A Study of Alternative TrkA Splicing Identifies TrkAIII as a Novel Potentially Targetable Participant in PitNET Progression.

Pituitary neuroendocrine tumors (PitNETs) are generally benign but comprise an aggressive, invasive, therapy-resistant, metastatic subset, underpinning a need for novel therapeutic targets. PitNETs exhibit low mutation rates but are associated with conditions linked to alternative splicing, an alternative oncogene pathway activation mechanism. PitNETs express the neurotrophin receptor TrkA, which exhibits oncogenic alternative TrkAIII splicing in other neuroendocrine tumors. We, therefore, assessed whether TrkAIII splicing represents a potential oncogenic participant in PitNETs. TrkAIII splicing was RT-PCR assessed in 53 PitNETs and TrkA isoform(s) expression and activation were assessed by confocal immunofluorescence. TrkAIII splicing was also compared to HIF1α, HIF2α, SF3B1, SRSF2, U2AF1, and JCPyV large T antigen mRNA expression, Xbp1 splicing, and SF3B1 mutation. TrkAIII splicing was detected in all invasive and most non-invasive PitNETs and was significantly elevated in invasive cases. In PitNET lineages, TrkAIII splicing was significantly elevated in invasive PIT1 PitNETs and high in invasive and non-invasive SF1 and TPIT lineages. Immunoreactivity consistent with TrkAIII activation characterized PitNET expressing TrkAIII mRNA, and invasive Pit1 PitNETs exhibited elevated HIF2α expression. TrkAIII splicing did not associate with SF3B1 mutations, altered SF3B1, SRSF2, and U2AF1 or JCPyV large T antigen expression, or Xbp1 splicing. Therefore, TrkAIII splicing is common in PitNETs, is elevated in invasive, especially PIT1 tumors, can result in intracellular TrkAIII activation, and may involve hypoxia. The data support a role for TrkAIII splicing in PitNET pathogenesis and progression and identify TrkAIII as a novel potential target in refractory PitNETs.

Clinical and Molecular Characteristics of Gonadotroph Pituitary Tumors According to the WHO Classification.

Since 2017, hormone-negative pituitary neuroendocrine tumors expressing the steroidogenic factor SF1 have been recognized as gonadotroph tumors (GnPT) but have been poorly studied. To further characterize their bio-clinical spectrum, 54 GnPT defined by immunostaining for FSH and/or LH (group 1, n = 41) or SF1 only (group 2, n = 13) were compared and studied for SF1, ßFSH, ßLH, CCNA2, CCNB1, CCND1, caspase 3, D2R, and AIP gene expression by qRT-PCR. Immunohistochemistry for AIP and/or D2R was performed in representative cases. Overall, patients were significantly younger in group 1 (P = 0.040 vs group 2), with a similar trend excluding recurrent cases (P = 0.078), and no significant difference in gender, tumor size, invasion or Ki67. SF1 expression was similar in both groups but negatively correlated with the patient's age (P = 0.013) and positively correlated with ßLH (P < 0.001) expression. Beta-FSH and AIP were significantly higher in group 1 (P = 0.042 and P = 0.024, respectively). Ki67 was unrelated to gonadotroph markers but positively correlated with CCNB1 (P = 0.001) and negatively correlated with CCND1 (P = 0.008). D2R and AIP were strongly correlated with each other (P < 0.001), and both positively correlated with SF1, ßFSH, ßLH, and CCND1. AIP immunopositivity was frequently observed in both groups, with a similar median score, and unrelated to Ki67. D2R immunostaining was best detected with a polyclonal antibody and mostly cytoplasmic. This study indicates that hormone-negative GnPT tend to occur in older patients but do not significantly differ from other GnPT in terms of invasion or proliferation. It also points out the current limits of D2R immunostaining in such tumors.

The clinical and therapeutic profiles of prolactinomas associated with germline pathogenic variants in the aryl hydrocarbon receptor interacting protein (AIP) gene.

