Targeted Next Generation Sequencing molecular profiling and its clinical application in adrenocortical cancer.

OBJECTIVE: Adrenal cortical carcinoma (ACC) is a rare malignancy with a generally poor but heterogeneous prognosis, especially depending on the tumour stage at diagnosis. Identification of somatic gene alterations combined with clinical/histopathological evaluation of the tumour can help improve prognostication. We applied a simplified targeted-Next-Generation Sequencing (NGS) panel to characterise the mutational profiles of ACCs, providing potentially relevant information for better patient management. DESIGN AND METHODS: Thirty frozen tumour specimens from a local ACC series were retrospectively analysed by a custom-NGS panel (CDKN2A, CTNNB1, DAXX, MED12, NF1, PRKAR1A, RB1, TERT, TP53, ZNRF3) to detect somatic prioritised single-nucleotide variants. This cohort was integrated with 86 patients from the ACC-TCGA series bearing point-mutations in the same genes and their combinations identified by our panel. Primary endpoints of the analysis on the total cohort (113 patients) were overall survival (OS) and progression-free survival (PFS), and hazard ratio (HR) for the different alterations grouped by the signalling pathways/combinations affected. RESULTS: Different PFS, OS, and HR were associated to the different pathways/combinations, being NF1 + TP53 and Wnt/ß-catenin + Rb/p53 combined mutations the most deleterious, with a statistical significance for progression HR which is retained only in low-(I/II) stages-NF1 + TP53 combination: HR = 2.96[1.01-8.69] and HR = 13.23[3.15-55.61], all and low stages, respectively; Wnt/ß-catenin + Rb/p53 combined pathways: HR = 6.47[2.54-16.49] and HR = 16.24[3.87-68.00], all and low-stages, respectively. CONCLUSIONS: A simplified targeted-NGS approach seems the best routinely applicable first step towards somatic genetic characterisation of ACC for prognostic assessment. This approach proved to be particularly promising in low-stage cases, suggesting the need for more stringent surveillance and personalised treatment.

The 3D in vitro Adrenoid cell model recapitulates the complexity of the adrenal gland.

The crosstalk between the chromaffin and adrenocortical cells is essential for the endocrine activity of the adrenal glands. This interaction is also likely important for tumorigenesis and progression of adrenocortical cancer and pheochromocytoma. We developed a unique in vitro 3D model of the whole adrenal gland called Adrenoid consisting in adrenocortical carcinoma H295R and pheochromocytoma MTT cell lines. Adrenoids showed a round compact morphology with a growth rate significantly higher compared to MTT-spheroids. Confocal analysis of differential fluorescence staining of H295R and MTT cells demonstrated that H295R organized into small clusters inside Adrenoids dispersed in a core of MTT cells. Transmission electron microscopy confirmed the strict cell-cell interaction occurring between H295R and MTT cells in Adrenoids, which displayed ultrastructural features of more functional cells compared to the single cell type monolayer cultures. Adrenoid maintenance of the dual endocrine activity was demonstrated by the expression not only of cortical and chromaffin markers (steroidogenic factor 1, and chromogranin) but also by protein detection of the main enzymes involved in steroidogenesis (steroidogenic acute regulatory protein, and CYP11B1) and in catecholamine production (tyrosine hydroxylase and phenylethanolamine N-methyltransferase). Mass spectrometry detection of steroid hormones and liquid chromatography measurement of catecholamines confirmed Adrenoid functional activity. In conclusion, Adrenoids represent an innovative in vitro 3D-model that mimics the spatial and functional complexity of the adrenal gland, thus being a useful tool to investigate the crosstalk between the two endocrine components in the pathophysiology of this endocrine organ.

Hypogonadism and sexual function in men affected by adrenocortical carcinoma under mitotane therapy.

Purpose: Adrenocortical carcinoma (ACC) is a rare and aggressive tumor. ACC male patients under adjuvant mitotane therapy (AMT) frequently develop hypogonadism, however sexual function has never been assessed in this setting. The aim of this retrospective study was to evaluate in AMT treated ACC patients the changes in Luteinizing hormone (LH), Sex Hormone Binding Globulin (SHBG), total testosterone (TT) and calculated free testosterone (cFT), the prevalence and type of hypogonadism and sexual function, the latter before and after androgen replacement therapy (ART). Methods: LH, SHBG, TT and cFT were assessed in ten ACC patients at baseline (T0) and six (T1), twelve (T2), and eighteen (T3) months after AMT. At T3, ART was initiated in eight hypogonadal patients, and LH, SHBG, TT and cFT levels were evaluated after six months (T4). In six patients, sexual function was evaluated before (T3) and after (T4) ART using the International Index of Erectile Function-15 (IIEF-15) questionnaire. Results: Under AMT we observed higher SHBG and LH and lower cFT levels at T1-T3 compared to T0 (all p<0.05). At T3, hypergonadotropic hypogonadism and erectile dysfunction (ED) were detected in 80% and 83.3% of cases. At T4, we observed a significant cFT increase in men treated with T gel, and a significant improvement in IIEF-15 total and subdomains scores and ED prevalence (16.7%) in men under ART. Conclusion: AMT was associated with hypergonatropic hypogonadism and ED, while ART led to a significant improvement of cFT levels and sexual function in the hypogonadal ACC patients. Therefore, we suggest to evaluate LH, SHBG, TT and cFT and sexual function during AMT, and start ART in the hypogonadal ACC patients with sexual dysfunction.