Psychological impact of von Hippel-Lindau genetic screening in patients with a previous history of hemangioblastoma of the central nervous system.

Von Hippel-Lindau (VHL) syndrome is a hereditary cancer syndrome characterized by a high risk of developing benign and malignant tumors, including central nervous system hemangioblastomas (CNS HBs). For an early diagnosis of VHL, before the occurrence of cancers (especially renal cell carcinoma), it is of huge importance to initiate VHL genetic testing in at-risk patients. The aim of the study was to assess the psychological impact of VHL genetic testing in patients previously diagnosed with a CNS HB. From 1999 until 2015, 55 patients underwent surgery for CNS HBs. Eleven patients were already screened for VHL mutations and 3 patients deceased before the start of the study. From the remaining 42 patients, 24 were accepted to be enrolled in the study. Assessment of psychological impact of VHL genetic testing was performed by measuring anxiety levels, mood disorders, quality of life, and psychological consequences of genetic screening. Twenty-one of the enrolled 24 patients underwent VHL genetic testing and 12 patients came back for the communication of positive genetic results. The baseline psychological status did not differ between these 2 groups. Patients who attended the visit of communication of genetic results had similar anxiety levels compared to those who had not. Furthermore, they also experienced an improvement in the level of anxiety and two QoL dimension scores compared to their baseline status. In summary, there is no evidence of a negative psychosocial impact of VHL genetic testing in patients with a previous history of CNS HB. We, therefore, recommend the recall of patients who have not been previously screened.

Somatic gain-of-function HIF2A mutations in sporadic central nervous system hemangioblastomas.

Central nervous system hemangioblastomas (CNS-HBs) occur sporadically or as a component of von Hippel-Lindau-VHL syndrome. CNS-HBs share some molecular similarities with pheochromocytomas/paragangliomas (PPGLs) and renal cell carcinomas (RCCs). Recently, hypoxia-inducible factors, particularly somatic HIF2A mutations, have been found to play an important role in the pathogenesis of PPGLs. Somatic mutations in HIF2A have been reported in PPGLs associated with polycythemia, which have been reported to also be present in patients with RCCs and HBs. However, whether CNS-HBs is associated with the presence of a HIF2A mutation is currently uknown. We analyzed somatic HIF2A and VHL mutations in a series of 28 sporadic CNS-HBs. We also investigated the expression of HIF target proteins and hypoxia-associated factor (HAF). Two sporadic CNS-HBs were found to have somatic HIF2A mutations. One tumor had 2 HIF2A missense mutations, one of which was previously described in a PPGL (c.1121 T>A, F374Y). The second patient had coexistence of somatic truncated mutations (c.1669 C>T, Q557*) in HIF2A together with a VHL mutation. Neither of the two patients had polycythemia at the time of diagnosis. We demonstrate that the novel truncated mutation in HIF2A (Q557*) affects HIF-2α prolyl hydroxylation with its reduced ubiquitination but intact transcriptional activity, resulting in an activating effect. Both CNS-HB samples showed positive expression of VEGFR2/CA9/Glut1 and HAF. Our data support the unique central role of the VHL/HIF-2α signaling pathway in the molecular pathogenesis of CNS-HBs and show for the first time the presence of HIF2A mutations in sporadic HB.

18F-FDG PET/CT as a predictor of hereditary head and neck paragangliomas.