Introduction: Prolactinomas are the most frequent type of pituitary adenoma encountered in clinical practice. Dopamine agonists (DA) like cabergoline typically provide sign/ symptom control, normalize prolactin levels and decrease tumor size in most patients. DA-resistant prolactinomas are infrequent and can occur in association with some genetic causes like MEN1 and pathogenic germline variants in the AIP gene (AIPvar). Methods: We compared the clinical, radiological, and therapeutic characteristics of AIPvar-related prolactinomas (n=13) with unselected hospital-treated prolactinomas ("unselected", n=41) and genetically-negative, DA-resistant prolactinomas (DA-resistant, n=39). Results: AIPvar-related prolactinomas occurred at a significantly younger age than the unselected or DA-resistant prolactinomas (p<0.01). Males were more common in the AIPvar (75.0%) and DA- resistant (49.7%) versus unselected prolactinomas (9.8%; p<0.001). AIPvar prolactinomas exhibited significantly more frequent invasion than the other groups (p<0.001) and exhibited a trend to larger tumor diameter. The DA-resistant group had significantly higher prolactin levels at diagnosis than the AIPvar group (p<0.001). Maximum DA doses were significantly higher in the AIPvar and DA-resistant groups versus unselected. DA-induced macroadenoma shrinkage (>50%) occurred in 58.3% in the AIPvar group versus 4.2% in the DA-resistant group (p<0.01). Surgery was more frequent in the AIPvar and DA- resistant groups (43.8% and 61.5%, respectively) versus unselected (19.5%: p<0.01). Radiotherapy was used only in AIPvar (18.8%) and DA-resistant (25.6%) groups. Discussion: AIPvar confer an aggressive phenotype in prolactinomas, with invasive tumors occurring at a younger age. These characteristics can help differentiate rare AIPvar related prolactinomas from DA-resistant, genetically-negative tumors.

Germline loss-of-function PAM variants are enriched in subjects with pituitary hypersecretion.

Introduction: Pituitary adenomas (PAs) are common, usually benign tumors of the anterior pituitary gland which, for the most part, have no known genetic cause. PAs are associated with major clinical effects due to hormonal dysregulation and tumoral impingement on vital brain structures. PAM encodes a multifunctional protein responsible for the essential C-terminal amidation of secreted peptides. Methods: Following the identification of a loss-of-function variant (p.Arg703Gln) in the peptidylglycine a-amidating monooxygenase (PAM) gene in a family with pituitary gigantism, we investigated 299 individuals with sporadic PAs and 17 familial isolated PA kindreds for PAM variants. Genetic screening was performed by germline and tumor sequencing and germline copy number variation (CNV) analysis. Results: In germline DNA, we detected seven heterozygous, likely pathogenic missense, truncating, and regulatory SNVs. These SNVs were found in sporadic subjects with growth hormone excess (p.Gly552Arg and p.Phe759Ser), pediatric Cushing disease (c.-133T>C and p.His778fs), or different types of PAs (c.-361G>A, p.Ser539Trp, and p.Asp563Gly). The SNVs were functionally tested in vitro for protein expression and trafficking by Western blotting, splicing by minigene assays, and amidation activity in cell lysates and serum samples. These analyses confirmed a deleterious effect on protein expression and/or function. By interrogating 200,000 exomes from the UK Biobank, we confirmed a significant association of the PAM gene and rare PAM SNVs with diagnoses linked to pituitary gland hyperfunction. Conclusion: The identification of PAM as a candidate gene associated with pituitary hypersecretion opens the possibility of developing novel therapeutics based on altering PAM function.

Germline loss-of-function PAM variants are enriched in subjects with pituitary hypersecretion.

