Molecular genetics of paediatric versus adult brain tumours

Tese de doutoramento em Ciências da Saúde (especialidade de Ciências da Saúde)Brain tumours are the leading cause of cancer-­‐related death in paediatric patients and are responsible for the greater part of the cancer-­‐related years of life lost across all age groups. The more malignant histologies are the major contributors to the high-­‐rates of mortality and morbidity of brain tumours. In the present thesis we focused our studies on high-­‐grade gliomas and medulloblastomas, the most common malignant brain tumours of adults and childhood patients, respectively. Childhood and adult patients share most of the different brain tumours’ histological types, despite a large variation in frequency across specific age groups. Nevertheless, there is increasing evidence that, despite being histologically similar, childhood and adult tumours have key clinical and molecular differences. The work summarized in this thesis aims to disclose molecular mechanisms particular to paediatric and adult high-­‐grade gliomas and medulloblastomas, attempting to better understand the biology of age-­‐specific, histologically identical, tumours. Brain tumours are characterized by multiple genetic alterations affecting receptor tyrosine kinase (RTK) pathways. As the EGFR RTK pathway is one of the most important signalling networks in high-­‐grade gliomas, we aimed to study EGFR molecular aberrations involved in protein activation, potentially relevant for tumour’s response to EGFR-­‐targeted therapy. The role of EGFR in adult high-­‐grade gliomas is well-­‐characterized, and in the present thesis we studied a unique series of Portuguese patients, aiming to understand whether the EGFR molecular alterations of these patients were in line with other populations in terms of potential biomarkers of EGFR-­‐targeted therapy. On the other hand, EGFR is thought to be less significant in childhood tumours, although there is limited published data. Accordingly, we aimed to study the frequency and role of EGFR molecular alterations in paediatric high-­‐grade gliomas, aiming to evaluate the presence of molecular signatures of response to existing drugs in these patients. We confirmed that EGFR represents one of the most frequently altered molecules in high-­‐grade glioma, particularly in adult glioblastoma, and that it is also true for Portuguese patients. In addition some paediatric tumours, particularly anaplastic oligodendrogliomas, frequently presented EGFR aberrations and therefore are also potential candidates for EGFR-­‐targeted therapy. Microsatellite instability (MSI) frequency in brain tumours remains a controversial research topic, and there is a lack of clarity in the published literature. In this context, we aimed to study MSI in high-­‐grade gliomas and medulloblastoma from adult and paediatric patients and identify MSI target genes potentially involved in MSI-­‐related tumorigenesis. Our findings show the presence of MSI in a fraction of medulloblastoma and high-­‐grade gliomas. Age-­‐specific differences in MSI frequency were not present in medulloblastoma, however MSI was significantly more frequent in paediatric high-­‐grade gliomas than in adults tumours. Moreover MSI-­‐positivity was associated with a stable genomic profile. Overall, of the 18 MSI target-­‐genes studied, only three were mutated, all in paediatric in MSI tumours, MBD4 in one medulloblastoma, and MSH6 and DNAPKcs in high-­‐grade glioma As we failed to find the MMR alteration responsible for the MSI phenotype, further research is critical to clarify this topic. Nevertheless, our studies provided evidence for a potential novel molecular pathway in a proportion of medulloblastoma and paediatric high-­‐grade gliomas, associated with the presence of MSI. Overall, the work summarized in this thesis contributed to the knowledge of the molecular mechanisms involved in the development of childhood and adult brain tumours, and confirms that, despite being histologically indistinguishable, these tumours can be molecularly distinctive.Os tumores cerebrais são a principal causa de morte por cancro em crianças, sendo também os principais responsáveis na diminuição de anos de vida em doentes oncológicos de todas as faixas etárias. Os tumores de maior malignidade sãos que mais contribuem para as altas taxas de mortalidade e morbilidade características dos tumores cerebrais. Nesta tese, centramos os nossos estudos em gliomas de alto grau e meduloblastomas, os tumores cerebrais malignos mais frequentes em doentes adultos e pediátricos, respectivamente. Apesar dos diversos tipos histológicos de tumores cerebrais serem comuns a doentes de diferentes idades, existe uma significativa diferença na frequência com que ocorrem em adultos e crianças. Além disso, há cada vez mais indícios de que, tumores histologicamente semelhantes, apresentam diferenças fundamentais a nível clínico e molecular, dependendo da idade dos doentes. Os tumores cerebrais são caracterizados por diversas alterações genéticas que afectam os receptores de tirosina cinase (RTK). Sendo a via de sinalização do RTK EGFR uma das mais importantes em gliomas de alto grau, estudámos alterações moleculares envolvidas na sua activação e potencialmente importantes na resposta tumoral à terapia dirigida. O papel desta molécula em gliomas de alto grau de doentes adultos tem sido amplamente descrito, pelo que avaliámos uma série de tumores de doentes Portugueses adultos. Neste trabalho pretendemos perceber se as alterações de EGFR nos tumores Portugueses se assemelham às descritas noutras populações, de forma a avaliar o seu potencial papel como biomarcador de terapia dirigida ao EGFR. Por outro lado, , apesar da escassez de dados publicados, pensa-­‐se que a importância do EGFR em tumores pediátricos seja limitada. Para melhor esclarecer este assunto, estudámos a presença de alterações moleculares do EGFR em gliomas pediátricos de alto grau, com o objectivo de avaliar quais os potenciais biomarcadores na resposta a fármacos anti-­‐EGFR. Confirmámos que o EGFR é uma das moléculas mais frequentemente alteradas em gliomas de alto grau, particularmente em tumores adultos, sendo isto também verdade para os doentes Portugueses. Igualmente os tumores pediátricos, em particular, os oligodendrogliomas anaplásicos, apresentam alterações nesta molécula e consequentemente, são também potenciais candidatos ao uso de fármacos anti-­‐EGFR. A ocorrência de instabilidade de microssatélites (MSI) em tumores cerebrais é um tópico de investigação controverso, sendo a literatura existente insuficiente para a esclarecer este assunto. Neste contexto, estudámos a presença de MSI em gliomas de alto grau e meduloblastomas de doentes adultos e pediátricos, assim como os genes-­‐alvo potencialmente envolvidos na tumorigénese relacionada com MSI. Os nossos resultados demonstram a presença de MSI numa fracção de meduloblastomas e gliomas de alto grau. Em meduloblastomas não se observaram variações na presença de MSI em doentes de diferentes idades, no entanto em gliomas de alto grau, a frequência de MSI é estatisticamente mais elevada nos tumores pediátricos . Além disso, em gliomas, a presença de MSI foi associada a um perfil genómico estável. Dos mais de 18 genes-­‐alvo analisados, foram encontradas mutações apenas em três e somente em tumores pediátricos: MBD4 num meduloblastoma e MSH6 e DNAPKcs em gliomas de alto grau. . O estudo das moléculas de “mismatch repair” não clarificou qual o seu papel no fenótipo de MSI, observado nestes doentes. No entanto, os nossos estudos evidenciam a presença de uma potencial nova via molecular em alguns meduloblastomas e gliomas de alto grau associada à presença de MSI. Concluindo, o trabalho resumido nesta tese, contribuiu para o conhecimento das mecanismos molecular envolvidos no desenvolvimento de tumores cerebrais pediátricos e adultos, e confirma que apesar de histologicamente semelhantes, podem ser molecularmente distintos