BACKGROUND: Hereditary head and neck paragangliomas (HNPGLs) account for at least 35% of all HNPGLs, most commonly due to germline mutations in SDHx susceptibility genes. Several studies about sympathetic paragangliomas have shown that (18)F-FDG PET/CT was not only able to detect and localize tumours, but also to characterize tumours ((18)F-FDG uptake being linked to SDHx mutations). However, the data concerning (18)F-FDG uptake specifically in HNPGLs have not been addressed. The aim of this study was to evaluate the relationship between (18)F-FDG uptake and the SDHx mutation status in HNPGL patients. METHODS: (18)F-FDG PET/CT from sixty HNPGL patients were evaluated. For all lesions, we measured the maximum standardized uptake values (SUVmax), and the uptake ratio defined as HNPGL-SUVmax over pulmonary artery trunk SUVmean (SUVratio). Tumour sizes were assessed on radiological studies. RESULTS: Sixty patients (53.3% with SDHx mutations) were evaluated for a total of 106 HNPGLs. HNPGLs-SUVmax and SUVratio were highly dispersed (1.2-30.5 and 1.0-17.0, respectively). The HNPGL (18)F-FDG uptake was significantly higher in SDHx versus sporadic tumours on both univariate and multivariate analysis (P = 0.002). We developed two models for calculating the probability of a germline SDHx mutation. The first one, based on a per-lesion analysis, had an accuracy of 75.5%. The second model, based on a per-patient analysis, had an accuracy of 80.0%. CONCLUSIONS: (18)F-FDG uptake in HNPGL is strongly dependent on patient genotype. Thus, the degree of (18)F-FDG uptake in these tumours can be used clinically to help identify patients in whom SDHx mutations should be suspected.

A novel TBX19 gene mutation in patients with isolated ACTH deficiency from distinct families with a common geographical origin.

Isolated ACTH deficiency (IAD) is a life-threatening condition, particularly in the neonatal period, while a main consequence of undiagnosed isolated ACTH deficiency in survivors is cognitive impairment. TBX19 is involved in the differentiation and proliferation of corticotropic cells and TBX19 mutations are responsible for more than 60% of neonatal cases of IAD. We describe a new variant of the main TBX19 transcript (NM 005149.3, c.840del (p.(Glu280Asp fs*27)), classified as pathogenic, whose pathogenicity is assumed to be due to nonsense mediated decay leading to non-expression of T-box transcription factor TBX19. Moreover we summarize the TBX19 mutations published as individual cases since our last large cohort. Interestingly, this pathogenic variant was identified in four patients from three apparently unrelated families. Two of these families were consanguineous, and after investigations all of three were discovered to have roots in the same mountainous region of northern Morocco, suggesting a founder effect. Early diagnosis, timely treatment (hydrocortisone therapy) and preventive education allowed normal development, growth and quality of life in all patients.

UMD-MEN1 Database: An Overview of the 370 MEN1 Variants Present in 1676 Patients From the French Population.

Context: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by mutations in the MEN1 gene characterized by a broad spectrum of clinical manifestations, of which the most frequent are primary hyperparathyroidism, pituitary adenomas, and neuroendocrine tumors. Objective: The aim of this work was to facilitate interpretation of variants and improve the genetic counseling and medical care of families of patients with MEN1. Design, Setting, and Patients: The TENGEN network (Oncogenetics Network of Neuroendocrine Tumors) has interpreted and collected all allelic variants and clinical characteristics of the MEN1-positive patients identified through genetic testing performed in the French population from 1997 to 2015. Patients and their variants were registered in the locus-specific UMD-MEN1 database (www.umd.be/MEN1/). Main Outcomes: Variant classification, age-related penetrance, and odds ratios. Results: A total of 370 distinct variants reported in 1676 patients, including 181 unpublished variants, have been registered. This database analysis revealed a low frequency (6.6%) of benign or likely benign missense variants in MEN1. Eight families (1.9%) had members with familial isolated hyperparathyroidism and harbored the same mutations as that found in families with authentic MEN1. An association existed between large rearrangements and an earlier onset of the disease, whereas no difference was observed between truncating and nontruncating variants. Conclusion: The UMD-MEN1 database provides an exhaustive overview of the MEN1 variants present in the French population. For each variant, a classification is publicly available. Clinical data collections allow the determination of genotype-phenotype correlation and age-related penetrance of lesions in the cohort.

ALK-TPM3 rearrangement in adult renal cell carcinoma: Report of a new case showing loss of chromosome 3 and literature review.