Pituitary adenomas (PAs) are common, usually benign tumors of the anterior pituitary gland which, for the most part, have no known genetic cause. PAs are associated with major clinical effects due to hormonal dysregulation and tumoral impingement on vital brain structures. Following the identification of a loss-of-function variant (p.Arg703Gln) in the PAM gene in a family with pituitary gigantism, we investigated 299 individuals with sporadic PAs and 17 familial isolated pituitary adenomas kindreds for PAM variants. PAM encodes a multifunctional protein responsible for the essential C-terminal amidation of secreted peptides. Genetic screening was performed by germline and tumor sequencing and germline copy number variation (CNV) analysis. No germline CNVs or somatic single nucleotide variants (SNVs) were identified. We detected seven likely pathogenic heterozygous missense, truncating, and regulatory SNVs. These SNVs were found in sporadic subjects with GH excess (p.Gly552Arg and p.Phe759Ser), pediatric Cushing disease (c.-133T>C and p.His778fs), or with different types of PAs (c.-361G>A, p.Ser539Trp, and p.Asp563Gly). The SNVs were functionally tested in vitro for protein expression and trafficking by Western blotting, for splicing by minigene assays, and for amidation activity in cell lysates and serum samples. These analyses confirmed a deleterious effect on protein expression and/or function. By interrogating 200,000 exomes from the UK Biobank, we confirmed a significant association of the PAM gene and rare PAM SNVs to diagnoses linked to pituitary gland hyperfunction. Identification of PAM as a candidate gene associated with pituitary hypersecretion opens the possibility of developing novel therapeutics based on altering PAM function.

Immunotherapy for Aggressive and Metastatic Pituitary Neuroendocrine Tumors (PitNETs): State-of-the Art.

Background: Aggressive and metastatic PitNETs are challenging conditions. Immune checkpoint inhibitors (ICIs) are currently considered in cases resistant to temozolomide (TMZ). However, clinical experience is essentially limited to case reports, with variable outcomes. Material and Methods: The effects of ICIs on 12 aggressive/metastatic PitNETs from the literature were reviewed and analyzed according to tumor characteristics, with the additional description of a silent-Pit1 metastatic tumor responding to pembrolizumab. Results: Most cases were metastatic (10/13: 6 corticotroph, 3 lactotroph, 1 silent Pit1); 3 were aggressive (2 corticotroph, 1 lactotroph). ICIS was used either as monotherapy or in combination. At last follow-up on ICI, a complete response (CR) was present in 3 cases and a partial response (PR) in 2 cases (4/5 metastatic). One sustained stable disease (SD) was reported. Progressive disease (PD) was observed in 7 cases, 3 of them after initial SD (n = 1) or PR (n = 3), with 2 reported deaths. PDL1 expression was studied in 10 cases and was high (>95%) in 2 Pit1-derived metastatic PitNETs (1 CR and 1 remarkable PR) but absent/low (<1%) in the remaining cases (including 1 CP and 2 PR). Elevated tumor mutation burden could be informative in corticotroph PitNETs, especially in mismatch repair-deficient tumors. Conclusion: Significant benefits from ICIs were documented in about half of TMZ-resistant PitNETS. High PDL1 expression was associated with remarkable responses but may be dispensable. Based on their acceptable tolerance and awaiting recognized predictors of response, ICIs may be considered a valuable option for such patients.

Alternative Lengthening of Telomeres (ALT) and Telomerase Reverse Transcriptase Promoter Methylation in Recurrent Adult and Primary Pediatric Pituitary Neuroendocrine Tumors.

