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Orientador: Antonio Guilherme Borges Neto, Ricardo Ramina / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T17:37:15Z (GMT). No. of bitstreams: 1 Cardoso_ArquimedesCavalcante_D.pdf: 2722209 bytes, checksum: 68aae3facfed4e8f5338b24a3994aadc (MD5) Previous issue date: 2007 / Resumo: O autor apresenta um estudo descritivo-retrospectivo, envolvendo 240 pacientes portadores de Schwannoma do Vestibular (SV) que foram operados em decúbito dorsal (posição de mastóide), monitorizados com doppler transtorácico, utilizando-se o acesso retrossigmóide transmeatal. A função do VII e VIII nervos cranianos foi monitorizada durante a cirurgia e a reconstrução da abertura do conduto auditivo interno (CAI) foi realizada com retalho vascularizado de duramáter, músculo e cola de fibrina. A exérese foi completa em 99% dos casos, com mortalidade de 1,6%. Houve preservação da função do nervo facial em 85% dos casos e da audição em 40% dos pacientes, com audição prévia e tumores menores de 1,5 cm. A incidência de fístula liquórica foi de 5,8% e meningite de 2,9%. Embolia gasosa foi registrada em 3% dos casos, não associada à mortalidade. O tratamento cirúrgico dos SV utilizando-se a posição de mastóide e o acesso retrossigmóide transmeatal tem várias vantagens, com baixa morbidade e mortalidade, permitindo elevadas taxas de remoção radical, com a cura do paciente. O estudo é comparado com outras séries cirúrgicas publicadas de grupos com grande experiência na cirurgia de SV, bem como em relação aos resultados de pacientes de outros grupos que foram submetidos a tratamento radioterápico. / Abstract: To evaluate the result of the surgical treatment of Vestibular Schwannoma (VS) operated in dorsal decubitus (mastoid position). Material and methods: 240 patients with VS underwent a retrosigmoid craniotomy for tumor resection in dorsal decubitus (mastoid position). The function of 7 and 8 cranial nerves was monitored during surgery and the opened internal auditory canal (ICA) was reconstructed using a vascularized dura flap, muscle and fibrin glue. Results: Complete tumor removal was achieved in 99% of the cases, with a mortality of 1.6%. The facial nerve function was preserved in 85% of cases and hearing in 40% of the patients (with preoperative hearing) with tumors of up 1, 5 cm in diameter. The incidence of cerebrospinal fluid leak was 5.8% and meningitis 2.9%. Venous air embolism was registered in 3% of cases; it was not associated to mortality. Conclusion: Surgical removal of VS in dorsal position has several advantages; the morbidity and mortality are very low. / Doutorado / Neurologia / Doutor em Ciências Médicas

In retrospektiv angelegten Untersuchungen soll der Einfluss unterschiedlicher Operationsstrategien auf die Morbidität bei der Behandlung des Akustikusneurinoms aufgezeigt werden. In einer ersten Untersuchung wurde der Einfluss eines doppelten Verschlusses der Dura mater nach subokzipitaler Kraniotomie zur mikrochirurgischen Exstirpation eines Akustikusneurinoms im Hinblick auf die Vermeidung einer Liquorfistel analysiert. Hier wurden zwei Patientengruppen miteinander verglichen. Zum einen Patienten mit einem einfachen Duraverschluss, Duranaht plus alloplastisches Material epidural. Zum anderen Patienten mit einem doppelten Duraverschluss. Hier epidurales und subdurales alloplastisches Material plus Duranaht. Untersucht wurden das Auftreten von Liquorfisteln sowie die Häufigkeit von Wundheilungsstörungen. Es konnte keine signifikante Überlegenheit einer Operationstechnik zur Vermeidung von postoperativen Liquorfisteln gezeigt werden. In einer zweiten Untersuchung wurde der Zusammenhang einer inkompletten Resektion des Tumors (Belassen eines Kapselrestes) mit dem Ziel der Schonung des Nervus facialis untersucht. Hierzu wurden die postoperative Facialisfunktion sowie ein möglicherweise erhöhtes Rezidivrisiko im Verlauf betrachtet. Die postoperative Funktion des N. facialis wurde zwischen den Patientengruppen mit Kapselrest (inkomplette Entfernung) und ohne Kapselrest (komplette Entfernung) verglichen. Es zeigte sich bei vergleichbarer postoperativer Facialisfunktion kein signifikanter Unterschied im Auftreten von Rezidiven nach inkompletter Resektion. Nur gut ein Drittel der inkomplett resezierten Tumore zeigten im Nachbeobachtungszeitraum (im Mittel 52 Monate) überhaupt ein Wachstum.

