DNA Methylation of Tumor Suppressor Genes in Pituitary Neuroendocrine Tumors.

CONTEXT: Epigenetic alterations may play a role in the development and behavior of pituitary neuroendocrine tumors (PitNETs). OBJECTIVE: To evaluate the effect of methylation of tumor suppressor genes (TSGs) on their gene expression and on the behavior of PitNETs. MATERIAL AND METHODS: We used methylation-specific multiplex ligation-dependent probe amplification and quantitative real-time PCR techniques to analyze the DNA-promoter hypermethylation and gene expression of 35 TSGs in 105 PitNETs. We defined functionality, size, and invasiveness of tumors according to their clinical manifestations, Hardy's classification, and MRI invasiveness of the cavernous sinus, respectively. RESULTS: We observed different methylation patterns among PitNET subtypes. The methylation status of TP73 correlated negatively with its gene expression in the overall series (P = 0.013) and in some subtypes. MSH6 and CADM1 showed higher methylation frequency in macroadenomas than in microadenomas in the overall series and in corticotroph PitNETs (all P ≤ 0.053). ESR1 and RASSF1 were more highly methylated in noninvasive than in invasive tumors in the overall series (P = 0.054 and P = 0.031, respectively) and in the gonadotroph subtype (P = 0.055 and P = 0.050, respectively). ESR1 and CASP8 appeared more hypermethylated in functioning than in silent corticotroph tumors (P = 0.034 and P = 0.034, respectively). CONCLUSIONS: DNA methylation of TSGs has a selective effect on their gene expression and on the growth and invasiveness of PitNETs. Its involvement in their functionality is biased because all silent operated tumors are macroadenomas, whereas all operated microadenomas are functioning ones. Therefore, the subtypes of PitNETs should be considered different entities.

Contribution of molecular analysis to the typification of the non-functioning pituitary adenomas.

AIM: The WHO Classification of Tumours of Endocrine Organs considers the inmunohistochemical characterization of pituitary adenomas (PA) as mandatory for patient diagnosis. Recent advances in the knowledge of the molecular patterns of these tumours could complement this classification with gene expression profiling. METHODS: Within the context of the Spanish Molecular Registry of Pituitary Adenomas (REMAH), a multicentre clinical-basic research project, we analysed the molecular phenotype of 142 PAs with complete IHC and clinical information. Gene expression levels of all pituitary hormones, type 1 corticotrophin-releasing hormone receptor, dopamine receptors and arginine vasopressin receptor 1b were measured by quantitative real-time polymerase chain reaction. In addition, we used three housekeeping genes for normalization and a pool of nine healthy pituitary glands from autopsies as calibration reference standard. RESULTS: Based on the clinically functioning PA (FPA: somatotroph, corticotroph, thyrotroph and lactotroph adenomas), we established the interquartile range of relative expression for all genes studied in each PA subtype. That allowed molecularly the different PA subtypes, including the clinically non-functioning PA (NFPA). Afterwards, we estimated the concordance of the molecular and immunohistochemical classification with clinical diagnosis in FPA and between them in NFPA. The kappa values were higher in molecular than in immunohistochemical classification in FPA and showed a bad concordance in all NFPA subtypes. CONCLUSIONS: According to these results, the molecular characterization of the PA complements the IHC analysis, allowing a better typification of the NFPA.

The Molecular Registry of Pituitary Adenomas (REMAH): A bet of Spanish Endocrinology for the future of individualized medicine and translational research. / El Registro Molecular de Adenomas Hipofisarios (REMAH): una apuesta de futuro de la Endocrinología española por la medicina individualizada y la investigación traslacional.

