Multi-chaperone function modulation and association with cytoskeletal proteins are key features of the function of AIP in the pituitary gland

Despite the well-recognized role of loss-of-function mutations of the aryl hydrocarbon receptor interacting protein gene (AIP) predisposing to pituitary adenomas, the pituitary-speci c function of this tumor suppressor remains an enigma. To determine the repertoire of interacting partners for the AIP protein in somatotroph cells, wild-type and variant AIP proteins were used for pull-down/quantitative mass spectrometry experiments against lysates of rat somatotropinoma-derived cells; relevant ndings were validated by co-immunoprecipitation and co-localization. Global gene expression was studied in AIP mutation positive and negative pituitary adenomas via RNA microarrays. Direct interaction with AIP was con rmed for three known and six novel partner proteins. Novel interactions with HSPA5 and HSPA9, together with known interactions with HSP90AA1, HSP90AB1 and HSPA8, indicate that the function/ stability of multiple chaperone client proteins could be perturbed by a de cient AIP co-chaperone function. Interactions with TUBB, TUBB2A, NME1 and SOD1 were also identi ed. The AIP variants p.R304* and p.R304Q showed impaired interactions with HSPA8, HSP90AB1, NME1 and SOD1; p.R304* also displayed reduced binding to TUBB and TUBB2A, and AIP-mutated tumors showed reduced TUBB2A expression. Our ndings suggest that cytoskeletal organization, cell motility/adhesion, as well as oxidative stress responses, are functions that are likely to be involved in the tumor suppressor activity of AIP

Ockham’s Razor for a Retinal Lesion and Acromegaly and Breaking the Vicious Circle

Acromegaly due to ectopic secretion of growth hormone-releasing hormone (GHRH) is rare. Treatment consists of surgical removal of the primary tumor, cytostatic therapy, “cold” or radioactive somatostatin analogue treatment, and medical therapy for acromegaly, if needed. A 53 year-old female had an ocular lesion noted on a routine optician visit, originally considered to be an ocular melanoma. She had a bronchial carcinoid successfully removed 22 years previously. She had acromegalic features with an enlarged pituitary gland on magnetic resonance imaging and, additionally, metastatic lesions in her bones, liver, and thyroid gland. Elevated GHRH levels (>250× upper limit of normal) suggested a metastatic lung neuroendocrine tumor secreting GHRH. Cold and radioactive somatostatin analogue therapy reduced both GHRH and insulin-like growth factor 1 (IGF-1) levels, but normalization of the biochemical markers of acromegaly was only achieved after pegvisomant was introduced. Complete control of IGF-1 was achieved, and this may have hindered the growth of the metastatic lesions as well, as the patient remains well 13 years after the diagnosis of metastatic disease and 35 years after the original lung operation. A gradual rise in prolactin levels over last 4 years was noted, which is likely due to the prolonged effect of GHRH on prolactin-secreting cells. The diagnosis of this case applied the law of parsimony from the Ockham’s razor principle. We consider that breaking the vicious circle of IGF-1 feeding the metastatic tumor was key for the long-term outcome of this case

Characterisation of myocardial structure and function in adult-onset growth hormone deficiency using cardiac magnetic resonance.

Growth hormone (GH) can profoundly influence cardiac function. While GH excess causes well-defined cardiac pathology, fewer data are available regarding the more subtle cardiac changes seen in GH deficiency (GHD). This preliminary study uses cardiac magnetic resonance imaging (CMR) to assess myocardial structure and function in GHD. Ten adult-onset GHD patients underwent CMR, before and after 6 and 12 months of GH replacement. They were compared to 10 age-matched healthy controls and sex-matched healthy controls. Left ventricular (LV) mass index (LVMi) increased with 1 year of GH replacement (53.8 vs. 57.0 vs. 57.3 g/m2, analysis of variance p = 0.0229). Compared to controls, patients showed a trend towards reduced LVMi at baseline (51.4 vs. 60.0 g/m2, p = 0.0615); this difference was lost by 1 year of GH treatment (57.3 vs. 59.9 g/m2, p = 0.666). Significantly reduced aortic area was observed in GHD (13.2 vs. 19.0 cm2/m2, p = 0.001). This did not change with GH treatment. There were no differences in other LV parameters including end-diastolic volume index (EDVi), end-systolic volume index, stroke volume index (SVi), cardiac index and ejection fraction. There was a trend towards reduced baseline right ventricular (RV)SVi (44.1 vs. 49.1 ml/m2, p = 0.0793) and increased RVEDVi over 1 year (70.3 vs. 74.3 vs. 73.8 ml/m2, p = 0.062). Two patients demonstrated interstitial expansion, for example with fibrosis, and three myocardial ischaemia as assessed by late gadolinium enhancement and stress perfusion. The increased sensitivity of CMR to subtle cardiac changes demonstrates that adult-onset GHD patients have reduced aortic area and LVMi increases after 1 year of GH treatment. These early data should be studied in larger studies in the future