Neoplastic cells acquire the ability to proliferate endlessly by maintaining telomeres via telomerase, or alternative lengthening of telomeres (ALT). The role of telomere maintenance in pituitary neuroendocrine tumors (PitNETs) has yet to be thoroughly investigated. We analyzed surgical samples of 24 adult recurrent PitNETs (including onset and relapses for 14 of them) and 12 pediatric primary PitNETs. The presence of ALT was assessed using telomere-specific fluorescence in situ hybridization, methylation of telomerase reverse transcriptase promoter (TERTp) by methylation-specific PCR, and ATRX expression by immunohistochemistry. Among the adult recurrent PitNETs, we identified 3/24 (12.5%) ALT-positive cases. ALT was present from the onset and maintained in subsequent relapses, suggesting that this mechanism occurs early in tumorigenesis and is stable during progression. ATRX loss was only seen in one ALT-positive case. Noteworthy, ALT was observed in 3 out of 5 aggressive PitNETs, including two aggressive corticotroph tumors, eventually leading to patient's death. ALT-negative tumors (87.5%) were classified according to their low (29.2%), medium (50%), and high (8.3%) telomere fluorescence intensity, with no significant differences emerging in their molecular, clinical, or pathological characteristics. TERTp methylation was found in 6/24 cases (25%), with a total concordance in methylation status between onset and recurrences, suggesting that this mechanism remains stable throughout disease progression. TERTp methylation did not influence telomere length. In the pediatric cohort of PitNETs, TERTp methylation was also observed in 4/12 cases (33.3%), but no case of ALT activation was observed. In conclusion, ALT is triggered at onset and maintained during tumor progression in a subset of adult PitNETs, suggesting that it could be used for clinical purposes, as a potential predictor of aggressive behavior.

Sleep Disorders in Patients With Craniopharyngioma: A Physiopathological and Practical Update.

Sleep disorders (SDs) represent an important issue in patients with craniopharyngioma (CP). Nearly 70% of these patients complain of sleep-wake cycle alterations and/or excessive diurnal somnolence due to sleep-related breathing disorders, such as obstructive sleep apnea (OSA) and/or central hypersomnia, including secondary narcolepsy. SDs may severely reduce quality of life, increase disease-related cardiorespiratory and cardiovascular morbidity, and finally play a major role in increased long-term mortality reported on patients with CP. A major risk factor for SDs is represented by the hypothalamic syndrome, which may develop because of direct hypothalamic damage by the tumor itself and/or complications of the treatments, neurosurgery and/or radiotherapy, and typically includes permanent neuroendocrine dysfunctions, morbid obesity, and secondary metabolic disorders. Despite increasing attention to SDs in the general population, and in particular to OSA as a risk factor for cardio-metabolic diseases and excessive daytime somnolence, sleep evaluation is still not routinely proposed to patients with CP. Hence, SDs are often underdiagnosed and undertreated. The aim of this paper is to update current knowledge of the pathogenesis and prevalence of SDs in patients with CP and propose practical algorithms for their evaluation and management in clinical practice. Particular attention is paid to screening and diagnostic tools for appropriate characterization of SDs, identification of risk factors, and potential role of hypothalamic sparing surgery in the prevention of morbid obesity and SDs. Available tools in sleep medicine, including lifestyle interventions, drugs, and respiratory devices, are discussed, as well as the importance of optimal hormone replacement and metabolic interventions. Current limits in the diagnosis and treatment of SDs in patients with CP and possible future avenues for research agenda are also considered.

Multivariable Prediction Model for Biochemical Response to First-Generation Somatostatin Receptor Ligands in Acromegaly.