Some patients suffer hearing loss in the early postoperative period following vestibular schwannoma (VS) excision despite having intact hearing immediately after surgery. As this phenomenon has rarely been documented or described, the putative mechanism remains vague. The objective of the current study was to document the patterns of change in behavioural and electrophysiological responses in patients following VS surgery to better describe the phenomenon of delayed hearing loss. In particular, we aimed to determine whether the impairment that eventually leads to delayed hearing loss is neural or cochlear in origin. Auditory function was monitored in six adult patients who underwent surgery at Christchurch Public Hospital for excision of unilateral vestibular schwannoma through the retrosigmoid approach. Patients were assessed pre- and postoperatively by puretone audiometry, speech audiometry, tympanometry, distortion product otoacoustic emissions (DPOAEs), and auditory brainstem response (ABR). When measurable hearing was demonstrated postoperatively, pure-tone audiometry, speech audiometry and ABR were assessed at 24 hour intervals following surgery. Transtympanic electrocochleography (ECochG) was carried out if wave I of the ABR was lost during the postoperative period. Postoperative monitoring revealed that 4 patients suffered permanent anacusis and the remaining 2 patients had permanent hearing preservation. There were no patients who experienced delayed hearing loss in the early postoperative period. A phenomenon similar to delayed hearing loss was observed in case 2 who demonstrated loss of ABR wave I on the 7th postoperative day. Postoperative ECochG recorded in this case showed an enhanced negative SP on the operated side. The findings of this study are discussed in detail with particular reference to the underlying pathophysiology.

Endogenous retroviruses (ERVs) are fossils of ancient retroviral infection in the germline. In primates they represent around 5% of the genome sequence. During time spent in the genome, being transmitted in a Mendelian fashion, copies of ERVs have accumulated mutations, which rendered them inactive. However, some of them (the most recently integrated ones) are still able to transcribe and produce viral proteins, although few are capable of re-infection. In the past often considered as unharmful 'junk DNA', recent evidence link ERVs with cancer and several inflammatory diseases. For example, a few reports demonstrate that ERVs are involved in tumour development using shRNA knock-down and over-expression systems, and their overexpression tends to correlate with inflammation status, generating the hypothesis that they can act as pathogen-associated molecular patterns (PAMPs) and bind to innate sensors. Focusing on the Human (Homo sapiens) and the rhesus macaque (Macaca mulatta), the main aims of this thesis are to look for further evidence linking ERVs to tumour development, with possible implications for therapies, and test the hypothesis that ERVs are PAMPs by seeing if individuals with higher levels of ERV expression exhibit a higher innate immune response. The work on ERVs in cancer involved the human ERV type-K HML2 lineage (HERV-K (HML2)), an ERV lineage found in humans, in Merlin-deficient tumours. These are schwannomas that arise from Schwann cells and for which effective drug therapy is urgently needed. The work on ERVs in inflammation involved the Papio cynocephalus ERV (PcEV), in rhesus macaques infected with simian immunodeficiency virus (SIV) infection. The main outcomes are as follows: regarding HERV-K (HML2) in human schwannomas, (i) HERV-K (HML2) proteins are overexpressed in schwannoma compared to Schwann cells; (ii) these proteins are released from the tumour; (iii) regulation of HERV-K (HML2) expression in the tumour appears to involve the transcription factor TEAD; (iv) schwannomas are potentially treatable using anti-HERV-K (HML2) monoclonal antibodies and antiretroviral drugs since both decreased proliferation in vitro. Regarding PcEV in SIV-infected macaques: (i) PcEV is transcriptionally active; (ii) PcEV can be retrieved at low levels in the blood of some macaque animals; (iii) the levels of PcEV in cells correlates strongly with the strength of the innate response as measured by cellular levels of STAT1 transcripts - an interferon-stimulated gene (ISG). Other recent research has shown that human ERV lineages, namely HERV-W and HERV-H, have been co-opted and are involved in placentation and pluripotency during development, respectively. The present work suggests that ERVs are involved in a wide range of biological process and supports the need for further research into the biological significance of ERVs for their hosts.