Acromegaly associated with GIST, non-small cell lung carcinoma, clear cell renal carcinoma, multiple myeloma, medulla oblongata tumour, adrenal adenoma, and follicular thyroid nodules

Acromegaly is associated with increased growth hormone (GH) and insulin-like growth factor-I (IGF-I) secretion which may support tumour development and growth. A 68-year-old woman was diagnosed with acromegaly due to typical clinical and hormonal characteristics. While contrast-enhanced MRI at diagnosis did not reveal a pituitary adenoma, a 5-mm lesion was identified on repeat scanning 13 months later. Abdominal and chest CT showed tumours of the stomach, right adrenal gland, and right lung. The CT also showed a hypodense lesion in the liver and heterogeneous echostructure of the thyroid gland with left lobe solid-cystic tumour. Somatostatin receptor scintigraphy revealed increased tracer accumulation in the right thyroid lobe. No tracer accumulation was noted at the location of the other tumours. The resected stomach, adrenal, chest, and thyroid lesions did not show GH secretion. The patient refused pituitary surgery, and her acromegaly is currently well-controlled with somatostatin analogue therapy. A CT scan 19 months later revealed a contrast-enhancing left kidney tumour that was a G1-grade clear cell carcinoma. Four years after the acromegaly diagnosis multiple myeloma were diagnosed with secondary renal amyloidosis. Genetic screening for a paraganglioma gene panel, AIP, MEN1, and CDKN1B mutations were negative. A next-generation cancer panel containing 94 cancer genes did not identify any possible unifying gene abnormality in her germline DNA. Coexistence of acromegaly and numerous other tumours suggests a common aetiology of these disorders. However, no genetic abnormality could be identified with the tests that have been performed.

Rapid proteasomal degradation of mutant proteins is the primary mechanism leading to tumorigenesis in patients with missense AIP mutations

CONTEXT The pathogenic effect of AIP mutations (AIPmuts) in pituitary adenomas is incompletely understood. We have identified the primary mechanism of loss of function for missense AIPmuts. OBJECTIVE To analyze the mechanism/speed of protein turnover of wild-type (WT) and missense AIP variants, correlating protein half-life with clinical parameters. DESIGN Half-life and protein-protein interaction experiments and cross-sectional analysis of AIPmut positive patients' data were performed. SETTING Clinical academic research institution. PATIENTS Data was obtained from our cohort of pituitary adenoma patients and literature-reported cases. INTERVENTIONS Protein turnover of endogenous AIP in two cell lines and fifteen AIP variants overexpressed in HEK293 cells was analyzed via cycloheximide chase and proteasome inhibition. GST pull-down and quantitative mass spectrometry identified proteins involved in AIP degradation; results were confirmed by co-immunoprecipitation and gene knockdown. Relevant clinical data was collected. MAIN OUTCOME MEASURES Half-life of WT and mutant AIP proteins and its correlation with clinical parameters. RESULTS Endogenous AIP half-life was similar in HEK293 and lymphoblastoid cells (43.5 and 32.7h). AIP variants were divided in stable proteins (median 77.7h [IQR 60.7-92.9]), and those with short (27h [21.6-28.7]) or very short (7.7h [5.6-10.5]) half-life; proteasomal inhibition rescued the rapid degradation of mutant proteins. The experimental half-life significantly correlated with age at diagnosis of acromegaly/gigantism (r=0.411, P=0.002). The FBXO3-containing SCF complex was identified as the E3 ubiquitin-ligase recognizing AIP. CONCLUSIONS AIP is a stable protein, driven to ubiquitination by the SCF complex. Enhanced proteasomal degradation is a novel pathogenic mechanism for AIPmuts, with direct implications for the phenotype

AIP Mutation-positive Pituitary Tumors

Context: Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene are responsible for a subset of familial isolated pituitary adenoma (FIPA) cases and sporadic pituitary neuroendocrine tumors (PitNETs). Objective: To compare prospectively diagnosed AIP mutation-positive (AIPmut) PitNET patients with clinically presenting patients and to compare the clinical characteristics of AIPmut and AIPneg PitNET patients. Design: 12-year prospective, observational study. Participants & Setting: We studied probands and family members of FIPA kindreds and sporadic patients with disease onset ≤18 years or macroadenomas with onset ≤30 years (n = 1477). This was a collaborative study conducted at referral centers for pituitary diseases. Interventions & Outcome: AIP testing and clinical screening for pituitary disease. Comparison of characteristics of prospectively diagnosed (n = 22) vs clinically presenting AIPmut PitNET patients (n = 145), and AIPmut (n = 167) vs AIPneg PitNET patients (n = 1310). Results: Prospectively diagnosed AIPmut PitNET patients had smaller lesions with less suprasellar extension or cavernous sinus invasion and required fewer treatments with fewer operations and no radiotherapy compared with clinically presenting cases; there were fewer cases with active disease and hypopituitarism at last follow-up. When comparing AIPmut and AIPneg cases, AIPmut patients were more often males, younger, more often had GH excess, pituitary apoplexy, suprasellar extension, and more patients required multimodal therapy, including radiotherapy. AIPmut patients (n = 136) with GH excess were taller than AIPneg counterparts (n = 650). Conclusions: Prospectively diagnosed AIPmut patients show better outcomes than clinically presenting cases, demonstrating the benefits of genetic and clinical screening. AIP-related pituitary disease has a wide spectrum ranging from aggressively growing lesions to stable or indolent disease course