SPINT2 deregulation in prostate carcinoma

SPINT2 is a tumor suppressor gene that inhibits proteases implicated in cancer progression, like HGFA, hepsin and matriptase. Loss of SPINT2 expression in tumors has been associated with gene promoter hypermethylation; however, little is known about the mechanisms of SPINT2 deregulation in prostate cancer (PCa). We aimed to analyze SPINT2 expression levels and understand the possible regulation by SPINT2 promoter hypermethylation in PCa. In a cohort of 57 cases including non-neoplastic and PCa tissues, SPINT2 expression and promoter methylation was analyzed by immunohistochemistry and methylation-specific PCR, respectively. Methylation status of the SPINT2 promoter was also evaluated by bisulfite sequencing and 5-aza-2’-deoxycytidine treatment. Oncomine and TCGA databases were used to perform in silico PCa analysis of SPINT2 mRNA and methylation levels. A reduction in SPINT2 expression levels from nonneoplastic to PCa tissues was observed; however, none of the cases exhibited SPINT2 promoter methylation. Both bisulfite sequencing and 5-aza demonstrated that SPINT2 promoter is not methylated in PCa cells. Bioinformatics approaches did not show downregulation of SPINT2 at the mRNA level and, in corroboration with our results, SPINT2 promoter region is reported to be unmethylated. Our study suggests an involvement of SPINT2 in PCa tumorigenesis, probably in association with a post-translational regulation of SPINT2.The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the ICVS internal research funds of participating authors and by FCT project, ref. PTDC/SAUONC/115513/2009. F.P. received fellowship from the FCT, ref. SFRH/BD/81369/2011 and M.VP from the ON.2 SR&TD Integrated Program (N-01-01-01-24-01-07), ref. UMINHO/ BPD/36/2013

