Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciência da Saúde, Programa de Pós-Graduação em Ciência da Saúde, 2014. / Submitted by Larissa Stefane Vieira Rodrigues (larissarodrigues@bce.unb.br) on 2014-11-18T15:37:56Z No. of bitstreams: 1 2014_OlíviaLaquisDeMoraes.pdf: 2089917 bytes, checksum: 3784e6f7d4b3f8c2057d7a208522f5b9 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2014-11-26T18:28:47Z (GMT) No. of bitstreams: 1 2014_OlíviaLaquisDeMoraes.pdf: 2089917 bytes, checksum: 3784e6f7d4b3f8c2057d7a208522f5b9 (MD5) / Made available in DSpace on 2014-11-26T18:28:47Z (GMT). No. of bitstreams: 1 2014_OlíviaLaquisDeMoraes.pdf: 2089917 bytes, checksum: 3784e6f7d4b3f8c2057d7a208522f5b9 (MD5) / INTRODUÇÃO: Feocromocitomas (FEO) e paragangliomas (PGL) são tumores neuroendócrinos, originários de células chromafins, localizados na medula supra-renal e em tecidos extra-adrenais, respectivamente. FEO e PGL são geralmente benignos, com morbidade e mortalidade relacionadas com a produção de catecolaminas. Malignidade é relatada em aproximadamente 10%, dos casos. Estudos recentes têm mostrado que, pelo menos, 25% de todos os casos de FEO / PGL podem ter uma base genética. MÉTODOS: 17 pacientes com FEO / PGL foram incluídos. Os dados clínicos, bioquímicos e radiológicos foram obtidos dos prontuários médicos. Estudos histopatológicos e imuno-histoquímico para marcadores neuroendócrinos foram realizados. DNA genômico foi extraído e as regiões codificantes dos genes VHL, SDHB, SDHC SDHD, MAX foram amplificados e seqüenciados automaticamente. A técnica de MLPA foi utilizada para a triagem de grandes deleções / inserções. A análise do gene RET foi realizada em um paciente com evidência clínica de neoplasia endócrina múltipla do tipo 2A (MEN2A). O consentimento livre e esclarecido foi obtido de todos os pacientes. RESULTADOS: Este estudo é composto por 6 casos de PGL e 11 FEO, 5 pacientes tiveram história familiar postivia e 4 apresentaram malignidade. A análise Imunohistoquímica confirmou a origem de neuroendócrina de todos os tumores. A análise genética revelou a mutação (p.Q164R) no gene VHL em uma paciente do sexo feminino com FEO e hemangioblastoma cerebelar e sua filha assintomática. Uma mutação no gene RET foi encontrada (p.C618R), em uma paciente do sexo feminino com FEO e carcinoma medular da tiróide (CMT; NEM2A) e seus 2 filhas com CMT. Uma grande dleção do exon 1 do gene SDHB foi encontrada em quatro pacientes: Duas irmãs com PGL paraaortico, e dois casos aparentemente esporádicos apresentando PGL. DISCUSSÃO: O diagnóstico molecular feito pelo seqüenciamento de Sanger juntamente com o MLPA constitui uma estratégia adequada para procurar tanto mutações pontuais quanto grandes deleções. Associações genótipo-fenótipo para VHL e RET são bem descritos, mas os efeitos fisiopatológicos das grandes deleções no SDHB ainda não estão bem elucidados. Correlações fenotípicas ainda não foram bem caracterizados já que grandes deleções no SDHB tem uma apresentação muito variável. O SDHB pode atuar como um gene supressor tumoral, e uma grande deleção pode conduzir a um fenótipo tumoral. Estudos genéticos em pacientes com FEO e PGL são recomendados, uma vez que as mutações podem também ser encontrados em casos aparentemente esporádicos, e que um resultado positivo pode influenciar a monitorização clínica e o aconselhamento genético dos pacientes e seus descendentes. ___________________________________________________________________________ ABSTRACT / INTRODUCTION: Pheochromocytomas (PCC) and Paragangliomas (PGL) are neuroendocrine tumors originating from chromafin cells, located in the adrenal medulla and extra-adrenal tissue, respectively. PCC and PGL are usually benign, with morbidity and mortality related to the production of catecholamines. Malignancy is reported in approximately 10%; of cases. Recent studies have shown that at least 25%; of all PCC/PGL cases may have a genetic basis. METHODS: 17 patients with PCC/PGL were included. Clinical, biochemical and radiological data were obtained from medical records. Histopathological and immunohistochemical studies for neuroendocrine markers were performed. Genomic DNA was extracted and the coding regions of VHL, SDHB, SDHC SDHD, MAX were amplified and automatically sequenced. Multiplex ligand-probe amplification (MLPA) was used for screening large deletions/insertions. RET gene analysis was also performed in one patient with clinical evidence of multiple endocrine neoplasia type 2A (MEN2A). Genuine consent was obtained from all. RESULTS: Our series consisted of 6 PGLs and 11 PCCs patients; 5 had familial history and 4 were malignant. Immunohistochemistry confirmed the neuroendocrine origin of all tumors. Genetic analysis revealed the (p.Q164R) VHL mutation in a woman with PCC and cerebellar hemangioblastoma and her asymptomatic daughter. A RET mutation was found (p.C618R) in a woman with PCC and thyroid medullary carcinoma (TMCa; MEN2A) and her 2 daughters with TMCa. A large deletion of SDHB exon 1 was found in 4 patients: Two sisters with paraortic PGL, and two apparently sporadic cases presenting with PGL. DISCUSSION: Molecular diagnosis done by Sanger’s sequencing combined with MLPA constitute an adequate strategy to search for both point mutations and large deletions. Genotype-phenotype associations for VHL and RET are well described, but the pathophysiological effects of large deletions on SDHB are still unclear. No phenotype correlations have been characterized for large SDHB deletions due to a highly variable presentation. SDHB may act as a tumor suppressor, and a large deletion may lead to a tumor phenotype. Rational recommendations for genetic studies in PCCs and PGLs are in progress given that mutations may also be found in apparently sporadic cases, and that a positive result might influence clinical monitoring and genetic counseling of patients and their offspring.

