An Update on the Genetic Drivers of Corticotroph Tumorigenesis.

The genetic landscape of corticotroph tumours of the pituitary gland has dramatically changed over the last 10 years. Somatic changes in the USP8 gene account for the most common genetic defect in corticotrophinomas, especially in females, while variants in TP53 or ATRX are associated with a subset of aggressive tumours. Germline defects have also been identified in patients with Cushing's disease: some are well-established (MEN1, CDKN1B, DICER1), while others are rare and could represent coincidences. In this review, we summarise the current knowledge on the genetic drivers of corticotroph tumorigenesis, their molecular consequences, and their impact on the clinical presentation and prognosis.

Aggressive Pituitary Tumors and Pituitary Carcinomas: From Pathology to Treatment.

Aggressive pituitary tumors (APTs) and pituitary carcinomas (PCs) are heterogeneous with regard to clinical presentation, proliferative markers, clinical course, and response to therapy. Half of them show an aggressive course only many years after the first apparently benign presentation. APTs and PCs share several properties, but a Ki67 index greater than or equal to 10% and extensive p53 expression are more prevalent in PCs. Mutations in TP53 and ATRX are the most common genetic alterations; their detection might be of value for early identification of aggressiveness. Treatment requires a multimodal approach including surgery, radiotherapy, and drugs. Temozolomide is the recommended first-line chemotherapy, with response rates of about 40%. Immune checkpoint inhibitors have emerged as second-line treatment in PCs, with currently no evidence for a superior effect of dual therapy compared to monotherapy with PD-1 blockers. Bevacizumab has resulted in partial response (PR) in few patients; tyrosine kinase inhibitors and everolimus have generally not been useful. The effect of peptide receptor radionuclide therapy is limited as well. Management of APT/PC is challenging and should be discussed within an expert team with consideration of clinical and pathological findings, age, and general condition of the patient. Considering that APT/PCs are rare, new therapies should preferably be evaluated in shared standardized protocols. Prognostic and predictive markers to guide treatment decisions are needed and are the scope of ongoing research.

Intact GR dimerization is critical for restraining plasma ACTH levels during chronic psychosocial stress.

Male C57BL/6N mice exposed to the chronic subordinate colony housing (CSC; 19 days) paradigm, a preclinically validated model of chronic psychosocial stress, are characterized by unaffected basal morning plasma corticosterone (CORT) concentrations despite adrenal and pituitary hyperplasia and increased adrenocorticotropic hormone (ACTH) plasma concentrations, compared with single-housed control (SHC) mice. However, as CSC mice are still able to show an increased CORT secretion towards novel heterotypic stressors, these effects might reflect an adaptation rather than a functional breakdown of general hypothalamus-pituitary-adrenal (HPA) axis functionality. In the present study we used male mice of a genetically modified mouse line, to investigate whether genetically-driven ACTH overexpression compromises adaptational processes occurring at the level of the adrenals during CSC exposure. Experimental mice carried a point mutation in the DNA binding domain of the glucocorticoid (GC) receptor (GR), attenuating dimerization of GR (GRdim), resulting in a congenially compromised negative feedback inhibition at the level of the pituitary. In line with previous studies, CSC mice in both the wild type (WT; GR+/+) and GRdim group developed adrenal enlargement. Moreover, compared with respective SHC and WT mice, CSC GRdim mice show increased basal morning plasma ACTH and CORT concentrations. Quantitative polymerase chain reaction (qPCR) analysis revealed neither a genotype effect, nor a CSC effect on pituitary mRNA expression of the ACTH precursor proopiomelanocortin (POMC). Finally, CSC increased anxiety-related behavior, active coping and splenocyte in vitro (re)activity in both WT and GRdim mice, while a CSC-induced increase in adrenal lipid vesicles and splenic GC resistance was detectable only in WT mice. Of note, lipopolysaccharide (LPS)-stimulated splenocytes of GRdim mice were resistant to the inhibitory effects of CORT. Together our findings support the hypothesis that pituitary ACTH protein concentration is negatively controlled by GR dimerization under conditions of chronic psychosocial stress, while POMC gene transcription is not dependent on intact GR dimerization under both basal and chronic stress conditions. Finally, our data suggest that adrenal adaptations during chronic psychosocial stress (i.e., ACTH desensitization), aiming at the prevention of prolonged hypercorticism, are protective only to a certain threshold of plasma ACTH levels.