Analysis of EGFR Overexpression, EGFR gene amplification and the EGFRvIII Mutation in portuguese high-grade gliomas

Background: Patients with malignant gliomas do not respond to any current therapy. Epidermal growth factor receptor (EGFR) controls several oncogenic processes, being frequently up-regulated in gliomas due to overexpression, gene amplification and gene mutation. EGFR inhibitors are being tried in gliomas, yet the molecular determinants of therapeutic response are unclear. Materials and Methods: EGFR overexpression, EGFRvIII mutation and EGFR amplification were determined by immunohistochemistry and chromogenic in situ hybridization (CISH) in 27 primary glioblastomas (GBM), 24 anaplastic oligodendrogliomas (AO) and four anaplastic oligoastrocytomas (AOA). Results: EGFR overexpression was associated with EGFR amplification, being found in 48% and 53% GBM, 33% and 40% AO and 75% and 67% AOA, respectively. EGFRvIII was found in 22% GBM, 8% AO and was absent in AOA. No association was observed between EGFR alterations and patient survival. Conclusion: We characterized, for the first time, EGFR molecular alterations in Portuguese patients with malignant glioma and identified a subpopulation of patients presenting putative biomarkers for EGFR-based therapies

Analysis of microsatellite instability in medulloblastoma

Medulloblastoma is the most common malignant brain tumor in children. The presence of microsatellite instability (MSI) in brain tumors, particularly medulloblastomas, has not been properly addressed. The aim of the present study was to evaluate the role of MSI in medulloblastoma carcinogenesis. MSI status was determined in 36 patients using a pentaplex PCR of quasimonomorphic markers (NR27, NR21, NR24, BAT25, and BAT26). Methylation status of mismatch repair (MMR) genes was achieved by methylation-specific multiplex ligation-dependent probe amplification (MLPA). In addition, MutS homolog 6 (MSH6) expression was determined by immunohistochemistry. Mutations of 10 MSI target genes (TCF4, XRCC2, MBD4, MRE11, ATR, MSH3, TGFBR2, RAD50, MSH6, and BAX) were studied by pentaplex PCR followed by analysis with GeneScan 3.7 software. Mutation analysis of hotspot regions of beta-catenin (CTNNB1) and BRAF (v-raf murine sarcoma viral oncogene homolog B1) oncogenes was performed by PCR single-strand conformation polymorphism analysis followed by direct sequencing. Among the 36 tumors, we found four (11%) cases with instability, one with high MSI and three with low MSI. Methylation analysis of MMR genes in cases presenting shifts on the MSI markers revealed mild hypermethylation of MSH6 in 75% of cases, yet MSH6 was expressed in all the tumors. The MSI target genes MBD4 (methyl-CpG binding domain protein 4) and MRE11 (meiotic recombination 11 homolog A) were mutated in two different tumors. No CTNNB1 or BRAF mutations were found. This study is the most comprehensive analysis of MSI in medulloblastomas to date. We observed the presence of MSI together with mutations of MSI target genes in a small fraction of cases, suggesting a new genetic pathway for a role in medulloblastoma development.M.V.-P. is the recipient of a Ph.D. fellowship (SFRH/BD/29145/2006), and I.A. is the recipient of a research fellowship (SFRH/BI/33160/2007) from Fundação para a Ciência e Tecnologia, Portugal. This study was partially supported by a grant from Clinical de Radioterapia do Porto, Portugal

