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Purpose: The epidermal growth factor receptor (EGFR) is amplified and overexpressed in adult glioblastoma, with response to targeted inhibition dependent on the underlying biology of the disease. EGFR has thus far been considered to play a less important role in pediatric glioma, although extensive data are lacking. We have sought to clarify the role of EGFR in pediatric high-grade glioma (HGG).Experimental Design: We retrospectively studied a total of 90 archival pediatric HGG specimens for EGFR protein overexpression, gene amplification, and mutation and assessed the in vitro sensitivity of pediatric glioma cell line models to the small-molecule EGFR inhibitor erlotinib.Results: Amplification was detected in 11% of cases, with corresponding overexpression of the receptor. No kinase or extracellular domain mutations were observed; however, 6 of 35 (17%) cases harbored the EGFRvIII deletion, including two anaplastic oligodendrogliomas and a gliosarcoma overexpressing EGFRvIII in the absence of gene amplification and coexpressing platelet-derived growth factor receptor α. Pediatric glioblastoma cells transduced with wild-type or deletion mutant EGFRvIII were not rendered more sensitive to erlotinib despite expressing wild-type PTEN. Phosphorylated receptor tyrosine kinase profiling showed a specific activation of platelet-derived growth factor receptor α/β in EGFRvIII-transduced pediatric glioblastoma cells, and targeted coinhibition with erlotinib and imatinib leads to enhanced efficacy in this model.Conclusions: These data identify an elevated frequency of EGFR gene amplification and EGFRvIII mutation in pediatric HGG than previously recognized and show the likely necessity of targeting multiple genetic alterations in the tumors of these children. (Clin Cancer Res 2009;15(18):5753–61)

Supplementary Data from EGFRvIII Deletion Mutations in Pediatric High-Grade Glioma and Response to Targeted Therapy in Pediatric Glioma Cell Lines

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Supplementary Table S3.

Purpose: As genome-scale technologies begin to unravel the complexity of the equivalent tumors in adults, we can attempt detailed characterization of high-grade gliomas in children, that have until recently been lacking. Toward this end, we sought to validate and extend investigations of the differences between pediatric and adult tumors.Experimental Design: We carried out copy number profiling by array comparative genomic hybridization using a 32K bacterial artificial chromosome platform on 63 formalin-fixed paraffin-embedded cases of high-grade glioma arising in children and young people (Results: The genomic profiles of these tumors could be subclassified into four categories: those with stable genomes, which were associated with a better prognosis; those with aneuploid and those with highly rearranged genomes; and those with an amplifier genotype, which had a significantly worse clinical outcome. Independent of this was a clear segregation of cases with 1q gain (more common in children) from those with concurrent 7 gain/10q loss (a defining feature of adults). Detailed mapping of all the amplification and deletion events revealed numerous low-frequency amplifications, including IGF1R, PDGFRB, PIK3CA, CDK6, CCND1, and CCNE1, and novel homozygous deletions encompassing unknown genes, including those at 5q35, 10q25, and 22q13. Despite this, aberrations targeting the “core signaling pathways” in adult glioblastomas are significantly underrepresented in the pediatric setting.Conclusions: These data highlight that although there are overlaps in the genomic events driving gliomagenesis of all ages, the pediatric disease harbors a distinct spectrum of copy number aberrations compared with adults. Clin Cancer Res; 16(13); 3368–77. ©2010 AACR.