CONTEXT: First-generation somatostatin receptor ligands (fg-SRLs) represent the mainstay of medical therapy for acromegaly, but they provide biochemical control of disease in only a subset of patients. Various pretreatment biomarkers might affect biochemical response to fg-SRLs. OBJECTIVE: To identify clinical predictors of the biochemical response to fg-SRLs monotherapy defined as biochemical response (insulin-like growth factor (IGF)-1 ≤ 1.3 × ULN (upper limit of normal)), partial response (>20% relative IGF-1 reduction without normalization), and nonresponse (≤20% relative IGF-1 reduction), and IGF-1 reduction. DESIGN: Retrospective multicenter study. SETTING: Eight participating European centers. METHODS: We performed a meta-analysis of participant data from 2 cohorts (Rotterdam and Liège acromegaly survey, 622 out of 3520 patients). Multivariable regression models were used to identify predictors of biochemical response to fg-SRL monotherapy. RESULTS: Lower IGF-1 concentration at baseline (odds ratio (OR) = 0.82, 95% confidence interval (CI) 0.72-0.95 IGF-1 ULN, P = .0073) and lower bodyweight (OR = 0.99, 95% CI 0.98-0.99 kg, P = .038) were associated with biochemical response. Higher IGF-1 concentration at baseline (OR = 1.40, (1.19-1.65) IGF-1 ULN, P ≤ .0001), the presence of type 2 diabetes (oral medication OR = 2.48, (1.43-4.29), P = .0013; insulin therapy OR = 2.65, (1.02-6.70), P = .045), and higher bodyweight (OR = 1.02, (1.01-1.04) kg, P = .0023) were associated with achieving partial response. Younger patients at diagnosis are more likely to achieve nonresponse (OR = 0.96, (0.94-0.99) year, P = .0070). Baseline IGF-1 and growth hormone concentration at diagnosis were associated with absolute IGF-1 reduction (ß = 0.90, standard error (SE) = 0.02, P ≤ .0001 and ß â€…= 0.002, SE = 0.001, P = .014, respectively). CONCLUSION: Baseline IGF-1 concentration was the best predictor of biochemical response to fg-SRL, followed by bodyweight, while younger patients were more likely to achieve nonresponse.

Are aggressive pituitary tumors and carcinomas two sides of the same coin? Pathologists reply to clinician's questions.

Pituitary adenohypophyseal tumors are considered as benign and termed "adenomas". However, many tumors are invasive and a proportion of these exhibit an "aggressive behavior" with premature death due to progressive growth. Only very rare (0.2%) tumors with metastases are considered malignant and termed "carcinomas". Taking into account this variability in behavior and the oncological definition, pathologists have proposed changing the term adenoma to tumor. Here we explain why use the term tumor instead of adenoma and identify tumor characteristics, associated with a high risk for poor prognosis. In a cohort of 125 tumors with aggressive behavior (APT) and 40 carcinomas with metastases (PC), clinical and pathological features were very similar. The comparison of this cohort (APT+PC) with a reference surgical cohort of 374 unselected patients clearly shows that the two cohorts differ greatly, especially the percentage of tumors with Ki67 ≥ 10% (35%vs3%; p < 0.001). A five-tiered prognostic classification, associating invasion and proliferation, identified grade 2b tumors (invasive and proliferative), with a high risk of recurrence/progression. Because half of the APT+ PC tumors have a Ki67 index ≥10%, and 80% of them show 2 or 3 positive markers of proliferation, we suggest that tumors that are clinically aggressive, invasive and highly proliferative with a Ki67 ≥ 10%, represent tumors with malignant potential. The percentage of grade 2b tumors, suspected of malignancy, which will become aggressive tumors or carcinomas is unknown. It is probably very low, but higher than 0.2% in surgical series. Early identification and active treatment of these aggressive tumors is needed to decrease morbidity and prolong survival.

How to Classify the Pituitary Neuroendocrine Tumors (PitNET)s in 2020.

Adenohypophyseal tumors, which were recently renamed pituitary neuroendocrine tumors (PitNET), are mostly benign, but may present various behaviors: invasive, "aggressive" and malignant with metastases. They are classified into seven morphofunctional types and three lineages: lactotroph, somatotroph and thyrotroph (PIT1 lineage), corticotroph (TPIT lineage) or gonadotroph (SF1 lineage), null cell or immunonegative tumor and plurihormonal tumors. The WHO 2017 classification suggested that subtypes, such as male lactotroph, silent corticotroph and Crooke cell, sparsely granulated somatotroph, and silent plurihormonal PIT1 positive tumors, should be considered as "high risk" tumors. However, the prognostic impact of these subtypes and of each morphologic type remains controversial. In contrast, the French five-tiered classification, taking into account the invasion, the immuno-histochemical (IHC) type, and the proliferative markers (Ki-67 index, mitotic count, p53 positivity), has a prognostic value validated by statistical analysis in 4 independent cohorts. A standardized report for the diagnosis of pituitary tumors, integrating all these parameters, has been proposed by the European Pituitary Pathology Group (EPPG). In 2020, the pituitary pathologist must be considered as a member of the multidisciplinary pituitary team. The pathological diagnosis may help the clinician to adapt the post-operative management, including appropriate follow-up and early recognition and treatment of potentially aggressive forms.