Study of hTERT and Histone 3 Mutations in Medulloblastoma

CNPq/Universal (475358/2011-2), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 2012/19590-0) and Fundação para a Ciência e Tecnologia (FCT; PTDC/SAU-ONC/115513/2009) grants to R.M.R. The project was cofinanced by Programa Operacional Regional do Norte (ON.2-O Novo Norte), Quadro de Referência Estratégico Nacional (QREN) and Fundo Europeu de Desenvol- vimento Regional (FEDER). M.V.-P. is the recipient of an FCT Post-Doctorate Research Fellowship (SFRH/BPD/104290/2014)Hotspot activating mutations of the telomerase reverse transcriptase (hTERT) promoter region were recently described in several tumor types. These mutations lead to enhanced expression of telomerase, being responsible for telomere maintenance and allowing continuous cell division. Additionally, there are alternative telomere maintenance mechanisms, associated with histone H3 mutations, responsible for disrupting the histone code and affecting the regulation of transcription. Here, we investigated the clinical relevance of these mechanistically related molecules in nnedulloblastoma. Sixty-nine medulloblastomas, formalin fixed and paraffin embedded, from a cohort of patients aged 1.5-70 years, were used to investigate the hotspot mutations of the hTERT promoter region, i.e. H3F3A and HIST1H3B, using Sanger sequencing. We successfully sequenced hTERT in all 69 medulloblastoma samples and identified a total of 19 mutated cases (27.5%). c.-124:G>A and c.-146:G>A mutations were detected, respectively, in 16 and 3 samples. Similar to previous reports, hTERT mutations were more frequent in older patients (p < 0.0001), being found only in 5 patients <20 years of age. In addition, hTERT-mutated tumors were more frequently recurrent (p = 0.026) and hTERT mutations were significantly enriched in tumors located in the right cerebellar hemisphere (p = 0.039). No mutations were found on the H3F3A or HIST1H3B genes. hTERT promoter mutations are frequent in medulloblastoma and are associated with older patients, prone to recurrence and located in the right cerebellar hemisphere. On the other hand, histone 3 mutations do not seem to be present in nnedulloblastoma.This study was partially supported by CNPq/Universal (475358/2011-2), Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP; 2012/19590-0) and Fundacao para a Ciencia e Tecnologia (FCT; PTDC/SAU-ONC/115513/2009) grants to R.M.R. The project was cofinanced by Programa Operacional Regional do Norte (ON.2-O Novo Norte), Quadro de Referencia Estrategico Nacional (QREN) and Fundo Europeu de Desenvolvimento Regional (FEDER). M.V.-P. is the recipient of an FCT Post-Doctorate Research Fellowship (SFRH/BPD/104290/2014).info:eu-repo/semantics/publishedVersio

Portuguese propolis antitumoral activity in melanoma involves ROS production and induction of apoptosis

Melanoma is the most aggressive and life-threatening skin cancer type. The melanoma genome is the most frequently mutated, with the BRAF mutation present in 40–60% of melanoma cases. BRAF-mutated melanomas are characterized by a higher aggressiveness and progression. Adjuvant targeted treatments, such as BRAF and MEK inhibitors, are added to surgical excision in BRAF-mutated metastatic melanomas to maximize treatment effectiveness. However, resistance remains the major therapeutic problem. Interest in natural products, like propolis, for therapeutic applications, has increased in the last years. Propolis healing proprieties offer great potential for the development of novel cancer drugs. As the activity of Portuguese propolis has never been studied in melanoma, we evaluated the antitumoral activity of propolis from Gerês (G18.EE) and its fractions (n-hexane, ethyl acetate (EtOAc), and n-butanol) in A375 and WM9 melanoma cell lines. Results from DPPH•/ABTS• radical scavenging assays indicated that the samples had relevant antioxidant activity, however, this was not confirmed in the cell models. G18.EE and its fractions decreased cell viability (SRB assay) and promoted ROS production (DHE/Mitotracker probes by flow cytometry), leading to activation of apoptotic signaling (expression of apoptosis markers). Our results suggest that the n-BuOH fraction has the potential to be explored in the pharmacological therapy of melanoma.Foundation for Science and Technology (FCT)—projects UIDB/04050/2020, UDBI/04033/2020, UIDB/50026/2020, UIDP/50026/2020, UIDB/50006/2020, UIDP/50006/2020, and by the project NORTE-01-0145-FEDER-000055, supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). S.P.C., C.B.M. and A.S.F. are recipients of FCT grants (2020.05779.BD, SFRH/BD/145955/2019, and PD/BD/128276/2017, respectively

