Loss of neuronatin expression is associated with promoter hypermethylation in pituitary adenoma

The imprinted gene, neuronatin (NNAT), is one of the most abundant transcripts in the pituitary and is thought to be involved in the development and maturation of this gland. In a recent whole-genome approach, exploiting a pituitary tumour cell line, we identified hypermethylation associated loss of NNAT. In this report, we determined the expression pattern of NNAT in individual cell types of the normal gland and within each of the different pituitary adenoma subtypes. In addition, we determined associations between expression and CpG island methylation and used colony forming efficiency assays (CFE) to gain further insight into the tumour-suppressor function of this gene. Immunohistochemical (IHC) co-localization studies of normal pituitaries showed that each of the hormone secreting cells (GH, PRL, ACTH, FSH and TSH) expressed NNAT. However, 33 out of 47 adenomas comprising, 11 somatotrophinomas, 10 prolactinomas, 12 corticotrophinomas and 14 non-functioning tumours, irrespective of subtype failed to express either NNAT transcript or protein as determined by quantitative real-time RT-PCR and IHC respectively. In normal pituitaries and adenomas that expressed NNAT the promoter-associated CpG island showed characteristics of an imprinted gene where approximately 50% of molecules were densely methylated. However, in the majority of adenomas that showed loss or significantly reduced expression of NNAT, relative to normal pituitaries, the gene-associated CpG island showed significantly increased methylation. Induced expression of NNAT in transfected AtT-20 cells significantly reduced CFE. Collectively, these findings point to an important role for NNAT in the pituitary and perhaps tumour development in this gland.

Genetic Basis of Pituitary Adenoma Predisposition

Much of the global cancer research is focused on the most prevalent tumors; yet, less common tumor types warrant investigation, since A rare disorder is not necessarily an unimportant one . The present work discusses a rare tumor type, the benign adenomas of the pituitary gland, and presents the advances which, during the course of this thesis work, contributed to the elucidation of a fraction of their genetic background. Pituitary adenomas are benign neoplasms of the anterior pituitary lobe, accounting for approximately 15% of all intracranial tumors. Pituitary adenoma cells hypersecrete the hormones normally produced by the anterior pituitary tissue, such as growth hormone (GH) and prolactin (PRL). Despite their non-metastasizing nature, these adenomas can cause significant morbidity and have to be adequately treated; otherwise, they can compromise the patient s quality of life, due to conditions provoked by hormonal hypersecretion, such as acromegaly in the case of GH-secreting adenomas, or due to compressive effects to surrounding tissues. The vast majority of pituitary adenomas arise sporadically, whereas a small subset occur as component of familial endocrine-related tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 is caused by germline mutations in the MEN1 tumor suppressor gene (11q13), whereas the majority of CNC cases carry germline mutations in the PRKAR1A gene (17q24). Pituitary adenomas are also encountered in familial settings outside the context of MEN1 and CNC, but unlike in the latter syndromes, their genetic background until recently remained elusive. Evidence in previous literature supported the notion that a tumor suppressor gene on 11q13, residing very close to but still distinct from MEN1, causes genetic susceptibility to pituitary tumors. The aim of the study was to identify the genetic cause of a low penetrance form of Pituitary Adenoma Predisposition (PAP) in families from Northern Finland. The present work describes the methodological approach that led to the identification of aryl hydrocarbon receptor interacting protein (AIP) as the gene causing PAP. Combining chip-based technologies (SNP and gene expression arrays) with traditional gene mapping methods and genealogy data, we showed that germline AIP mutations cause PAP in familial and sporadic settings. PAP patients were diagnosed with mostly adenomas of the GH/PRL-secreting cell lineage. In Finland, two AIP mutations accounted for 16% of all patients diagnosed with GH-secreting adenomas, and for 40% of patients being younger than 35 years of age at diagnosis. AIP is suggested to act as a tumor suppressor gene, a notion supported by the nature of the identified mutations (most are truncating) and the biallelic inactivation of AIP in the tumors studied. AIP has been best characterized as a cytoplasmic interaction partner of aryl hydrocarbon receptor (AHR), also known as dioxin receptor, but it has other partners as well. The mechanisms that underlie AIP-mediated pituitary tumorigenesis are to date largely unknown and warrant further investigation. Because AIP was identified in the genetically homogeneous Finnish population, it was relevant to examine its contribution to PAP in other, more heterogeneous, populations. Analysis of pituitary adenoma patient series of various ethnic origins and differing clinical settings revealed germline AIP mutations in all cohorts studied, albeit with low frequencies (range 0.8-7.4%). Overall, PAP patients were typically diagnosed at a young age (range 8-41 years), mainly with GH-secreting adenomas, without strong family history of endocrine disease. Because many PAP patients did not display family history of pituitary adenomas, detection of the condition appeared challenging. AIP immunohistochemistry was tested as a molecular pre-screening tool on mutation-positive versus mutation-negative tumors, and proved to be a potentially useful predictor of PAP. Mutation screening of a large cohort of colorectal, breast, and prostate tumors did not reveal somatic AIP mutations. These tumors, apart from being the most prevalent among men and women worldwide, have been associated with acromegaly, particularly colorectal neoplasia. In this material, AIP did not appear to contribute to the pathogenesis of these common tumor types and other genes seem likely to play a role in such tumorigenesis. Finally, the contribution of AIP in pediatric onset pituitary adenomas was examined in a unique population-based cohort of sporadic pituitary adenoma patients from Italy. Germline AIP mutations may account for a subset of pediatric onset GH-secreting adenomas (in this study one of seven GH-secreting adenoma cases or 14.3%), and appear to be enriched among young (≤25 years old) patients. In summary, this work reveals a novel tumor susceptibility gene, namely AIP, which causes genetic predisposition to pituitary adenomas, in particular GH-secreting adenomas. Moreover, it provides molecular tools for identification of individuals predisposed for PAP. Further elaborate studies addressing the functional role of AIP in normal and tumor cells will hopefully expand our knowledge on endocrine neoplasia and reveal novel cellular mechanisms of pituitary tumorigenesis, including potential drug targets.