Background: The epidermal growth factor receptor (EGFR) regulates important cellular processes and is frequently implicated in human tumors. Three EGFR polymorphisms have been described as having a transcriptional regulatory function: two single-nucleotide polymorphisms in the essential promoter region, −216G/T and −191C/A, and a polymorphic (CA)n microsatellite sequence in intron 1. We aimed to elucidate the roles of these EGFR polymorphisms in glioma susceptibility and prognosis.Methods: We conducted a case–control study with 196 patients with glioma and 168 cancer-free controls. Unconditional multivariate logistic regression models were used to calculate ORs and 95% confidence intervals. A Cox regression model was used to evaluate associations with patient survival. False-positive report probabilities were also assessed.Results: None of the EGFR −216G/T variants was significantly associated with glioma risk. The −191C/A genotype was associated with higher risk for glioma when the (CA)n alleles were classified as short for ≤16 or ≤17 repeats. Independently of the (CA)n repeat cutoff point used, shorter (CA)n repeat variants were significantly associated with increased risk for glioma, particularly glioblastoma and oligodendroglioma. In all tested models with different (CA)n cutoff points, only −191C/A genotype was consistently associated with improved survival of patients with glioblastoma.Conclusions: Our findings implicate EGFR −191C/A and the (CA)n repeat polymorphisms as risk factors for gliomas, and suggest −191C/A as a prognostic marker in glioblastoma.Impact: Our data support a role of these EGFR polymorphisms in determining glioma susceptibility, with potential relevance for molecularly based stratification of patients with glioblastoma for individualized therapies. Cancer Epidemiol Biomarkers Prev; 20(12); 2610–7. ©2011 AACR.

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Aim: To investigate correlations between microsatellite instability (MSI) and the phenotype, clinicopathological features, and overall survival (OS) in Moroccan gastric cancer (GC) patients. We eva...

High-grade gliomas (HGG) remain one of the most aggressive tumors, which is primarily due to its diffuse infiltrative nature. Serine proteases and metalloproteases are known to play key roles in cellular migration and invasion mechanisms. SPINT2, also known as HAI-2, is an important serine protease inhibitor that can affect MET signaling. SPINT2 has been found to be frequently downregulated in various tumors, whereby hypermethylation of its promoter appears to serve as a common mechanism. Here, we assessed the clinical relevance of SPINT2 expression and promoter hypermethylation in pediatric and adult HGG and explored its functional role. A series of 371 adult and 77 pediatric primary HGG samples was assessed for SPINT2 protein expression (immunohistochemistry) and promoter methylation (methylation-specific PCR) patterns. After SPINT2 knockdown and knock-in in adult and pediatric HGG cell lines, a variety of in vitro assays was carried out to determine the role of SPINT2 in glioma cell viability and invasion, as well as their mechanistic associations with metalloprotease activities. We found that SPINT2 protein expression was frequently absent in adult (85.3%) and pediatric (100%) HGG samples. The SPINT2 gene promoter was found to be hypermethylated in approximately half of both adult and pediatric gliomas. Through functional assays we revealed a suppressor activity of SPINT2 in glioma cell proliferation and viability, as well as in their migration and invasion. These functions appear to be mediated in part by MMP2 expression and activity. We conclude that dysregulation of SPINT2 is a common event in both pediatric and adult HGG, in which SPINT2 may act as a tumor suppressor.

Introduction. The Cancer Genome Atlas (TCGA) project and Asian Cancer Research Group (ACRG) recently categorized gastric cancer into molecular subtypes. Nevertheless, these classification systems require high cost and sophisticated molecular technologies, preventing their widespread use in the clinic. This study is aimed to generating molecular subtypes of gastric cancer using techniques available in routine diagnostic practice in a series of Moroccan gastric cancer patients. In addition, we assessed the associations between molecular subtypes, clinicopathological features, and prognosis. Methods. Ninety-seven gastric cancer cases were classified according to TCGA, ACRG, and integrated classifications using a panel of four molecular markers (EBV, MSI, E-cadherin, and p53). HER2 status and PD-L1 expression were also evaluated. These markers were analyzed using immunohistochemistry (E-cadherin, p53, HER2, and PD-L1), in situ hybridization (EBV and HER2 equivocal cases), and multiplex PCR (MSI). Results. Our results showed that the subtypes presented distinct clinicopathological features and prognosis. EBV-positive gastric cancers were found exclusively in male patients. The GS (TCGA classification), MSS/EMT (ACRG classification), and E-cadherin aberrant subtype (integrated classification) presented the Lauren diffuse histology enrichment and tended to be diagnosed at a younger age. The MSI subtype was associated with a better overall survival across all classifications (TCGA, ACRG, and integrated classification). The worst prognosis was observed in the EBV subtype (TCGA and integrated classification) and MSS/EMT subtype (ACRG classification). Discussion/Conclusion. We reported a reproducible and affordable gastric cancer subtyping algorithms that can reproduce the recently recognized TCGA, ACRG, and integrated gastric cancer classifications, using techniques available in routine diagnosis. These simplified classifications can be employed not only for molecular classification but also in predicting the prognosis of gastric cancer patients.