Expression of Peroxisome Proliferator-Activated Receptor Alpha (PPARα) in Non-Somatotroph Pituitary Tumours and the Effects of PPARα Agonists on MMQ Cells.

Peroxisome proliferator-activated receptor alpha (PPARα) has been involved in the regulation of somatotroph tumour cells and may be targeted by different drugs, some of them are in current clinical use. The aim of this study was to investigate the expression of PPARα in additional phenotypes of pituitary adenomas (PA), the relationship between PPARα and its potential molecular partner aryl hydrocarbon receptor interacting protein (AIP) in these tumours, and the effects of PPARα agonists on lactotroph cells. Seventy-five human PA - 57 non-functioning (NFPA) and 18 prolactinomas (PRL-PA) - were characterised for PPARα and AIP expression by real time RT-PCR and/or immunohistochemistry (IHC), and the effects of fenofibrate and WY 14 643 on MMQ cells were studied in vitro. PPARα was expressed in a majority of PA. PPARα immunostaining was observed in 93.7% PRL-PA vs. 60.6% NFPA (p=0.016), the opposite being found for AIP (83.3% in NFPA vs. 43.7% in PRL-PA, p=0.003). PPARα expression was unrelated to gonadotroph differentiation in NFPA, but positively correlated with tumour volume in PRL-PA. Both drugs significantly reduced MMQ cell growth at high concentrations (100-200 µM). At the same time, despite modest stimulating effects on PRL secretion were observed, these were overcome by the reduction in cell number. In conclusion, PPARα is commonly expressed by PRL-PA and NFPA, regardless of AIP, and may represent a new target of PPARα agonists.

Acromegaly at diagnosis in 3173 patients from the Liège Acromegaly Survey (LAS) Database.

Acromegaly is a rare disorder caused by chronic growth hormone (GH) hypersecretion. While diagnostic and therapeutic methods have advanced, little information exists on trends in acromegaly characteristics over time. The Liège Acromegaly Survey (LAS) Database, a relational database, is designed to assess the profile of acromegaly patients at diagnosis and during long-term follow-up at multiple treatment centers. The following results were obtained at diagnosis. The study population consisted of 3173 acromegaly patients from ten countries; 54.5% were female. Males were significantly younger at diagnosis than females (43.5 vs 46.4 years; P < 0.001). The median delay from first symptoms to diagnosis was 2 years longer in females (P = 0.015). Ages at diagnosis and first symptoms increased significantly over time (P < 0.001). Tumors were larger in males than females (P < 0.001); tumor size and invasion were inversely related to patient age (P < 0.001). Random GH at diagnosis correlated with nadir GH levels during OGTT (P < 0.001). GH was inversely related to age in both sexes (P < 0.001). Diabetes mellitus was present in 27.5%, hypertension in 28.8%, sleep apnea syndrome in 25.5% and cardiac hypertrophy in 15.5%. Serious cardiovascular outcomes like stroke, heart failure and myocardial infarction were present in <5% at diagnosis. Erythrocyte levels were increased and correlated with IGF-1 values. Thyroid nodules were frequent (34.0%); 820 patients had colonoscopy at diagnosis and 13% had polyps. Osteoporosis was present at diagnosis in 12.3% and 0.6-4.4% had experienced a fracture. In conclusion, this study of >3100 patients is the largest international acromegaly database and shows clinically relevant trends in the characteristics of acromegaly at diagnosis.

T2-weighted MRI signal predicts hormone and tumor responses to somatostatin analogs in acromegaly.