Pheochromocytomas and paragangliomas are neuroendocrine tumors that arise from neural crest-derived cells of the adrenal medulla and the extra-adrenal paraganglia. They cause hypertension due to an abnormally high production of catecholamines (mainly adrenaline and noradrenaline), with symptoms including recurrent episodes of headache, palpitations and sweating, and an increased risk of cardiovascular disease. Malignancy in the form of distant metastases occurs in 10-15% of the patients. The malignant cases are difficult to predict and cure, and have a poor prognosis. About a third of pheochromocytomas and paragangliomas are caused by hereditary mutations in a growing list of known susceptibility genes. However, the cause of the remaining, sporadic, tumors is still largely unknown. The aim of this thesis project has been to further characterize the genetic background of pheochromocytomas and paragangliomas, with a focus on the sporadic tumors. First, we investigated the role of the genes known from the familial tumors in the sporadic form of the disease. By studying mutations, copy number variations, DNA methylation and gene expression, we found that many of the known susceptibility genes harbor somatic alterations in sporadic pheochromocytomas. Particularly, we found that the NF1 gene, which plays an important role in suppressing cell growth and proliferation by regulating the RASMAPK pathway, was inactivated by mutations in more than 20% of the cases. The mutations occurred together with deletions of the normal allele and were associated with a reduced NF1 gene expression and a specific hormone profile. We also detected activating mutations in the gene EPAS1, which encodes HIF-2α, in a subset of sporadic cases. Microarray analysis of gene expression showed that several genes involved in angiogenesis and cell metabolism were upregulated in EPAS1-mutated tumors, which is in agreement with the role of HIF-2α in the cellular response to hypoxia. In order to comprehensively investigate all the known susceptibility genes in a larger patient cohort, we designed a targeted next-generation sequencing approach and could conclude that it was fast and cost-efficient for genetic testing of pheochromocytomas and paragangliomas. The results showed that about 40% of the sporadic cases had mutations in the tested genes. The majority of the mutations were somatic, but some apparently sporadic cases in fact carried germline mutations. Such knowledge of the genetic background can be of importance to facilitate early detection and correct treatment of pheochromocytomas, paragangliomas and potential co-occurring cancers, and also to identify relatives that might be at risk. By sequencing all the coding regions of the genome, the exome, we then identified recurrent activating mutations in a novel gene, in which mutations have previously only been reported in subgroups of brain tumors. The identified mutations are proposed to cause constitutive activation of the encoded receptor tyrosine kinase, resulting in the activation of downstream kinase signaling pathways that promote cell growth and proliferation. In summary, the studies increase our biological understanding of pheochromocytoma and paraganglioma, and possibly also co-occurring cancers in which the same genes and pathways are involved. Together with the findings of other scientific studies, our results may contribute to the development of future treatment options.