Seven cases of translocation-associated renal cell carcinoma involving ALK (ALK-tRCC) were referenced in the last World Health Organization's classification (2016), in a group of emerging/provisional RCC. The first three cases were pediatric, medullary-based, associated with sickle-cell trait and showed a fusion of ALK with VCL. Thirteen cases have been further described. They displayed clinical, morphological and genomic heterogeneity. Most of them occurred in adults. None of the patients was affected by sickle-cell disease. We report a new case of ALK-tRCC in a 55-year-old woman. Genomic profile showed losses of chromosomes 3, 9 and 14, anomalies often observed in clear cell RCC. VHL mutation or morphological features suggesting a clear cell RCC were not detected. We identified an unbalanced rearrangement of ALK and TPM3. Review of the literature identified similar features in our case and previously published cases: heterogeneous solid architecture, eosinophilic cells, mucinous cytoplasmic elements, rhabdoid cells and intracytoplasmic lumina. These elements may constitute the basis of a pathological definition of ALK-tRCC. Their observation in a RCC should lead to perform molecular detection of ALK rearrangement. This may have a crucial importance for metastatic patients treatment since ALK rearrangements confer sensitivity to tyrosine kinases inhibitors such as crizotinib.

Paraganglioma of the organ of Zuckerkandl associated with a somatic HIF2α mutation: A case report.

Paragangliomas of the organ of Zuckerkandl (OZ-PGL) are rare tumors that, in >70% of cases, occur in association with succinate dehydrogenase complex iron sulfur subunit B (SDHB) or SDHD gene mutations. The aim of the current study was to determine whether a somatic genetic defect in the hypoxia-inducible factor 2α (HIF2α) gene was present in a case of sporadic OZ-PGL. A 32-year-old African female presented with uncontrolled hypertension during the first trimester of pregnancy. A diagnostic hysteroscopy was performed 3 months after delivery, precipitating a hypertensive crisis. Thereafter, the patient was diagnosed with noradrenaline-secreting OZ-PGL. A complete blood count identified mild normocytic anemia of an inflammatory origin. Surgical removal of the tumor resulted in normalization of plasma and urinary normetanephrine levels. Genetic testing for germline mutations (including large deletions) in the von Hippel-Lindau tumor suppressor, SDHB, SDHC and SDHD genes was normal. However, a heterozygous missense mutation (c.1589Cys>Tyr) was detected in exon 12 of HIF2α, which results in a substitution of alanine 530 with valine (Ala530Val) in the HIF2α protein. A germline mutation was excluded based on the negative results of blood DNA testing. A three-dimensional homology model of Ala530Val was constructed, which showed impaired HIF2α/VHL interaction and decreased HIF2α ubiquitination. 1H-high-resolution magic-angle-spinning nuclear magnetic resonance spectroscopy detected low succinate levels and high α and ß glucose levels. To the best of our knowledge, the present case represents the first of its kind to associate a somatic HIF2α gain-of-function mutation with OZ-PGL. It is therefore recommended that patients without germline SDHx mutations should be tested for HIF2α mutations.

Regulation of the RAP1/RAF-1/extracellularly regulated kinase-1/2 cascade and prolactin release by the phosphoinositide 3-kinase/AKT pathway in pituitary cells.

In pituitary cells, prolactin (PRL) synthesis and release are controlled by multiple transduction pathways. In the GH4C1 somatolactotroph cell line, we previously reported that MAPK ERK-1/2 are a point of convergence between the pathways involved in the PRL gene regulation. In the present study, we focused on the involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the MAPK ERK-1/2 regulation and PRL secretion in pituitary cells. Either specific pharmacological PI3K and Akt inhibitors (LY294002, Akt I, and phosphoinositide analog-6) or Akt dominant-negative mutant (K179M) enhanced ERK-1/2 phosphorylation in unstimulated GH4C1 cells. Under the same conditions, PI3K and Akt inhibition also both increased Raf-1 kinase activity and the levels of GTP-bound (active form) monomeric G protein Rap1, which suggests that a down-regulation of the ERK-1/2 cascade is induced by the PI3K/Akt signaling pathway in unstimulated cells. On the contrary, ERK-1/2 phosphorylation, Raf-1 activity, and Rap1 activation were almost completely blocked in IGF-I-stimulated cells previously subjected to PI3K or Akt inhibition. Although the PRL promoter was not affected by either PI3K/Akt inhibition or activation, PRL release increased in response to the pharmacological PI3K/Akt inhibitors in unstimulated GH4C1 and rat pituitary primary cells. The IGF-I-stimulated PRL secretion was diminished, on the contrary, by the pharmacological PI3K/Akt inhibitors. Taken together, these findings indicate that the PI3K/Akt pathway exerts dual regulatory effects on both the Rap1/Raf-1/ERK-1/2 cascade and PRL release in pituitary cells, i.e. negative effects in unstimulated cells and positive ones in IGF-I-stimulated cells.