Pituitary adenomas are uncommon, difficult to diagnose tumors whose heterogeneity and low incidence complicate large-scale studies. The Molecular Registry of Pituitary Adenomas (REMAH) was promoted by the Andalusian Society of Endocrinology and Nutrition (SAEN) in 2008 as a cooperative clinical-basic multicenter strategy aimed at improving diagnosis and treatment of pituitary adenomas by combining clinical, pathological, and molecular information. In 2010, the Spanish Society of Endocrinology and Nutrition (SEEN) extended this project to national level and established 6 nodes with common protocols and methods for sample and clinical data collection, molecular analysis, and data recording in a common registry (www.remahnacional.com). The registry combines clinical data with molecular phenotyping of the resected pituitary adenoma using quantitative real-time PCR of expression of 26 genes: Pituitary hormones (GH-PRL-LH-FSH-PRL-ACTH-CGA), receptors (somatostatin, dopamine, GHRH, GnRH, CRH, arginine-vasopressin, ghrelin), other markers (Ki67, PTTG1), and control genes. Until 2015, molecular information has been collected from 704 adenomas, out of 1179 patients registered. This strategy allows for comparative and relational analysis between the molecular profile of the different types of adenoma and the clinical phenotype of patients, which may provide a better understanding of the condition and potentially help in treatment selection. The REMAH is therefore a unique multicenter, interdisciplinary network founded on a shared database that provides a far-reaching translational approach for management of pituitary adenomas, and paves the way for the conduct of combined clinical-basic innovative studies on large patient samples.

Severe sleep apnea-hypopnea syndrome is related to left ventricle dysfunction and hypertrophy in acromegalic patients.

OBJECTIVE: To assess whether sleep apnea-hypopnea syndrome (SAHS) is a risk factor for development of acromegalic cardiomyopathy. METHODS: A descriptive, cross-sectional study of 32 patients with acromegaly (15 categorized as non-controlled-NCA and 17 as controlled-CA) compared to 20 matched controls (by sex, age, and BMI) referred to the pulmonology department for suspected SAHS. Polysomnography, echocardiography (M-mode, 2-dimensional, and Doppler), and 12-lead electrocardiography were performed in all participants. Development of cardiac morbidity (ischemia heart disease or heart failure) was evaluated after 7 years. RESULTS: SAHS was diagnosed in 81.3% of patients with acromegaly and 85% of controls. Mild SAHS was more common in CA than in NCA patients (31.3% vs. 0%, p=0.048). There was a trend to greater prevalence of left ventricular diastolic dysfunction (LVDD) in acromegalic patients as compared to controls (58.1% vs. 30%, p=0.05). Presence of severe SAHS in patients with acromegaly was related to greater risk of LVDD (90.9% vs. 40%, p=0.008; OR 2.3 [1.3-4.0]), LV hypertrophy (55.6% vs. 10.5%, p=0.02; OR 5.3 [1.3-22.2]), and cardiac events (87.5% vs. 35.6%; p=0.01; OR 7.53 [1.07-53.24]). CONCLUSION: SAHS is highly prevalent in patients with acromegaly. Only in these patients was severe SAHS associated to hypertrophy, LV diastolic dysfunction, and cardiac events.

Pituitary tumor transforming gene and insulin-like growth factor 1 receptor expression and immunohistochemical measurement of Ki-67 as potential prognostic markers of pituitary tumors aggressiveness.

INTRODUCTION AND OBJECTIVE: The ability to predict recurrence of pituitary adenoma (PA) after surgery may be helpful to determine follow-up frequency and the need for adjuvant treatment. The purpose of this study was to assess the prognostic capacity of pituitary tumor transforming gene (PTTG), insulin-like growth factor 1 receptor (IGF1R), and Ki-67. MATERIALS AND METHODS: In this retrospective study, the normalized copy number (NCN) of PTIG and IGF1R mRNA was measured using RT-PCR, and the Ki-67 index was measured by immunohistochemistry in 46 PA samples. Clinical data, histological subtype, and radiographic characteristics were collected to assess associations between variables and tumor behavior. Progression of tumor remnants and its association to markers was also studied in 14 patients with no adjuvant treatment after surgery followed up for 46±36 months. RESULTS: Extrasellar tumors had a lower PTTG expression as compared to sellar tumors (0.065 [1st-3rd quartile: 0.000-0.089] NCN vs. 0.135 [0.105-0.159] NCN, p=0.04). IGF1R expression changed depending on histological subtype (p=0.014), and was greater in tumor with remnant growth greater than 20% during follow-up (10.69±3.84 NCN vs. 5.44±3.55 NCN, p=0.014). CONCLUSIONS: Our results suggest that the IGF1R is a more helpful molecular marker than PTTG in PA management. Ki-67 showed no association to tumor behavior. However, the potential of these markers should be established in future studies with standardized methods and on larger samples.