Prevalence and clinical correlations of SF3B1 variants in lactotroph tumours.

OBJECTIVE: A somatic mutational hotspot in the SF3B1 gene was reported in lactotroph tumours. The aim of our study was to examine the prevalence of driver SF3B1 variants in a multicentre independent cohort of patients with lactotroph tumours and correlate with clinical data. DESIGN AND METHODS: This was a retrospective, multicentre study involving 282 patients with lactotroph tumours (including 6 metastatic lactotroph tumours) from 8 European centres. We screened SF3B1 exon 14 hotspot for somatic variants using Sanger sequencing and correlated with clinicopathological data. RESULTS: We detected SF3B1 variants in seven patients with lactotroph tumours: c.1874G > A (p.Arg625His) (n = 4, 3 of which metastatic) and a previously undescribed in pituitary tumours variant c.1873C > T (p.Arg625Cys) (n = 3 aggressive pituitary tumours). In two metastatic lactotroph tumours with tissue available, the variant was detected in both primary tumour and metastasis. The overall prevalence of likely pathogenic SF3B1 variants in lactotroph tumours was 2.5%, but when we considered only metastatic cases, it reached the 50%. SF3B1 variants correlated with significantly larger tumour size; higher Ki67 proliferation index; multiple treatments, including radiotherapy and chemotherapy; increased disease-specific death; and shorter postoperative survival. CONCLUSIONS: SF3B1 variants are uncommon in lactotroph tumours but may be frequent in metastatic lactotroph tumours. When present, they associate with aggressive tumour behaviour and worse clinical outcome.

Improved pasireotide response in USP8 mutant corticotroph tumours in vitro.

Cushing's disease is a rare but devastating and difficult to manage condition. The somatostatin analogue pasireotide is the only pituitary-targeting pharmaceutical approved for the treatment of Cushing's disease but is accompanied by varying efficacy and potentially severe side effects. Finding means to predict which patients are more likely to benefit from this treatment may improve their management. More than half of corticotroph tumours harbour mutations in the USP8 gene, and there is evidence of higher somatostatin receptor 5 (SSTR5) expression in the USP8-mutant tumours. Pasireotide has a high affinity for SSTR5, indicating that these tumours may be more sensitive to treatment. To test this hypothesis, we examined the inhibitory action of pasireotide on adrenocorticotrophic hormone synthesis in primary cultures of human corticotroph tumour with assessed USP8 mutational status and in immortalized murine corticotroph tumour cells overexpressing human USP8 mutants frequent in Cushing's disease. Our in vitro results demonstrate that pasireotide exerts a higher antisecretory response in USP8-mutant corticotroph tumours. Overexpressing USP8 mutants in a murine corticotroph tumour cell model increased endogenous somatostatin receptor 5 (Sstr5) transcription. The murine Sstr5 promoter has two binding sites for the activating protein 1 (AP-1) and USP8 mutants possibly to mediate their action by stimulating AP-1 transcriptional activity. Our data corroborate the USP8 mutational status as a potential marker of pasireotide response and describe a potential mechanism through which USP8 mutants may regulate SSTR5 gene expression.

TP53 mutations in functional corticotroph tumors are linked to invasion and worse clinical outcome.