Catecholamines are stress hormones that are produced and released by a rare tumor called pheochromocytoma. This tumor can cause hypertension which if undiagnosed and untreated leads to death. Since good therapy is available, it is important to find the tumor in time. The most common way to diagnose the tumor is measurement of the biochemical markers; catecholamines and their metabolites, methylated catecholamines. After observation that almost all normetanephrine results for women were higher than the upper reference limit and therefore pathological, the accuracy of the present reference intervals was questioned. Therefore new reference intervals for both urinary catecholamines and methylated catecholamines were developed by analysis of 46 samples using HPLC. Creatinine was analysed in acidified urine in order to see if the results became the same as when analysed in non-acidified urine. Urinary catecholamines and methylated catecholamines were analysed using HPLC. Comparison between measurement of creatinine in acidified urine and non-acidified urine with an enzymatic method was performed using Architect ci 8200, Abbott. As suspected, there was a difference between the present and new intervals. Therefore the new intervals will be used for future diagnosis. There was no difference between the two treatments of creatinine samples wherefore it can be measured in both.In conclusion reference intervals determind in this study will be used and it was shown that creatinine can be measured in acidified urine.

Ectopic thyroid gland is a developmental anomaly that results from the arrest of thyroid tissue along its path of descent from the floor of mouth to the pre tracheal position in the lower neck. It is typically found along the thyroglossal duct with the base of the tongue being the most common site. Apart from mediastinal extension of goiter, the incidence of true intrathoracic ectopic thyroid tissue is rare. Presence of ectopic thyroid has been reported not only in the chest but also in the abdomen and pelvis. Pericardial and intracardiac locations are extremely uncommon and right ventricle location is predominant among the described cases. We describe a case of incidentally detected ectopic thyroid tissue in a rarer location—adjacent to the left atrium. The patient, who had undergone a nephrectomy for renal oncocytoma 5 years ago, presented with unintentional weight loss and left sided flank pain, prompting a workup to rule out abdominal malignancy. Findings on the computed tomography (CT) scan of the abdomen and pelvis prompted further investigation including a chest CT which showed a heterogeneously enhancing mass near the left atrium. Given its location, further radiological investigations played an important role in eliminating the differential diagnosis of paraganglioma. The mass was surgically resected and discovered to be a hyperplastic thyroid nodule on histologic examination.

Urinary bladder paraganglioma (UBP) is a rare neuroendocrine neoplasm. It accounts for less than 1% of urinary bladder tumors and less than 6% among all types of paragangliomas. More commonly, UBP occurs in the female population aged 20-40 years old. UBP is classified into functional and non-functional types, and the majority is functional, leading to symptoms and signs of excess catecholamine, including hypertension, palpitation, syncope, and headache. Non-functional UBP comprises about 15% of UBPs and lacks the excess secretion of catecholamine, which often leads to misdiagnosis as urothelial cancer due to its rarity and nonspecific symptoms - increased urinary frequency/urgency and painless gross hematuria. Here, we present a rare case of a non-functional UBP. A 65-year-old male with BPH presented to ER with a 6-month history of urinary retention. He also was experiencing intermittent hematuria and dysuria during this time but otherwise remained asymptomatic without headache, dyspnea, wheezing, or diarrhea. Physical exam showed normal BP and no suprapubic tenderness on palpation. UA showed gross hematuria. Subsequent cystoscopy showed thickening of the bladder dome and an 8 mm lesion. Transurethral resection of bladder tumor (TURBT) was performed, and pathology showed 1 cm tumor confined to submucosa with questionable margins. Chromogranin, synaptophysin, CD56, GATA3, CD10 were stained positive; cytokeratin AE1/AE3, cytokeratin 34betaE12, SOX10, S100, and calretinin were negative. From morphology and immunochemistry, the diagnosis of UBP was made. Free metanephrine, plasma normetanephrine, 24-hour urine metanephrine and normetanephrine levels were not elevated. Post-TURBT MRI abdomen showed no other suspicious lesions. A wide re-resection of the bladder dome was performed due to the questionable margins from the initial surgery, and pathology showed benign bladder tissue with unremarkable immunostains, indicating no overt features of residual paraganglioma. Due to its paucity and uncertain biological behavior, the prognosis of UBP is not well established. While most UBP are benign, 10-15% of cases are malignant. High expression of VEGF and or abnormal vessel architecture in the tumor cells raise suspicion of malignancy. However, typically, definitive evidence of malignancy in paraganglioma is its invasion of adjacent organs or distant metastasis. The local recurrence rate ranges from 5-15%, thus necessitating long-term surveillance for 10-years. Systemic chemotherapy, including cyclophosphamide, vincristine, dacarbazine (CVD), or temozolomide, is necessary for distant metastatic or symptomatic disease. Iobenguane I-131 or Lutathera can be utilized with positive MIBG or 68Ga DOTATATE scan, respectively. Otherwise, surgical extirpation remains the choice of curative intent, and a multidisciplinary approach consisting of urologists, medical/radiation oncologists, and endocrinologists would be warranted for this rare entity of disease.