GH-secreting pituitary adenomas can be hypo-, iso- or hyper-intense on T2-weighted MRI sequences. We conducted the current multicenter study in a large population of patients with acromegaly to analyze the relationship between T2-weighted signal intensity on diagnostic MRI and hormonal and tumoral responses to somatostatin analogs (SSA) as primary monotherapy. Acromegaly patients receiving primary SSA for at least 3 months were included in the study. Hormonal, clinical and general MRI assessments were performed and assessed centrally. We included 120 patients with acromegaly. At diagnosis, 84, 17 and 19 tumors were T2-hypo-, iso- and hyper-intense, respectively. SSA treatment duration, cumulative and mean monthly doses were similar in the three groups. Patients with T2-hypo-intense adenomas had median SSA-induced decreases in GH and IGF-1 of 88% and 59% respectively, which were significantly greater than the decreases observed in the T2-iso- and hyper-intense groups (P < 0.001). Tumor shrinkage on SSA was also significantly greater in the T2-hypo-intense group (38%) compared with the T2-iso- and hyper-intense groups (8% and 3%, respectively; P < 0.0001). The response to SSA correlated with the calculated T2 intensity: the lower the T2-weighted intensity, the greater the decrease in random GH (P < 0.0001, r = 0.22), IGF-1 (P < 0.0001, r = 0.14) and adenoma volume (P < 0.0001, r = 0.33). The T2-weighted signal intensity of GH-secreting adenomas at diagnosis correlates with hormone reduction and tumor shrinkage in response to primary SSA treatment in acromegaly. This study supports its use as a generally available predictive tool at diagnosis that could help to guide subsequent treatment choices in acromegaly.

Somatic mosaicism underlies X-linked acrogigantism syndrome in sporadic male subjects.

Somatic mosaicism has been implicated as a causative mechanism in a number of genetic and genomic disorders. X-linked acrogigantism (XLAG) syndrome is a recently characterized genomic form of pediatric gigantism due to aggressive pituitary tumors that is caused by submicroscopic chromosome Xq26.3 duplications that include GPR101 We studied XLAG syndrome patients (n= 18) to determine if somatic mosaicism contributed to the genomic pathophysiology. Eighteen subjects with XLAG syndrome caused by Xq26.3 duplications were identified using high-definition array comparative genomic hybridization (HD-aCGH). We noted that males with XLAG had a decreased log2ratio (LR) compared with expected values, suggesting potential mosaicism, whereas females showed no such decrease. Compared with familial male XLAG cases, sporadic males had more marked evidence for mosaicism, with levels of Xq26.3 duplication between 16.1 and 53.8%. These characteristics were replicated using a novel, personalized breakpoint junction-specific quantification droplet digital polymerase chain reaction (ddPCR) technique. Using a separate ddPCR technique, we studied the feasibility of identifying XLAG syndrome cases in a distinct patient population of 64 unrelated subjects with acromegaly/gigantism, and identified one female gigantism patient who had had increased copy number variation (CNV) threshold for GPR101 that was subsequently diagnosed as having XLAG syndrome on HD-aCGH. Employing a combination of HD-aCGH and novel ddPCR approaches, we have demonstrated, for the first time, that XLAG syndrome can be caused by variable degrees of somatic mosaicism for duplications at chromosome Xq26.3. Somatic mosaicism was shown to occur in sporadic males but not in females with XLAG syndrome, although the clinical characteristics of the disease were similarly severe in both sexes.

Expression of Peroxisome Proliferator-Activated Receptor alpha (PPARα) in somatotropinomas: Relationship with Aryl hydrocarbon receptor Interacting Protein (AIP) and in vitro effects of fenofibrate in GH3 cells.

