CXCR4 and CXCR7 in both local tumors and metastases suggests that some ACC patients might benefit from CXCR4/CXCR7-targeted therapy. KW - chemokine receptor KW - prognosis KW - adrenocortical carcinoma KW - CXCR4 KW - CXCR7 Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216494 SN - 1664-2392 VL - 11 ER - TY - JOUR A1 - Altieri, Barbara A1 - Sbiera, Silviu A1 - Della Casa, Silvia A1 - Weigand, Isabel A1 - Wild, Vanessa A1 - Steinhauer, Sonja A1 - Fadda, Guido A1 - Kocot, Arkadius A1 - Bekteshi, Michaela A1 - Mambretti, Egle M. A1 - Rosenwald, Andreas A1 - Pontecorvi, Alfredo A1 - Fassnacht, Martin A1 - Ronchi, Cristina L. T1 - Livin/BIRC7 expression as malignancy marker in adrenocortical tumors JF - Oncotarget N2 - Livin/BIRC7 is a member of the inhibitors of apoptosis proteins family, which are involved in tumor development through the inhibition of caspases. Aim was to investigate the expression of livin and other members of its pathway in adrenocortical tumors and in the adrenocortical carcinoma (ACC) cell line NCI-H295R. The mRNA expression of livin, its isoforms α and β, XIAP, CASP3 and DIABLO was evaluated by qRT-PCR in 82 fresh-frozen adrenal tissues (34 ACC, 25 adenomas = ACA, 23 normal adrenal glands = NAG). Livin protein expression was assessed by immunohistochemistry in 270 paraffin-embedded tissues (192 ACC, 58 ACA, 20 NAG). Livin, CASP3 and cleaved caspase-3 were evaluated in NCI-H295R after induction of livin overexpression. Relative livin mRNA expression was significantly higher in ACC than in ACA and NAG (0.060 ± 0.116 vs 0.004 ± 0.014 and 0.002 ± 0.009, respectively, p < 0.01), being consistently higher in tumors than in adjacent NAG and isoform β more expressed than α. No significant differences in CASP3, XIAP and DIABLO levels were found among these groups. In immunohistochemistry, livin was localized in both cytoplasm and nuclei. The ratio between cytoplasmic and nuclear staining was significantly higher in ACC (1.51 ± 0.66) than in ACA (0.80 ± 0.35) and NAG (0.88 ± 0.27; p < 0.0001). No significant correlations were observed between livin expression and histopathological parameters or clinical outcome. In NCI-H295R cells, the livin overexpression slightly reduced the activation of CASP3, but did not correlate with cell viability. In conclusion, livin is specifically over-expressed in ACC, suggesting that it might be involved in adrenocortical tumorigenesis and represent a new molecular marker of malignancy. KW - cancer KW - livin KW - BIRC7 KW - adrenocortical cancer KW - adrenal tumor KW - caspase-3 Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171887 VL - 8 IS - 6 ER - TY - JOUR A1 - Ronchi, Cristina L. A1 - Sbiera, Silviu A1 - Volante, Marco A1 - Steinhauer, Sonja A1 - Scott-Wild, Vanessa A1 - Altieri, Barbara A1 - Kroiss, Matthias A1 - Bala, Margarita A1 - Papotti, Mauro A1 - Deutschbein, Timo A1 - Terzolo, Massimo A1 - Fassnacht, Martin A1 - Allolio, Bruno T1 - CYP2W1 Is Highly Expressed in Adrenal Glands and Is Positively Associated with the Response to Mitotane in Adrenocortical Carcinoma N2 - Background Adrenocortical tumors comprise frequent adenomas (ACA) and rare carcinomas (ACC). Human cytochrome P450 2W1 (CYP2W1) is highly expressed in some cancers holding the potential to activate certain drugs into tumor cytotoxins. Objective To investigate the CYP2W1 expression in adrenal samples and its relationship with clinical outcome in ACC. Material and Methods CYP2W1 expression was investigated by qRT-PCR in 13 normal adrenal glands, 32 ACA, 25 ACC, and 9 different non-adrenal normal tissue samples and by immunohistochemistry in 352 specimens (23 normal adrenal glands, 33 ACA, 239 ACC, 67 non-adrenal normal or neoplastic samples). Results CYP2W1 mRNA expression was absent/low in normal non-adrenal tissues, but high in normal and neoplastic adrenal glands (all P<0.01 vs non-adrenal normal tissues). Accordingly, CYP2W1 immunoreactivity was absent/low (H-score 0–1) in 72% of non-adrenal normal tissues, but high (H-score 2–3) in 44% of non-adrenal cancers, in 65% of normal adrenal glands, in 62% of ACAs and in 50% of ACCs (all P<0.001 vs non-adrenal normal tissues), being significantly increased in steroid-secreting compared to non-secreting tumors. In ACC patients treated with mitotane only, high CYP2W1 immunoreactivity adjusted for ENSAT stage was associated with longer overall survival and time to progression (P<0.05 and P<0.01, respectively), and with a better response to therapy both as palliative (response/stable disease in 42% vs 6%, P<0.01) or adjuvant option (absence of disease recurrence in 69% vs 45%, P<0.01). Conclusion CYP2W1 is highly expressed in both normal and neoplastic adrenal glands making it a promising tool for targeted therapy in ACC. Furthermore, CYP2W1 may represent a new predictive marker for the response to mitotane treatment. KW - CYP2W1 KW - cancer treatment KW - adrenal glands KW - carcinomas KW - drug therapy KW - hormones KW - immune response KW - immunohistochemistry techniques KW - surgical oncology Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113096 ER -