Diffuse gliomas are the most common malignant primary brain tumors and remain incurable. A better knowledge of the tumor etiology is required. Specific single nucleotides polymorphisms (SNPs) rs4977756 (CDKN2A/B), rs6010620 (RTEL1), rs498872 (PHLDB1), rs2736100 (TERT), and rs4295627 (CCDC26) have been associated with glioma susceptibility and are potential risk biomarkers. This study aimed to analyze five SNPs associated with glioma susceptibility, in the Portuguese population. SNPs were genotyped using the Sequenom MassARRAY platform in 127 gliomas and 180 controls. Unconditional logistic regression models were used to calculate odds ratio (OR) and 95% confidence intervals. The false-positive report probability was also assessed. The associations between polymorphisms and survival were evaluated using the log-rank test. It was found that the AG and GG genotypes of the rs4977756 (CDKN2A/B) were associated with an increased risk of gliomas (OR 1.85 and OR 2.38) and glioblastomas (OR 2.77 and OR 3.94). The GA genotype of the rs6010620 (RTEL1) was associated with a decreased risk of glioblastomas (OR 0.45). We also observed that the GA genotype of the rs498872 (PHLDB1) was associated with an increased risk of gliomas (OR 2.92) and glioblastomas (OR 2.39). No significant risk associations were found for the rs2736100 (TERT) and rs4295627 (CCDC26). In addition, the genotype AA of the rs498872 (PHLDB1) was associated with poor overall survival of gliomas patients (AA vs. GA, p = 0.037). The rs6010620 (RTEL1), rs4977756 (CDKN2A/B), and rs498872 (PHLDB1) are associated with glioma risk in the Portuguese population and these data may contribute to understanding gliomas etiology.

Abnormal expression of the long non-coding RNA HOX transcript antisense intergenic RNA (HOTAIR) is oncogenic in several human cancers, including gliomas. The HOTAIR single nucleotide polymorphisms (SNPs) rs920778 (C > T) and rs12826786 (C > T) present in the intronic enhancer and promoter regions of HOTAIR, respectively, are associated with expression, cancer susceptibility, and patient prognosis in some tumor types. However, the relevance of these HOTAIR SNPs has not been studied in glioma. Here, we report a case-control study comprising 177 Portuguese glioma patients and 199 cancer-free controls. All subjects were genotyped by PCR and restriction fragment length polymorphism (RFLP). No statistically significant differences were found in the genotype or allele distributions of either rs920778 or rs12826786 between glioma patients and controls, suggesting these SNPs are not associated with glioma risk. No significant associations were found between rs920778 variants and HOTAIR expression levels, while rs12826786 CT genotype was associated with increased intratumoral HOTAIR RNA levels when compared to TT genotype (p-value = 0.04). Univariate (Log-rank) and multivariate (Cox proportional) analyses showed both rs920778 CT and rs12826786 CT genotypes were significantly associated with longer overall survival of WHO grade III anaplastic oligodendroglioma patients. Our results suggest that HOTAIR SNPs rs920778 and rs12826786 do not play a significant role in glioma susceptibility, but may be important prognostic factors in anaplastic oligodendroglioma patients. Future studies are warranted to validate and expand these findings, and to further dissect the importance of these SNPs in glioma., Fundação para a Ciência e Tecnologia (PTDC/SAU-GMG/113795/2009 and IF/00601/2012 to B.M.C.; SFRH/BD/88220/2012 to A.X.M.; SFRH/BD/52287/2013 to A.I.O.; SFRH/BD/88121/2012 to J.V.C.; SFRH/BD/81042/2011 to M.P.; SFRH/BD/92786/2013 to C.S.G.; SFRH/BD/51996/2012 to T.L.; SFRH/BPD/104290/2014 to M.V.P.; PTDC/SAU-ONC/115513/2009 to R.M.R., Fundação Calouste Gulbenkian (B.M.C.), Liga Portuguesa Contra o Cancro (B.M.C.) and Inter-University Doctoral Programme in Ageing and Chronic Disease (PhDOC; to A.X.M. and A.I.O.). Project co-financed by Programa Operacional Regional do Norte (ON.2—O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), Fundo Europeu de Desenvolvimento Regional (FEDER)