Los paragangliomas (PGL) de cabeza y cuello son tumores poco frecuentes que pueden ser esporádicos o familiares. En los PGL familiares hasta el momento se han identificado 4 locus cromosómicos relacionados con la enfermedad: 11q23 (PGL1), 11q13 (PGL2), 1q21 (PGL3) y 1p36.1p35 (PGL4). No obstante, el hecho de tener la mutación no significa necesariamente desarrollar la enfermedad, ya que su penetrancia es del 70% a los 50 años de edad. Por otra parte, los pacientes con PGL deben ser examinados genéticamente con el fin de determinar si la enfermedad es hereditaria y en ese caso extender a sus familiares el estudio genético, lo que conlleva un coste importante. La presente tesis trata de arrojar luz sobre las causas por las que en portadores de la mutación se desarrolla o no la enfermedad, así como las bases clínicas para realizar en primer lugar el estudio del gen más probablemente mutado, lo que ahorraría costes en el estudio de un paciente al evitar el análisis de los genes menos probable Con la primera finalidad, se realizó en primer lugar un cribaje de las alteraciones cromosómicas mediante la técnica de microarray-CGH, estudiándose además las mutaciones en los genes SDHB, SDHC, SDHD, RET Y VHL en pacientes que presentaban PGL de cabeza y cuello. Una vez realizado el análisis mutacional de todos los genes implicados en los PGLs familiares y establecida la correlación con los datos clínicos, se estudió la secuencia ideal de análisis de los distintos genes con la finalidad de estudiar primero aquel gen que tuviera probabilidad de estar mutado de acuerdo con las características clínicas.Del total de 72 pacientes estudiados, únicamente se objetivó mutación germinal patogénica en los genes SDHB o SDHD en 26 (36%) casos: 14 casos con mutación en el gen SDHB, y 12 en el SDHD. 14 de estos paciente con mutación germinal eran casos familiares y 12 supuestamente esporádicos ("familiares ocultos") con mutación en los genes SDH, lo que contribuyó a conocer el carácter hereditario de la enfermedad. Se observaron cuatro mutaciones no descritas previamente en la literatura, y por el contrario no se objetivaron mutaciones o delecciones en exones de los genes SDHC, VHL o RET. Estos datos sugieren que los tumores múltiples y con historia familiar positiva son altamente predictivos para mutaciones en el gen SDHD. De igual modo, los tumores únicos y malignos se asocian con mutaciones en el gen SDHB. En los pacientes analizados mediante microarrays-CGH se detectaron anomalías en el 45% de los casos, consistentes más frecuentemente en pérdidas cromosómicas (84%) que en ganancias. Los cromosomas que presentaban pérdidas o delecciones con mayor frecuencia eran el 1, el 11, el 22 y el 21. Los tumores que presentaban mutación germinal en los genes SDH tenían más alteraciones cromosómicas. La metodología utilizada en la realización del trabajo es adecuada desde la perspectiva del rigor científico, tanto en lo que se refiere a los procedimientos de laboratorio (microarrays-CGH, estudio de las mutaciones, etc.), el tamaño muestral (muy grande dada la rareza de la enfermedad) y el análisis estadístico de los resultados, incluyendo las correlaciones clínico-patológicas. Así, se comprobó la utilidad de los parámetros clínicos (edad <40 años, sexo varón, el hecho de presentar un feocromocitoma o tumores múltiples o presentar antecedentes familiares) para ayudar a predecir las mutaciones y su utilidad para priorizar el análisis de las mismas en los genes SDH y reducir los costes en el estudio de los pacientes con PGL.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Thesis (Ph.D.)--Georgetown University, 2009. / Includes bibliographical references.

Thesis (doctoral)--Katholieke Universiteit te Nijmegen.

No description available.