PURPOSE: To search for a possible role of Peroxisome Proliferator-Activated Receptor α (PPARα), a molecular partner of the Aryl hydrocarbon receptor Interacting Protein (AIP), in somatotropinomas. METHODS: Tumours from 51 acromegalic patients were characterized for PPARα and AIP expression by immunohistochemistry (IHC) and/or Real Time RT-PCR. Data were analysed according to tumour characteristics and pre-operative treatment with somatostatin analogues (SSA). The effects of fenofibrate were studied in GH3 cells in vitro. RESULTS: PPARα was expressed in most somatotropinomas. A modest relationship was found between PPARα and AIP expression, both being significantly higher in the presence of pre-operative SSA. However, only AIP expression was influenced by the response to treatment. Dual effects of fenofibrate were observed in GH3 cells, consisting of cell growth inhibition and an increase in GH secretion inhibited by octreotide. CONCLUSIONS: PPARα is a new player in somatotropinomas. Potential interactions between PPARα agonists and SSA may deserve further investigation.

A constitutive active MAPK/ERK pathway due to BRAFV600E positively regulates AHR pathway in PTC.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor mediating the toxicity and tumor-promoting properties of dioxin. AHR has been reported to be overexpressed and constitutively active in a variety of solid tumors, but few data are currently available concerning its role in thyroid cancer. In this study we quantitatively explored a series of 51 paired-normal and papillary thyroid carcinoma (PTC) tissues for AHR-related genes. We identified an increased AHR expression/activity in PTC, independently from its nuclear dimerization partner and repressor but strictly related to a constitutive active MAPK/ERK pathway. The AHR up-regulation followed by an increased expression of AHR target genes was confirmed by a meta-analysis of published microarray data, suggesting a ligand-independent active AHR pathway in PTC. In-vitro studies using a PTC-derived cell line (BCPAP) and HEK293 cells showed that BRAFV600E may directly modulate AHR localization, induce AHR expression and activity in an exogenous ligand-independent manner. The AHR pathway might represent a potential novel therapeutic target for PTC in the clinical practice.

Clinical and genetic characterization of pituitary gigantism: an international collaborative study in 208 patients.

Despite being a classical growth disorder, pituitary gigantism has not been studied previously in a standardized way. We performed a retrospective, multicenter, international study to characterize a large series of pituitary gigantism patients. We included 208 patients (163 males; 78.4%) with growth hormone excess and a current/previous abnormal growth velocity for age or final height >2 s.d. above country normal means. The median onset of rapid growth was 13 years and occurred significantly earlier in females than in males; pituitary adenomas were diagnosed earlier in females than males (15.8 vs 21.5 years respectively). Adenomas were ≥10 mm (i.e., macroadenomas) in 84%, of which extrasellar extension occurred in 77% and invasion in 54%. GH/IGF1 control was achieved in 39% during long-term follow-up. Final height was greater in younger onset patients, with larger tumors and higher GH levels. Later disease control was associated with a greater difference from mid-parental height (r=0.23, P=0.02). AIP mutations occurred in 29%; microduplication at Xq26.3 - X-linked acrogigantism (X-LAG) - occurred in two familial isolated pituitary adenoma kindreds and in ten sporadic patients. Tumor size was not different in X-LAG, AIP mutated and genetically negative patient groups. AIP-mutated and X-LAG patients were significantly younger at onset and diagnosis, but disease control was worse in genetically negative cases. Pituitary gigantism patients are characterized by male predominance and large tumors that are difficult to control. Treatment delay increases final height and symptom burden. AIP mutations and X-LAG explain many cases, but no genetic etiology is seen in >50% of cases.

Downregulation of miR-410 targeting the cyclin B1 gene plays a role in pituitary gonadotroph tumors.

MicroRNAs (miRNAs) are small noncoding RNAs that act as posttranscriptional regulators of gene expression, and are frequently altered in human neoplasias. Here, we have analyzed the miRNA expression profile of human gonadotroph adenomas versus normal pituitary tissue using a miRNACHIP microarray. We demonstrate that miRNA-410 is downregulated in gonadotroph adenomas when compared with normal pituitary gland. We validate CCNB1 as target of miRNA-410 since its overexpression reduces CCNB1 at protein and mRNA levels, decreasing cell proliferation. In conclusion, our study suggess that the downregulation of miRNA-410 plays a role in the behavior of gonadotroph tumors.