Molecular and Clinical Characteristics of Pituitary Adenoma Predisposition (PAP)

Pituitary adenomas are common benign neoplasms. Although most of them are sporadic, a minority occurs in familial settings. Heterozygous germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were found to underlie familial pituitary adenomas, a condition designated as pituitary adenoma predisposition (PAP). PAP confers incomplete penetrance of mostly growth hormone (GH) secreting adenomas in young patients, who often lack a family history of pituitary adenomas. This thesis work aimed to clarify the molecular and clinical characteristics of PAP. Applying the multiplex ligation-dependent probe amplification assay (MLPA), we found large genomic AIP deletions to account for a subset of PAP. Therefore, MLPA could be considered in PAP suspected patients with no AIP mutations found with conventional sequencing. We generated an Aip mouse model to examine pituitary tumorigenesis in vivo. The heterozygous Aip mutation conferred complete penetrance of pituitary adenomas that were mostly GH-secreting, rendering the phenotype of the Aip mouse similar to that of PAP patients. We suggest that AIP may function as a candidate gatekeeper gene in somatotrophs. To clarify molecular mechanisms of tumorigenesis, we elucidated the expression of AIP-related molecules in human and mouse pituitary tumors. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT) was reduced in mouse Aip-deficient adenomas, and similar ARNT reduction was also evident in human AIP mutation positive adenomas. This suggests that in addition to participating in the hypoxia pathway, estrogen receptor signaling and xenobiotic response pathways, ARNT may play a role in AIP-related tumorigenesis. We also studied the characteristics and the response to therapy of PAP patients and found them to have an aggressive disease phenotype with young age at onset. Therefore, improvement in treatment outcomes of PAP patients would require their efficient identification and earlier diagnosis of the pituitary adenomas. The possible role of the RET proto-oncogene in tumorigenesis of familial AIP mutation negative pituitary adenomas was evaluated, but none of the found RET germline variants were considered pathogenic. Surprisingly, RET immunohistochemistry suggested possible underexpression of RET in AIP mutation positive pituitary adenomas an observation that merits further investigation.

Frequency of multiple endocrine neoplasia type 1 in a group of patients with pituitary adenoma: genetic study and familial screening

The purpose of this study it was to evaluate the frequency of Multiple Endocrine Neoplasia type 1 (MEN1) in patients with pituitary adenoma and to perform genetic analysis and familial screening of those individuals afflicted with MEN1. 144 patients with pituitary adenoma at Botucatu Medical School, UNESP-Univ Estadual Paulista, were assessed retrospectively for MEN1 during the years of 2005-2011. The patients were evaluated for the presence of primary hyperparathyroidism (PHP) and enteropancreatic tumors. Genetic analysis was performed for the individuals with clinically diagnosed MEN1. Thirteen patients met the diagnostic criteria for MEN1, but three individuals belong to the same family and they were considered as a single MEN1 event, revealing 7.7 % frequency of MEN1 in this patient group. Genetic analysis showed MEN1 mutations in four index cases: IVS4+1 G>A, IVS3-6 C>T, c.1547insC and a new D180A mutation. One patient did not agree to participate in the genetic study and another one was referred for follow up in other hospital. Only polymorphisms were found in the other individuals, one of which was novel. We identified a high frequency of MEN1 in pituitary adenoma patients. Since PHP is one of the most common MEN1 tumor and patients are mostly asymptomatic, we suggest that all pituitary adenoma patients have their calcium profile analyzed. © 2013 Springer Science+Business Media New York.