Over-expression of vascular endothelial growth factor in pituitary adenomas is associated with extrasellar growth and recurrence.

Some pituitary adenomas (PA) demonstrate aggressive behavior with local invasion and recurrences. Angiogenesis is regarded as an essential step in the formation of solid tumors. The aim of this study is to find out whether angiogenic factors may have information about the aggressiveness of PA that could be useful in determining the frequency of follow-up and whether adjuvant therapy is necessary. In this retrospective descriptive study, we evaluated vascular endothelial growth factors (VEGF) and VEGF receptor (KDR) mRNA expression by RT-PCR analysis on 46 human PA samples. Clinical data, histological subtype and radiologic characteristics were studied to determine the associations between the variables and the pre-operative behavior of the tumor. In addition, we monitored 12 patients without adjuvant post-operative therapies over 46 months after surgery, determining progression of tumor remnants and its association with these markers. VEGF expression correlates with KDR expression (r = 0.40, p = 0.006). VEGF demonstrates different expression between histological subtypes (p = 0.036). The extension at magnetic resonance imaging showed that VEGF expression was related to suprasellar extension (p = 0.007), being expressed more on tumors with extrasellar growth than intrasellar ones (p = 0.008). Our results demonstrate a 27.5 times increased risk of extrasellar growth when VEGF expression exceeds 0.222 normalized copy number (NCN) (p = 0.002). Likewise, tumors with KDR greater than 0.750 NCN had less recurrence-free survival time (p = 0.032). Our results suggest that the expression of VEGF and its receptor could be a marker for poor outcome after partial tumor resection. These data should be considered in future studies evaluating angiogenic factors as therapeutic targets in patients with PA.

Association of mammalian target of rapamycin with aggressive type II endometrial carcinomas and poor outcome: a potential target treatment.

The classification of endometrial carcinoma divided into types I and II has shown clinical usefulness. Molecular alterations of PTEN and Wnt/ß-catenin have been identified in this neoplasia. However, the role of mammalian target of rapamycin according to subcellular localization in the pathogenesis of this neoplasia and its prognostic significance are not well defined. We studied the expression of phosphorylated mammalian target of rapamycin, PTEN, and ß-catenin and their relationship with clinicopathologic features, molecular factors (microsatellite instability, mismatch repair, and BRAF genes) and patients' survival in a series of 260 nonconsecutive endometrial carcinomas. Tissue microarrays were manually constructed, and genomic DNA was extracted from paraffin-embedded cylinders (1 mm thick) from preselected tumor areas. The mammalian target of rapamycin in the nuclei (mTORC2; 47%) or cytoplasm (mTORC1; 48%) were seen in type II endometrial carcinoma, the latter also in advanced stages (P ≤ .046). PTEN loss (58%) was detected in type I endometrial carcinoma of grade 1, at early stage, with mismatch repair gene loss (24.4%) and microsatellite instability-positive status (22%; P ≤ .05). Nuclear ß-catenin (16%) was found in type I tumors of younger patients (P ≤ .003). In contrast, BRAF-V600E mutations were not detected (0%). Mammalian target of rapamycin cytoplasmic high expression implied poorer prognosis (P = .02; Kaplan-Meier, log-rank test), but grade 3 tumors, vascular invasion, advanced stage, or PTEN presence correlated independently with a negative impact on survival (all P ≤ .036; Cox analysis). Our results show that mammalian target of rapamycin, PTEN, and ß-catenin are independently involved in different molecular subtypes of endometrial carcinoma with diverse patients' prognosis and support their distinctive treatment based on targeted drugs.

Increased insulin-like growth factor-1 receptor mRNA expression predicts poor survival in immunophenotypes of early breast carcinoma.