Some patients suffer hearing loss in the early postoperative period following vestibular schwannoma (VS) excision despite having intact hearing immediately after surgery. As this phenomenon has rarely been documented or described, the putative mechanism remains vague. The objective of the current study was to document the patterns of change in behavioural and electrophysiological responses in patients following VS surgery to better describe the phenomenon of delayed hearing loss. In particular, we aimed to determine whether the impairment that eventually leads to delayed hearing loss is neural or cochlear in origin. Auditory function was monitored in six adult patients who underwent surgery at Christchurch Public Hospital for excision of unilateral vestibular schwannoma through the retrosigmoid approach. Patients were assessed pre- and postoperatively by puretone audiometry, speech audiometry, tympanometry, distortion product otoacoustic emissions (DPOAEs), and auditory brainstem response (ABR). When measurable hearing was demonstrated postoperatively, pure-tone audiometry, speech audiometry and ABR were assessed at 24 hour intervals following surgery. Transtympanic electrocochleography (ECochG) was carried out if wave I of the ABR was lost during the postoperative period. Postoperative monitoring revealed that 4 patients suffered permanent anacusis and the remaining 2 patients had permanent hearing preservation. There were no patients who experienced delayed hearing loss in the early postoperative period. A phenomenon similar to delayed hearing loss was observed in case 2 who demonstrated loss of ABR wave I on the 7th postoperative day. Postoperative ECochG recorded in this case showed an enhanced negative SP on the operated side. The findings of this study are discussed in detail with particular reference to the underlying pathophysiology.

Neurofibromatosis type 2 (NF2) is an autosomal dominant inherited condition that predisposes individuals to develop multiple nervous system tumours, primarily schwannoma, meningioma and ependymoma. NF2 is characterised by loss of the tumour suppressor protein Merlin, caused by bi-allelic mutations of the encoding gene NF2 or by loss of heterozygosity. These tumours can occur either sporadically or as part of the NF2 condition. The majority are slow growing and display benign features, but this benignancy renders them largely unresponsive to classic chemotherapeutic agents leaving surgery and radiosurgery as the only remaining treatment options. Depending on their location, NF2-related tumours can cause a number of side effects, including nausea, balance problems, and in some cases hearing and/or vision loss. Phosphorylation is a key regulatory mechanism leading to changes in cell signalling. By identifying phosphoproteins that are significantly activated in tumour cells, novel therapies can be developed aiming to specifically target the phosphorylated status of these proteins thus ‘switching off’ the signalling cascade. The objective of this study is to identify and validate common targets in both Merlin-deficient meningioma and schwannoma to eventually exploit in novel therapeutic approaches. Using phosphoprotein purification followed by mass spectrometry analysis, we identified Signal Transducer and Activator of Transcription 1 (STAT1), phosphorylated at Serine (S) 727 and Tyrosine (Y) 701, PDZ and LIM domain protein 2 (PDLIM2), Heat Shock 70kDa Protein 1A (HSPA1A) and Filamin B (FLNB) as potential common, novel therapeutic targets. We validated these candidates in human primary meningioma and schwannoma tumour cells using a variety of techniques. We also showed that specific 7 knockdown of STAT1 and PDLIM2 was related to a significant decrease in cellular proliferation. Additionally, we performed co-immunoprecipitation using PDLIM2 as the bait protein and identified STAT1, HSPA1A and FLNB as binding partners, suggesting a novel interaction network involving all of the potential targets identified in this study. We also identified activation of several pathways and/ or biological processes in both tumour types that warrant further investigation i.e. endocytosis in schwannoma and the proteasome in meningioma. In conclusion, with our approach we substantially increased the overall body of knowledge regarding the proteome and phosphoproteome of meningioma and schwannoma. We generated a comprehensive set of data that highlighted several potential therapeutic targets and dysregulated pathways which will be further investigated.