Endoscopic endonasal approach for pituitary adenoma: surgical complications in 301 patients

The authors investigate the complications of transnasal transsphenoidal endoscopic surgery in the treatment of 301 patients with pituitary adenomas. A retrospective analysis of complications in 301 patients submitted to transsphenoidal transnasal endoscopic surgery at the General Hospital of Fortaleza, Brazil between January 1998 and December 2009. The complications were divided in two groups: anatomical (oronasofacial, sphenoid sinus, intrasellar, suprasellar and parasellar) and endocrinological complications (anterior and posterior pituitary dysfunctions). We observed a total of 81 complications (26.9%) in our series. Anatomical complications occurred in 8.97% (27 cases): 8 CSF postoperative leaks (2.6%), 6 cases (1.9%) of delayed nasal bleeding, 5 cases (1.6%) of sphenoidal sinusitis, 3 cases (0.9%) of carotid artery lesion, 2 cases of meningitis (0.6%) and one case (0.3%) of each of the uncommon following complications: intrasella-suprasella hematoma, pontine hematoma and chiasmaplexy. Endocrinological complications occurred in 17.9% (54 cases): additional postoperative anterior lobe insufficiency in 35 cases (11.6%), and postoperative diabetes insipidus in 19 cases (6.3%). In our series, 3 cases of deaths (not directly related to the procedure) were also observed. Endoscopic transsphenoidal surgery represents an effective option for the treatment of patients with pituitary tumor. Complications still occur and must be reduced as much as possible. Successful endoscopic pituitary surgery requires extensive training in the use of an endoscope and careful planning of the surgery. Additional improvement can be expected with greater experience and new technical developments.

Somatotroph pituitary adenoma with acromegaly and autosomal dominant polycystic kidney disease: SSTR5 polymorphism and PKD1 mutation

A 39-year-old woman with autosomal dominant polycystic kidney disease (ADPKD) presented with acromegaly and a pituitary macroadenoma. There was a family history of this renal disorder. She had undergone surgery for pituitary adenoma 6 years prior. Physical examination disclosed bitemporal hemianopsia and elevation of both basal growth hormone (GH) 106 ng/mL (normal 0-5) and insulin-like growth factor (IGF-1) 811 ng/mL (normal 48-255) blood levels. A magnetic resonance imaging scan disclosed a 3.0 cm sellar and suprasellar mass with both optic chiasm compression and left cavernous sinus invasion. Pathologic, cytogenetic, molecular and in silico analysis was undertaken. Histologic, immunohistochemical and ultrastructural studies of the lesion disclosed a sparsely granulated somatotroph adenoma. Standard chromosome analysis on the blood sample showed no abnormality. Sequence analysis of the coding regions of PKD1 and PKD2 employing DNA from both peripheral leukocytes and the tumor revealed the most common PKD1 mutation, 5014_5015delAG. Analysis of the entire SSTR5 gene disclosed the variant c.142C > A (p.L48M, rs4988483) in the heterozygous state in both blood and tumor, while no pathogenic mutations were noted in the MEN1, AIP, p27Kip1 and SSTR2 genes. To our knowledge, this is the fourth reported case of a GH-producing pituitary adenoma associated with ADPKD, but the first subjected to extensive morphological, ultrastructural, cytogenetic and molecular studies. The physical proximity of the PKD1 and SSTR5 genes on chromosome 16 suggests a causal relationship between ADPKD and somatotroph adenoma.

Selective inferior petrosal sinus sampling without venous outflow diversion in the detection of a pituitary adenoma in Cushing's syndrome

Conventional MRI may still be an inaccurate method for the non-invasive detection of a microadenoma in adrenocorticotropin (ACTH)-dependent Cushing's syndrome (CS). Bilateral inferior petrosal sinus sampling (BIPSS) with ovine corticotropin-releasing hormone (oCRH) stimulation is an invasive, but accurate, intervention in the diagnostic armamentarium surrounding CS. Until now, there is a continuous controversial debate regarding lateralization data in detecting a microadenoma. Using BIPSS, we evaluated whether a highly selective placement of microcatheters without diversion of venous outflow might improve detection of pituitary microadenoma.