In vitro impact of pegvisomant on growth hormone-secreting pituitary adenoma cells.

Pegvisomant (PEG), an antagonist of growth hormone (GH)-receptor (GHR), normalizes insulin-like growth factor 1 (IGF1) oversecretion in most acromegalic patients unresponsive to somatostatin analogs (SSAs) and/or uncontrolled by transsphenoidal surgery. The residual GH-secreting tumor is therefore exposed to the action of circulating PEG. However, the biological effect of PEG at the pituitary level remains unknown. To assess the impact of PEG in vitro on the hormonal secretion (GH and prolactin (PRL)), proliferation and cellular viability of eight human GH-secreting tumors in primary cultures and of the rat somatolactotroph cell line GH4C1. We found that the mRNA expression levels of GHR were characterized in 31 human GH-secreting adenomas (0.086 copy/copy ß-Gus) and the GHR was identified by immunocytochemistry staining. In 5/8 adenomas, a dose-dependent inhibition of GH secretion was observed under PEG with a maximum of 38.2±17% at 1µg/mL (P<0.0001 vs control). A dose-dependent inhibition of PRL secretion occurred in three mixed GH/PRL adenomas under PEG with a maximum of 52.8±11.5% at 10µg/mL (P<0.0001 vs control). No impact on proliferation of either human primary tumors or GH4C1 cell line was observed. We conclude that PEG inhibits the secretion of GH and PRL in primary cultures of human GH(/PRL)-secreting pituitary adenomas without effect on cell viability or cell proliferation.

Case report of GNAS epigenetic defect revealed by a congenital hypothyroidism.

Pseudohypoparathyroidism (PHP) is a group of disorders characterized by end-organ resistance to the parathyroid hormone (PTH). PHP type 1A includes multihormone resistance syndrome, Albright's hereditary osteodystrophy, and obesity and is caused by mutations in GNAS exon 1 through 13. PHP type 1B (PHP1B), caused by epigenetic changes in the GNAS locus, was initially described as an isolated resistance to PTH. Epigenetic changes in GNAS have also been reported in patients who display mild Albright's hereditary osteodystrophy or mild thyroid-stimulating hormone (TSH) resistance without mutation of GNAS. Here we report a case of PHP caused by epigenetic changes in GNAS in a patient with congenital hypothyroidism. The patient was referred for a positive newborn screening for hypothyroidism (TSH 50 mIU/L). She exhibited severe clinical features of congenital hypothyroidism. The thyroid was in place, and etiologic explorations were negative. TSH was normalized under L-thyroxin, and the symptoms disappeared, except for a macroglossia. In childhood, PHP was suspected in addition to elevated PTH, obesity, brachydactyly, and a rounded face. Sequencing, methylation analysis, and large deletion research were performed in GNAS. No genetic mutations were found. Methylation analysis revealed a broad epigenetic defect without deletion in GNAS consistent with sporadic PHP1B. The multilocus methylation analysis were negative. This finding expands the known onsets of PHP1B and emphasizes the need for a new PHP classification system. This case report has important consequences for the etiologic diagnosis of congenital hypothyroidism because it adds a new cause of the disease.

A MEN1 syndrome with a paraganglioma.