Genetic variants of vascular endothelial growth factor predict risk and survival of gliomas

The vascular endothelial growth factor regulates angiogenesis that is increased in glioma. VEGF polymorphisms are thought to modulate vascular endothelial growth factor plasma levels and therefore may be implicated in glioma risk. We aimed to clarify the role of VEGF and von Willebrand factor polymorphisms in glioma susceptibility and prognosis. A case-control study of 126 glioma patients and 180 cancer-free controls was performed. Using Sequenom MassARRAY platform, 11 VEGF and 1 VWF polymorphisms were genotyped. Unconditional multivariate logistic regression models were used to calculate odds ratios and 95% confidence intervals. The associations between polymorphisms and survival were evaluated using a Cox regression model. Bonferroni's adjustment was used to correct for multiple testing. The VEGF polymorphism rs833061 was strongly associated with increased risk for glioma (odds ratio = 164.85) and glioblastoma (odds ratio = 155.66), confirmed after Bonferroni correction. Also, the VEGF polymorphisms rs3024994, rs2010963, and particularly the homozygous carriers of rs1005230 were associated with a worse prognosis for glioma and glioblastoma. Our data support a role of VEGF and VWF polymorphisms as glioma biomarkers, with additional potential relevance for molecular stratification of patients for anti-angiogenic therapies.FCT -Fundação para a Ciência e a Tecnologia(NORTE-01-0145-FEDER-000013)info:eu-repo/semantics/publishedVersio

Copy number profiling of Brazilian astrocytomas

Copy number alterations (CNA) are one of the driving mechanisms of glioma tumorigenesis, and are currently used as important biomarkers in the routine setting. Therefore, we performed CNA profiling of 65 astrocytomas of distinct malignant grades (WHO grade I-IV) of Brazilian origin, using array-CGH and microsatellite instability analysis (MSI), and investigated their correlation with TERT and IDH1 mutational status and clinico-pathological features. Furthermore, in silico analysis using the Oncomine database was performed to validate our findings and extend the findings to gene expression level. We found that the number of genomic alterations increases in accordance with glioma grade. In glioblastomas (GBM), the most common alterations were gene amplifications (PDGFRA, KIT, KDR, EGFR, and MET) and deletions (CDKN2A and PTEN). Log-rank analysis correlated EGFR amplification and/or chr7 gain with better survival of the patients. MSI was observed in 11% of GBMs. A total of 69% of GBMs presented TERT mutation, whereas IDH1 mutation was most frequent in diffuse (85.7%) and anaplastic (100%) astrocytomas. The combination of 1p19q deletion and TERT and IDH1 mutational status separated tumor groups that showed distinct age of diagnosis and outcome. In silico validation pointed to less explored genes that may be worthy of future investigation, such as CDK2, DMRTA1, and MTAP. Herein, using an extensive integrated analysis, we indicated potentially important genes, not extensively studied in gliomas, that could be further explored to assess their biological and clinical impact in astrocytomas.This study was partially supported by the Universal/National Counsel of Technological and Scientific Development (CNPq) (475358/2011-2 – R.M.R.), São Paulo Research Foundation (FAPESP) (2012/19590-0 and 2016/09105-8 – R.M.R.) and the Fundação para a Ciência e a Tecnologia (FCT) (PTDC/SAU-ONC/115513/2009-FCMO-01-0124FEDER-015949). L.T.B. was recipient of FAPESP fellowships (2011/ 08523-7 and 2012/08287-4), N.C.C.was recipient of a FAPESP fellowship (2013/25787-3), M.L.S. was recipient of a CNPq/Programa Institucional de Bolsas de Iniciação Científica (PIBIC) fellowship (100707/ 2014-9), W.M. was recipient of FAPESP (2013/15515-6) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)/ Programa de Suporte à Pós-Graduação de Instituições de Ensino Particulares (Prosup) fellowships, and M.V.P. was a Postdoctoral research fellow under the FCT project PTDC/SAU-ONC/115513/2009. R.M.R. has a CNPq scholarship. C.J. and A.M. acknowledge National Health Service funding to the National Institute for Health Research Biomedical Research Centre at The Royal Marsden and the Institute of Cancer Research.info:eu-repo/semantics/publishedVersio