Glucocorticoid Replacement and Mortality in Patients with Nonfunctioning Pituitary Adenoma

Current treatment guidelines generally suggest using lower and weight-adjusted glucocorticoid replacement doses in patients with insufficiency of the hypothalamic-pituitary-adrenal (HPA) axis. Although data in patients with acromegaly revealed a positive association between glucocorticoid dose and mortality, no comparable results exist in patients with nonfunctioning pituitary adenomas (NFPA).

Transient MR changes and symptomatic epilepsy following gamma knife treatment of a residual GH-secreting pituitary adenoma in the cavernous sinus

To report a rare side effect of gamma knife treatment of pituitary macroadenoma.

Peptide receptor radionuclide therapy in a patient with disabling non-functioning pituitary adenoma

Non-functioning pituitary adenoma (NFPA) with higher proliferation index (WHO II) are often a therapeutical challenge. Low somatostatin receptor expression in these tumors usually prevents a treatment with somatostatin analogs. In 1996, a 55-year-old patient was referred due to right-sided headache. A pituitary macroadenoma with infiltration into the right cavernous sinus was diagnosed. There was no visual field deficit and the clinical and biochemical work up was consistent with a NFPA. The patient underwent transsphenoidal surgery. Residual adenoma remained in the right cavernous sinus. Histologically, a null-cell adenoma with a high proliferation index was documented (MIB-1: 11.6 %, WHO II). Somatostatin receptor autoradiography was performed in the surgical specimen showing a homogenous expression of sst2 receptors. Radiosurgery was completed with stable disease for 8 years. In 2004, the patient was diagnosed with an incomplete palsy of the right oculomotorius nerve and a significant increase in the volume of the adenoma in the right cavernous sinus. After a positive Octreoscan(®) the patient consented to an experimental therapy approach using Lutetium DOTATOC (3 × 200 mCi). The palsy of the oculomotorius nerve improved and remained stable until today (March 2013), the follow-up MRI scans demonstrated stable disease. This is the first case of a patient with a NFPA (WHO II) in whom PRRT successfully improved the local complications of the tumor for more than 8 years after ineffective surgery and gamma knife therapy. The determination of sst2 in vitro using autoradiography and in vivo by Octreoscan was instrumental to administer this therapy in a challenging situation.

Association of secondary glioblastoma and pituitary adenoma : case report

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Sellar collision tumor involving pituitary gonadotroph adenoma and chondroma: a potential clinical diagnosis

We report on a 74-year-old male patient who presented with progressive neuroophthalmologic symptoms soon after the administration of a long-acting gonadotropin-releasing hormone agonist for treatment of a prostate cancer. Imaging revealed a destructively growing and extensively calcified sellar mass inconsistent with a pituitary adenoma. A transseptal transsphenoidal tumor mass reduction yielded a histological diagnosis of a collision tumor comprised of a gonadotroph adenoma intermingled with osteochondroma. We discuss a potential causal relationship between the administration of the long-acting gonadotropin-releasing hormone agonist and the sudden appearance of the previously unsuspected sellar lesion. Although the association of these two tumors is very likely coincidental, the possibility of causal relationship is addressed.

Genome-wide analysis in a murine Dnmt1 knockdown model identifies epigenetically silenced genes in primary human pituitary tumors

DNA methylation at promoter CpG islands (CGI) is an epigenetic modification associated with inappropriate gene silencing in multiple tumor types. In the absence of a human pituitary tumor cell line, small interfering RNA-mediated knockdown of the maintenance methyltransferase DNA methyltransferase (cytosine 5)-1 (Dnmt1) was used in the murine pituitary adenoma cell line AtT-20. Sustained knockdown induced reexpression of the fully methylated and normally imprinted gene neuronatin (Nnat) in a time-dependent manner. Combined bisulfite restriction analysis (COBRA) revealed that reexpression of Nnat was associated with partial CGI demethylation, which was also observed at the H19 differentially methylated region. Subsequent genome-wide microarray analysis identified 91 genes that were significantly differentially expressed in Dnmt1 knockdown cells (10% false discovery rate). The analysis showed that genes associated with the induction of apoptosis, signal transduction, and developmental processes were significantly overrepresented in this list (P < 0.05). Following validation by reverse transcription-PCR and detection of inappropriate CGI methylation by COBRA, four genes (ICAM1, NNAT, RUNX1, and S100A10) were analyzed in primary human pituitary tumors, each displaying significantly reduced mRNA levels relative to normal pituitary (P < 0.05). For two of these genes, NNAT and S100A10, decreased expression was associated with increased promoter CGI methylation. Induced expression of Nnat in stable transfected AtT-20 cells inhibited cell proliferation. To our knowledge, this is the first report of array-based "epigenetic unmasking" in combination with Dnmt1 knockdown and reveals the potential of this strategy toward identifying genes silenced by epigenetic mechanisms across species boundaries.