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 medulloblastoma. 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 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 medulloblastoma.

Human hotspot TERT promoter (TERTp) mutations have been reported in a wide range of tumours. Several studies have shown that TERTp mutations are associated with clinicopathological features; in some instances, TERTp mutations were considered as biomarkers of poor prognosis. The rs2853669 SNP, located in the TERT promoter region, was reported to modulate the increased TERT expression levels induced by the recurrent somatic mutations. In this study we aimed to determine the frequency and prognostic value of TERTp mutations and TERT rs2853669 SNP in 504 gliomas from Portuguese and Brazilian patients. TERTp mutations were detected in 47.8% of gliomas (216/452). Glioblastomas (GBM) exhibited the highest frequency of TERTp mutations (66.9%); in this glioma subtype, we found a significant association between TERTp mutations and poor prognosis, regardless of the population. Moreover, in a multivariate analysis, TERTp mutations were the only independent prognostic factor. Our data also showed that the poor prognosis conferred by TERTp mutations was restricted to GBM patients carrying the rs2853669 A allele and not in those carrying the G allele. In conclusion, the presence of TERTp mutations was associated with worse prognosis in GBM patients, although such association depended on the status of the rs2853669 SNP. The status of the rs2853669 SNP should be taken in consideration when assessing the prognostic value of TERTp mutations in GBM patients. TERTp mutations and the rs2853669 SNP can be used in the future as biomarkers of glioma prognosis.

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/publishedVersion

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/publishedVersion

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 medulloblastoma. 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:GA and c.-146:GA mutations were detected, respectively, in 16 and 3 samples. Similar to previous reports, hTERT mutations were more frequent in older patients (p0.0001), being found only in 5 patients20 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 medulloblastoma.

Sensitivity to temozolomide is restricted to a subset of glioblastoma patients, with the major determinant of resistance being a lack of promoter methylation of the gene encoding the repair protein DNA methyltransferase MGMT, although other mechanisms are thought to be active. There are, however, limited preclinical data in model systems derived from pediatric glioma patients. We screened a series of cell lines for temozolomide efficacy in vitro, and investigated the differential mechanisms of resistance involved. In the majority of cell lines, a lack of MGMT promoter methylation and subsequent protein overexpression were linked to temozolomide resistance. An exception was the pediatric glioblastoma line KNS42. Expression profiling data revealed a coordinated upregulation of HOX gene expression in resistant lines, especially KNS42, which was reversed by phosphoinositide 3-kinase pathway inhibition. High levels of HOXA9/HOXA10 gene expression were associated with a shorter survival in pediatric high-grade glioma patient samples. Combination treatment in vitro of pathway inhibition and temozolomide resulted in a highly synergistic interaction in KNS42 cells. The resistance gene signature further included contiguous genes within the 12q13-q14 amplicon, including the Akt enhancer PIKE, significantly overexpressed in the KNS42 line. These cells were also highly enriched for CD133 and other stem cell markers. We have thus shown an in vitro link between phosphoinositide 3-kinase-mediated HOXA9/HOXA10 expression, and a drug-resistant, progenitor cell phenotype in MGMT-independent pediatric glioblastoma., Cancer Research UK (C1178/A10294, C309/A2187, C309/A8274), the Oak Foundation (L. Marshall), and La Fondation de France (N. Gaspar). We acknowledge NHS funding to the NIHR Biomedical Research Centre. P. Workman is a Cancer Research UK Life Fellow

