Expression and mutational status of USP8 in tumors causing ectopic ACTH secretion syndrome.

One of the most common paraneoplastic syndromes is the ectopic ACTH secretion syndrome (EAS), a sustained hypercortisolism driven by tumors of extra-pituitary origin that secrete either adrenocorticotropic hormone (ACTH), its precursor proopiomelanocortin (POMC, unprocessed or partially cleaved) or, less frequently, corticotropin-releasing hormone (CRH). Bronchial carcinoids and small cell carcinomas of the lung (SCLC) are the most frequent causes of EAS (Alexandraki & Grossman 2010). This syndrome is commonly associated with an increased risk of morbidity and mortality since patients with hypercortisolism usually exhibit worse response to chemotherapy and suffer from severe infections and metabolic complications during treatment (Terzolo et al. 2001). The mechanisms underlying the ectopic production of ACTH are still not well understood. Recently, we and others have reported that the gene coding for the human ubiquitin-specific protease 8 (USP8) is recurrently mutated in 50% of all the investigated ACTH-producing corticotroph adenomas causing sporadic Cushing’s disease (Ma et al. 2015, Perez-Rivas et al. 2015, Reincke et al. 2015). Mutant USP8 triggers POMC transcription and ACTH secretion through the stabilization of the epidermal growth factor receptor (EGFR) (Reincke et al. 2015). As USP8 mutations are considered causative for Cushing’s disease, we investigated whether USP8 is involved in the ectopic production of ACTH by endocrine tumors. Therefore, we performed a retrospective, single-center analysis using formalin-fixed, paraffin-embedded samples of 15 patients (7 females and 8 males) who developed EAS and were treated and followed up in our hospital since 1986. Patients were included when presenting with persistent ACTH-dependent hypercortisolism (Cushing’s syndrome) of confirmed non-pituitary origin. Cushing’s syndrome was diagnosed based on the clinical signs and symptoms of hypercortisolism (easy bruising, facial plethora, proximal muscle weakness, purple striae, low-impact bone fractures, mood changes, irregular menstruation, infertility or impotence, hypokalemic hypertension, myopathy and hyperglycemia) together with increased levels of latenight salivary cortisol (>145 ng/dL), urinary free cortisol (>150 μg/24 h) and/or lack of suppression of cortisol (>1.8 μg/dL) after administration of dexamethasone, following the recommendations of the Endocrine Society (Nieman et al. 2008). Non-pituitary ACTH dependency was confirmed by levels of basal plasma ACTH >15 pg/mL, less than 50% suppression of serum cortisol during an 8 mg high-dose dexamethasone test, lack of response of ACTH and cortisol to CRH and/or a negative inferior petrosal sinus sampling. The tumor was considered to be the cause of ECS when tumor tissue stained positive for ACTH immunoreactivity and/or the patient reached hormonal remission following resection. The most common signs were weight gain, typical Cushing’s stigmata, muscle weakness and hypertension. The thorax was the most prevalent tumor location (11/15 patients). Six tumors were classified as typical and 2 as atypical bronchial carcinoids (according to the WHO classification of 2004; Travis et al. 2004), 3 were smallcell lung carcinomas (SCLC), 1 low-grade mediastinal paraganglioma, 1 choriocarcinoma and 2 were classified as carcinoma of unknown primary (CUP) tumor. Tissue was available only from liver metastasis in 2 cases. Overall, metastases were detected in 10 patients (3 with SCLC, 1 with the germ cell tumor, 2 with CUP, 2 with atypical and 2 with typical bronchial carcinoid) and were rare in patients with well-differentiated tumors (5 bronchial carcinoids and 1 paraganglioma). Sanger sequencing showed that all 15 cases were wildtype for the USP8 hotspot located in exon 14. USP8 immunoreactivity (1:1000, Sigma) was detected in all