Neurofibromatosis type two (NF2) is a genetic disorder predisposing those affected to the development of multiple benign tumors in their central and peripheral nervous systems. This is due to the absence of the tumor suppressor protein merlin, which is encoded by the NF2 gene. In nearly all NF2 cases, patients present with bilateral schwannomas of the vestibulocochlear nerve, in addition to other schwannomas throughout the central and peripheral nervous systems, as well as meningiomas and ependymomas. Currently, no therapeutic alternatives to surgical removal and radiation therapy are available for NF2 patients. This study investigated cobimetinib, an inhibitor of the often-deregulated mitogen activated protein kinase (MAPK) pathway in NF2 tumors, and its in vitro mechanism of action in both mouse and human NF2 schwannoma model cell lines. It was demonstrated that the drug decreased 70% and 60% of the viability at 10μM in the mouse and human merlin-deficient cell lines, respectively. It was further demonstrated that this decrease in viability was due to cytostatic and cytotoxic effects of cobimetinib in the case of the mouse NF2 schwannoma model but only due to cytostatic effects of cobimetinib in the human NF2 schwannoma model. These results show promise in targeting the MAPK pathway in NF2 tumors, and the promise of cobimetinib specifically, supporting further cytometric flow and in vivo testing of the inhibitor.

Yes / Merlin has broad tumor-suppressor functions as its mutations have been identified in multiple benign tumors and malignant cancers. In all schwannomas, the majority of meningiomas and 1/3 of ependymomas Merlin loss is causative. In neurofibromatosis type 2, a dominantly inherited tumor disease because of the loss of Merlin, patients suffer from multiple nervous system tumors and die on average around age 40. Chemotherapy is not effective and tumor localization and multiplicity make surgery and radiosurgery challenging and morbidity is often considerable. Thus, a new therapeutic approach is needed for these tumors. Using a primary human in vitro model for Merlin-deficient tumors, we report that the Ras/Raf/mitogen-activated protein, extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) scaffold, kinase suppressor of Ras 1 (KSR1), has a vital role in promoting schwannomas development. We show that KSR1 overexpression is involved in many pathological phenotypes caused by Merlin loss, namely multipolar morphology, enhanced cell-matrix adhesion, focal adhesion and, most importantly, increased proliferation and survival. Our data demonstrate that KSR1 has a wider role than MEK1/2 in the development of schwannomas because adhesion is more dependent on KSR1 than MEK1/2. Immunoprecipitation analysis reveals that KSR1 is a novel binding partner of Merlin, which suppresses KSR1's function by inhibiting the binding between KSR1 and c-Raf. Our proteomic analysis also demonstrates that KSR1 interacts with several Merlin downstream effectors, including E3 ubiquitin ligase CRL4(DCAF1). Further functional studies suggests that KSR1 and DCAF1 may co-operate to regulate schwannomas formation. Taken together, these findings suggest that KSR1 serves as a potential therapeutic target for Merlin-deficient tumors.