Germline mutations of the MEN1 gene cause multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disorder characterized by tumors of the parathyroids, the pancreas, and the anterior pituitary. Paraganglioma (PGL) is a rare endocrine tumor, which can be sporadic or genetically determined. To date, PGL has never been reported as a feature of MEN1.We report here a patient presenting three features of MEN1 syndrome (hyperparathyroidism, pancreatic neuroendocrine tumor, and adrenocortical adenoma) associated with PGL. Genetic analysis of MEN1 gene revealed a new missense mutation in exon 5 (AGGAAG), causing the substitution of arginine by lysine at codon 275. Screening for other genetic disorders (SDHx, TMEM127, MAX, CDKN1B) causing PGL was negative. Immunohistochemical analyses showed normal levels of succinate dehydrogenase (SDH)A and SDHB in the PGL. The proband's sister, bearing the mutation, had primary hyperparathyroidism. It was the first typical MEN1 syndrome reported with an extra-adrenal PGL.

Genetic analysis in young patients with sporadic pituitary macroadenomas: besides AIP don't forget MEN1 genetic analysis.

CONTEXT: Germline mutations in the aryl hydrocarbon receptor interacting protein gene (AIP) have been identified in young patients (age ≤30 years old) with sporadic pituitary macroadenomas. Otherwise, there are few data concerning the prevalence of multiple endocrine neoplasia type 1 (MEN1) mutations in such a population. OBJECTIVE: We assessed the prevalence of both AIP and MEN1 genetic abnormalities (mutations and large gene deletions) in young patients (age ≤30 years old) diagnosed with sporadic and isolated macroadenoma, without hypercalcemia and/or MEN1-associated lesions. DESIGN: The entire coding sequences of AIP and MEN1 were screened for mutations. In cases of negative sequencing screening, multiplex ligation-dependent probe amplification was performed for the detection of large genetic deletions. PATIENTS AND SETTINGS: One hundred and seventy-four patients from endocrinology departments of 15 French University Hospital Centers were eligible for this study. RESULTS: Twenty-one out of 174 (12%) patients had AIP (n=15, 8.6%) or MEN1 (n=6, 3.4%) mutations. In pediatric patients (age ≤18 years old), AIP/MEN1 mutation frequency reached nearly 22% (n=10/46). AIPmut and MEN1mut were identified in 8/79 (10.1%) and 1/79 (1.2%) somatotropinoma patients respectively; they each accounted for 4/74 (5.4%) prolactinoma (PRL) patients with mutations. Half of those patients (n=3/6) with gigantism displayed mutations in AIP. Interestingly, 4/12 (33%) patients with non-secreting adenomas bore either AIP or MEN1 mutations, whereas none of the eight corticotroph adenomas or the single thyrotropinoma case had mutations. No large gene deletions were observed in sequencing-negative patients. CONCLUSION: Mutations in MEN1 can be of significance in young patients with sporadic isolated pituitary macroadenomas, particularly PRL, and together with AIP, we suggest genetic analysis of MEN1 in such a population.

Ras and Rap1 govern spatiotemporal dynamic of activated ERK in pituitary living cells.

The Ras/Raf/MEK/ERK is a conserved signalling pathway involved in the control of fundamental cellular processes. Despite extensive research, how this pathway can process a myriad of diverse extracellular inputs into substrate specificity to determine biological outcomes is not fully understood. It has been established that the ERK1/2 pathway is an integrative point in the control of the pituitary function exerted by various extracellular signals. In addition we previously established that the GTPases Ras and Rap1 play a key role in the regulation of ERK1/2-dependent prolactin transcription by EGF or the cAMP-dependent neuropeptide VIP. In this report, using the FRET-based biosensor of ERK activity (EKAR) in the pituitary GH4C1 cell line, we show that both EGF and VIP tightly control the spatiotemporal dynamic of activated ERK with different magnitude and duration. Importantly, we provide the first evidence of a differential control of cytoplasmic and nuclear pools of activated ERK by the GTPases Ras and Rap1. Ras is required for nuclear magnitude and duration of EGF-dependent ERK activation, whereas it is required for both VIP-activated cytoplasmic and nuclear ERK pools. Rap1 is exclusively involved in VIP-activated ERK nuclear pool. Moreover, consistent with the control of the nuclear pool of activated ERK by the GTPases, we observe the same differential role of Ras and Rap1 on ERK nuclear translocation triggered by EGF or VIP. Together these findings identify Ras and Rap1 as determinant partners in shaping nuclear and cytoplasmic ERK kinetics in response to EGF and VIP, which in turn should control pituitary secretion.