Risk category system to identify pituitary adenoma patients with AIP mutations

Background Predictive tools to identify patients at risk for gene mutations related to pituitary adenomas are very helpful in clinical practice. We therefore aimed to develop and validate a reliable risk category system for aryl hydrocarbon receptor-interacting protein (AIP) mutations in patients with pituitary adenomas. Methods A n international cohort of 2227 subjects were consecutively recruited between 2007 and 2016, including patients with pituitary adenomas (familial and sporadic) and their relatives. All probands (n=1429) were screened for AIP mutations, and those diagnosed with a pituitary adenoma prospectively, as part of their clinical screening (n=24), were excluded from the analysis. Univariate analysis was performed comparing patients with and without AIP mutations. Based on a multivariate logistic regression model, six potential factors were identified for the development of a risk category system, classifying the individual risk into low-risk, moderate-risk and high-risk categories. An internal cross-validation test was used to validate the system. Results 1405 patients had a pituitary tumour, of which 43% had a positive family history, 55.5% had somatotrophinomas and 81.5% presented with macroadenoma. Overall, 134 patients had an AIP mutation (9.5%). We identified four independent predictors for the presence of an AIP mutation: age of onset providing an odds ratio (OR) of 14.34 for age 0-18 years, family history (OR 10.85), growth hormone excess (OR 9.74) and large tumour size (OR 4.49). In our cohort, 71% of patients were identified as low risk (<5% risk of AIP mutation), 9.2% as moderate risk and 20% as high risk (≥20% risk). Excellent discrimination (c-statistic=0.87) and internal validation were achieved. Conclusion We propose a user-friendly risk categorisation system that can reliably group patients into high-risk, moderate-risk and low-risk groups for the presence of AIP mutations, thus providing guidance in identifying patients at high risk of carrying an AIP mutation. This risk score is based on a cohort with high prevalence of AIP mutations and should be applied cautiously in other populations

The clinical outcomes of imaging modalities for surgical management Cushing’s disease – A systematic review and meta-analysis

INTRODUCTION: Cushing’s disease presents major diagnostic and management challenges. Although numerous preoperative and intraoperative imaging modalities have been deployed, it is unclear whether these investigations have improved surgical outcomes. Our objective was to investigate whether advances in imaging improved outcomes for Cushing’s disease. METHODS: Searches of PubMed and EMBASE were conducted. Studies reporting on imaging modalities and clinical outcomes after surgical management of Cushing’s disease were included. Multilevel multivariable meta-regressions identified predictors of outcomes, adjusting for confounders and heterogeneity prior to investigating the effects of imaging. RESULTS: 166 non-controlled single-arm studies were included, comprising 13181 patients over 44 years. The overall remission rate was 77.0% [CI: 74.9%-79.0%]. Cavernous sinus invasion (OR: 0.21 [CI: 0.07-0.66]; p=0.010), radiologically undetectable lesions (OR: 0.50 [CI: 0.37–0.69]; p<0.0001), previous surgery (OR=0.48 [CI: 0.28–0.81]; p=0.008), and lesions ≥10mm (OR: 0.63 [CI: 0.35–1.14]; p=0.12) were associated with lower remission. Less stringent thresholds for remission was associated with higher reported remission (OR: 1.37 [CI: 1.1–1.72]; p=0.007). After adjusting for this heterogeneity, no imaging modality showed significant differences in remission compared to standard preoperative MRI. The overall recurrence rate was 14.5% [CI: 12.1%-17.1%]. Lesion ≥10mm was associated with greater recurrence (OR: 1.83 [CI: 1.13–2.96]; p=0.015), as was greater duration of follow-up (OR: 1.53 (CI: 1.17–2.01); p=0.002). No imaging modality was associated with significant differences in recurrence. Despite significant improvements in detection rates over four decades, there were no significant changes in the reported remission or recurrence rates. CONCLUSION: A lack of controlled comparative studies makes it difficult to draw definitive conclusions. Within this limitation, the results suggest that despite improvements in radiological detection rates of Cushing’s disease over the last four decades, there were no changes in clinical outcomes. Advances in imaging alone may be insufficient to improve surgical outcomes. SYSTEMATIC REVIEW REGISTRATION: https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42020187751