Os tumores extra-axias do sistema nervoso são de localização extra-cerebral e na maioria das vezes benignos; meningiomas, schwanomas e metástases fazem parte deste grupo. O aparecimento de um tumor ocorre a partir do acúmulo de alterações genéticas e epigenéticas nas células. Para entender o mecanismo molecular da progressão tumoral e a formação de metástases é indispensável identificar os genes que acumulam essas alterações. Sendo assim, este trabalho teve como objetivo analisar o perfil de metilação dos genes TP16, TP53, DAL-1, GSTP-1, MEN-1, NDRG2 e das DNA metiltransferases 3A, 3B e 3L e sua associação com os tumores extra-axiais e ainda, avaliar, através de um estudo caso-controle, a influência dos SNPs TP53 Pro47Ser e Arg72Pro, EGF + 61, GSTP-1 Ile105Val e WRN Cys1367Arg no desenvolvimento e prognóstico desses tumores. A técnica utilizada para a análise de hipermetilação foi a MSP, e através dela observamos que a atividade das DNMTs não está associada à metilação dos tumores extra-axiais e ainda, os perfis de metilação das DNMTs de novo não estão associados com alterações no padrão de metilação dos genes TP16, TP53, DAL-1, GSTP-1, MEN- 1 e NDRG2. Observamos que a metilação do gene TP53 está associada principalmente aos tumores de maior grau de malignidade, a uma deficiência na resposta a tratamentos e, conseqüentemente, a um maior número de óbitos. A metilação do gene TP16 está envolvida mais freqüentemente na formação de schwanomas e a de NDRG2 na progressão dos meningiomas. A análise de polimorfismos foi realizada através da técnica de PCR-RFLP e observamos diferenças nas distribuições genotípicas entre pacientes e controles nos SNPs TP53 Pro47Ser e Arg72Pro, EGF + 61 e GSTP-1 Ile105Val, onde as variantes Ser47, Pro72, EGF G61 e Val105 foram observadas com maior freqüência entre os portadores de tumores extra-axiais. Dessa forma, estas variantes podem ser fatores de susceptibilidade para o desenvolvimento dos tumores. / The extra-axial brain tumors have extra-brain localization and in most of the time they are benign, meningiomas, schwannomas and metastasis are included in this group. The appearance of a tumor occurs because of the accumulation of genetic and epigenetic alterations in the cells. In order to understand the molecular mechanism of the tumor progression and the metastasis formation it is important to identify the genes that accumulate the alterations. Thereby, the objective of this study was to analyze the methylation profile of the genes TP16, TP53, DAL-1, GSTP-1, MEN-1, NDRG2 and the DNA methyltransferases 3A, 3B and 3L and their association with the extra-axial brain tumors. Another purpose was to determine, in a case-control study, the roles of the TP53 Pro47Ser and Arg72Pro, EGF + 61, GSTP-1 Ile105Val and WRN Cys1367Arg SNPs in the development and prognosis of these tumors. We used the MSP to screen the hypermethylation profile and we observed no association between the DNMTs activity and the hypermethylation of the tumors. We also did not find association between the methylation of the DNMTs de novo and alterations in the methylation profile of the genes TP16, TP53, DAL-1, GSTP-1, MEN-1 and NDRG2. We observed that TP53 hypermethylation was associated with the high grade tumors, a poor response to the treatments and, consequently, the high number of obits. The TP16 methylation was involved with the shwannomas formation and the NDRG2 gene was involved in the meningiomas progression. For the polymorphism analysis, we used the PCR-RFLP technique and we observed differences in the genotype distributions between cases and controls of TP53 Pro47Ser and Arg72Pro, EGF + 61 and GSTP-1 Ile105Val SNPs, where the variants Ser47, Pro72, EGF G61 and Val105 were more frequent in patients than in controls. Thus, these variants can be important factors of susceptibility to the tumor development.