The biology of breast carcinoma shows a great variation, reflected by the recent classification of phenotypes based on DNA microarrays or immunohistochemistry. The aim of this study was to determine the prevalence of insulin-like growth factor-1 receptor (IGF1R) in breast carcinoma subtypes and the impact on the outcome. We studied 197 consecutive breast carcinoma patients in stage I-II treated conservatively. Phenotypes were assessed on the basis of the expressions of ER/PR, HER2, Ki67, p53, Bcl2, CK5/6 and EGFR. Moreover, IGF1R expression (α-subunit and ß-phosphorylated/active form) was evaluated by immunohistochemistry, IGF1R mRNA levels by quantitative RT-PCR and IGF1R mutations by direct DNA sequencing. Overall, 40% (78/197) of tumors were luminal A, 24% (48/197) luminal B, 19% (37/197) HER2-positive and 17% (34/197) basal/triple-negative. Luminal A tumors were predominantly of low grade, without necrosis, presenting in older patients as a ≤2-cm unilateral mass (all P ≤ 0.046). α-IGF1R overexpression was observed more frequently in luminal A (49%) cases, followed by luminal B (20%), HER2-positive area under the curve (22%) and basal/triple-negative cases (9%) (P = 0.01) with similar results for mRNA levels (53, 24, 13 and 10%, respectively) (P = 0.038), but without differences for mutations (P = NS). High IGF1R mRNA correlated with poor patient survival among subtypes (P = 0.004) (Kaplan-Meier; log-rank test). For overall survival, only histological grade and IGF1R mRNA emerged as significant predictors (P ≤ 0.034; Cox regression). Increased IGF1R mRNA implies poorer patient prognosis among the different subtypes, and that may be associated with the lack of responsiveness to tamoxifen in cases with a positive hormone receptor status. Our results highlight the biological and clinical relevance of IGF1R in early breast carcinoma subtypes, and provide knowledge to assist in treatment decision.

[Rol of pituitary tumour-transforming gene (PTTG) in the pituitary adenomas]. / Papel de pituitary tumour-transforming gene (PTTG) en los adenomas hipofisarios.

The pathogenesis of pituitary tumours is far to be understood. Pituitary transforming tumour gene (PTTG), a gen that induces aneuploidy, genetic instability, cellular proliferation and to stimulate angiogenesis, has been involved in neoplasic transformation and shown overexpressed in many neoplasm as lung, breast, endometrium, thyroid and colon malignant tumours. On the other hand, PTTG has been inconsistently studied in pituitary tumours. The majority of studies have been performed in animals and there is a great variability in the methods used in its determination. The goal of this review is to resume the role of PTTG in tumourogenesis and critically to revise the studies published in humans in order to advance in the knowledge of the pathogenesis of pituitary adenomas and to find clinical useful predictors of the behavior of these tumours.

Low activation of Insulin-like Growth Factor 1-Receptor (IGF1R) is associated with local recurrence in early breast carcinoma.

Background The predictive value of IGF1R on local recurrence in invasive breast carcinoma (BC) is not well known. Methods In a series of 197 lymph-node negative BC patients treated with breast-conserving surgery and radiation therapy, we performed immunohistochemistry for alpha-IGF1R, beta-IGF1R (phosphorylated/active form) and Estrogen/Progesterone receptors. We further evaluated the IGF1R mRNA expression by quantitative RT-PCR and IGF1R mutations by direct DNA sequencing (exons 19 and 21) in 85 primary BC (42 control cases, 31 with local recurrence and 12 with distant metastasis) and in 31 local recurrences. Unconditional logistic regression analyses were performed to identify risk factors for recurrence. Results Local recurrences were associated with high-grade tumors, PR-negative and low active-IGF1R, which emerged as independent breast relapse predictors by multivariate analysis. Conclusion Patients with early BC treated with lumpectomy and radiation who have low-grade tumors and favorable markers (increased content of active IGF1R and PR-positive) have a low risk of local recurrence. Therefore, do not benefit from a boost dose on the surgical scar.