Corticotroph macroadenomas are rare but difficult to manage intracranial neoplasms. Mutations in the two Cushing's disease mutational hotspots USP8 and USP48 are less frequent in corticotroph macroadenomas and invasive tumors. There is evidence that TP53 mutations are not as rare as previously thought in these tumors. The aim of this study was to determine the prevalence of TP53 mutations in corticotroph tumors, with emphasis on macroadenomas, and their possible association with clinical and tumor characteristics. To this end, the entire TP53 coding region was sequenced in 86 functional corticotroph tumors (61 USP8 wild type; 66 macroadenomas) and the clinical characteristics of patients with TP53 mutant tumors were compared with TP53/USP8 wild type and USP8 mutant tumors. We found pathogenic TP53 variants in 9 corticotroph tumors (all macroadenomas and USP8 wild type). TP53 mutant tumors represented 14% of all functional corticotroph macroadenomas and 24% of all invasive tumors, were significantly larger and invasive, and had higher Ki67 indices and Knosp grades compared to wild type tumors. Patients with TP53 mutant tumors had undergone more therapeutic interventions, including radiation and bilateral adrenalectomy. In conclusion, pathogenic TP53 variants are more frequent than expected, representing a relevant amount of functional corticotroph macroadenomas and invasive tumors. TP53 mutations associated with more aggressive tumor features and difficult to manage disease.

Inherited Forms of Primary Hyperaldosteronism: New Genes, New Phenotypes and Proposition of A New Classification.

Primary aldosteronism is a common cause of endocrine hypertension. It results from the excess production of aldosterone by the adrenal cortex and is related to increased morbidity and mortality. Most cases of PA are sporadic but inherited patterns of the disease have been reported in the literature. Four forms of familial hyperaldosteronism (FH-I- FH-IV) are currently recognized, and the genetic basis has been clarified in recent years. In FH-I patients, aldosterone excess is produced by a CYP11B1/CYP11B2 fusion gene and it is suppressed by glucocorticoid treatment. FH-II is caused by mutations in the inwardly rectifying chloride channel CLCN2. FH-III is caused by mutations in KCNJ5, a gene coding for an inward rectifier K+ channel and mutations in the T-type calcium channel subunit CACNA1H cause FH-IV. In this review we summarize the knowledge on inherited forms of primary aldosteronism, the genetic alterations that cause them and the implications it may have for the classification. Based on current evidence, we propose the term "familial hyperaldosteronism" to refer only to inherited forms of primary aldosteronism with a known genetic basis.

Heat Shock Protein 90 as a Prognostic Marker and Therapeutic Target for Adrenocortical Carcinoma.

Background: Adrenocortical carcinoma (ACC) is a rare tumor entity with restricted therapeutic opportunities. HSP90 (Heat Shock Protein 90) chaperone activity is fundamental for cell survival and contributes to different oncogenic signaling pathways. Indeed, agents targeting HSP90 function have shown therapeutic efficacy in several cancer types. We have examined the expression of HSP90 in different adrenal tumors and evaluated the use of HSP90 inhibitors in vitro as possible therapy for ACC. Methods: Immunohistochemical expression of HSP90 isoforms was investigated in different adrenocortical tumors and associated with clinical features. Additionally, a panel of N-terminal (17-allylamino-17-demethoxygeldanamycin (17-AAG), luminespib, and ganetespib) and C-terminal (novobiocin and silibinin) HSP90 inhibitors were tested on various ACC cell lines. Results: Within adrenocortical tumors, ACC samples exhibited the highest expression of HSP90ß. Within a cohort of ACC patients, HSP90ß expression levels were inversely correlated with recurrence-free and overall survival. In functional assays, among five different compounds tested luminespib and ganetespib induced a significant decrease in cell viability in single as well as in combined treatments with compounds of the clinically used EDP-M scheme (etoposide, doxorubicin, cisplatin, mitotane). Inhibition of cell viability correlated furthermore with a decrease in proliferation, in cell migration and an increase in apoptosis. Moreover, analysis of cancer pathways indicated a modulation of the ERK1/2-and AKT-pathways by luminespib and ganetespib treatment. Conclusions: Our findings emphasize HSP90 as a marker with prognostic impact and promising target with N-terminal HSP90 inhibitors as drugs with potential therapeutic efficacy toward ACC.

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

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.