Aims: Gastrointestinal stromal tumours (GISTs) are commonly driven by oncogenic mutations in KIT and PDGFRA. However, 10-40% of these patients are wild-type for these genes. The prognostic significance of wild-type GISTs is controversial, and they rarely respond to imatinib. The aim of this study was to elucidate the molecular lesions underlying wild-type GISTs tumorigenesis. Methods and results: Twenty-nine KIT and PDGFRA wild-type GISTs were re-assessed for the presence of 'cryptic'KIT exon 11 duplications. Using a specific polymerase chain reaction assay, three previously undetected mutations were identified. In the remaining 26 wild-type GISTs, KIT, stem cell factor (SCF), phospho-KIT and phospho-ERK expression was evaluated by immunohistochemistry. Samples were screened for gain-of-function mutations in the mitogen-activated protein kinase (MAPK) cascade. KIT and SCF co-expression associated with KIT activation was observed in approximately 30% of cases. Furthermore, phospho-ERK expression showed that MAPK is activated in approximately 30% of cases. None of RAS family (H-, K- and N-RAS) oncogenes exhibited activating mutations, whereas BRAF mutations were found in approximately 4% of cases. Conclusions: In the absence of RAS mutations, MAPK could be activated through SCF/KIT autocrine/paracrine mechanisms and/or mutated BRAF in a subset of KIT/PDGFRA wild-type GISTs., O.M. is the recipient of a PhD fellowship (SFRH ⁄ BD ⁄ 36463 ⁄ 2007) from FCT, Portugal. This study was partially supported by Novartis Oncology, Portugal.

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.

Background: Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer death worldwide. Most cancer cells display high rates of glycolysis with production of lactic acid, which is then exported to the microenvironment by monocarboxylate transporters (MCTs). The main aim of this study was to evaluate the significance of MCT expression in a comprehensive series of primary CRC cases, lymph node and hepatic metastasis. Methods: Expressions of MCT1, MCT4, CD147 and GLUT1 were studied in human samples of CRC, lymph node and hepatic metastasis, by immunohistochemistry. Results: All proteins were overexpressed in primary CRC, lymph node and hepatic metastasis, when compared with non-neoplastic tissue, with exception of MCT1 in lymph node and hepatic metastasis. MCT1 and MCT4 expressions were associated with CD147 and GLUT1 in primary CRC. These markers were associated with clinical pathological features, reflecting the putative role of these metabolism-related proteins in the CRC setting. Conclusion: These findings provide additional evidence for the pivotal role of MCTs in CRC maintenance and progression, and support the use of MCTs as biomarkers and potential therapeutic targets in primary and metastatic CRC., This work was supported by the Fundação para a Ciência e a Tecnologia (FCT) grant ref. PTDC/SAU-FCF/104347/2008, under the scope of ‘Programa Operacional Temático Factores de Competitividade’ (COMPETE) of ‘Quadro Comunitário de Apoio III’ and co-financed by the Fundo Europeu De Desenvolvimento Regional (FEDER). Ricardo Amorim was recipient of the fellowship SFRH/BD/98002/2013, from Fundação para a Ciência e a Tecnologia (FCT Portugal)., info:eu-repo/semantics/publishedVersion

Human hotspot TERT promoter (TERTp) mutations have been reported in a wide range of tumours. Several studies have shown that TERTp mutations are associated with clinicopathological features; in some instances, TERTp mutations were considered as biomarkers of poor prognosis. The rs2853669 SNP, located in the TERT promoter region, was reported to modulate the increased TERT expression levels induced by the recurrent somatic mutations. In this study we aimed to determine the frequency and prognostic value of TERTp mutations and TERT rs2853669 SNP in 504 gliomas from Portuguese and Brazilian patients. TERTp mutations were detected in 47.8% of gliomas (216/452). Glioblastomas (GBM) exhibited the highest frequency of TERTp mutations (66.9%); in this glioma subtype, we found a significant association between TERTp mutations and poor prognosis, regardless of the population. Moreover, in a multivariate analysis, TERTp mutations were the only independent prognostic factor. Our data also showed that the poor prognosis conferred by TERTp mutations was restricted to GBM patients carrying the rs2853669 A allele and not in those carrying the G allele. In conclusion, the presence of TERTp mutations was associated with worse prognosis in GBM patients, although such association depended on the status of the rs2853669 SNP. The status of the rs2853669 SNP should be taken in consideration when assessing the prognostic value of TERTp mutations in GBM patients. TERTp mutations and the rs2853669 SNP can be used in the future as biomarkers of glioma prognosis.

Transforming growth factor beta (TGF-ß) plays an important role in carcinogenesis. Two polymorphisms in the TGF-ß1 gene (-509C/T and 869T/C) were described to influence susceptibility to gastric and breast cancers. The 869T/C polymorphism was also associated with overall survival in breast cancer patients. In the present study, we investigated the relevance of these TGF-ß1 polymorphism in glioma risk and prognosis. A case-control study that included 114 glioma patients and 138 cancer-free controls was performed. Single nucleotide polymorphisms (SNPs) were evaluated by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP). Univariate and multivariate logistic regression analyses were used to calculate odds ratio (OR) and 95 % confidence intervals (95 % CI). The influence of TGF-ß1 -509C/T and 869T/C polymorphisms on glioma patient survival was evaluated by a Cox regression model adjusted for patients' age and sex and represented in Kaplan-Meier curves. Our results demonstrated that TGF-ß1 gene polymorphisms -509C/T and 869T/C are not significantly associated with glioma risk. Survival analyses showed that the homozygous -509TT genotype associates with longer overall survival of glioblastoma (GBM) patients when compared with patients carrying CC + CT genotypes (OR, 2.41; 95 % CI, 1.06-5.50; p = 0.036). In addition, the homozygous 869CC genotype is associated with increased overall survival of GBM patients when compared with 869TT + TC genotypes (OR, 2.62; 95 % CI, 1.11-6.17; p = 0.027). In conclusion, this study suggests that TGF-ß1 -509C/T and 869T/C polymorphisms are not significantly associated with risk for developing gliomas but may be relevant prognostic biomarkers in GBM patients., This work was supported by Fundação para a Ciência e Tecnologia, Portugal (PTDC/SAU-GMG/113795/2009 and SFRH/BPD/33612/2009 to B.M.C.; SFRH/BD/88121/2012 to J.V.C.; SFRH/BD/92786/2013 to C.S.G.; PTDC/SAU-ONC/115513/2009 to R.R.).

Both genetic and environmental factors are thought to be causal in gliomagenesis. Several genes have been implicated in glioma development, but the putative role of a major immunity-related gene complex member, immunoglobulin heavy chain ? (IGHG) has not been evaluated. Prior observations that IGHG-encoded ? marker (GM) allotypes exhibit differential sensitivity to an immunoevasion strategy of cytomegalovirus, a pathogen implicated as a promoter of gliomagenesis, has lead us to hypothesize that these determinants are risk factors for glioma. To test this hypothesis, we genotyped the IGHG locus comprising the GM alleles, specifically GM alleles 3 and 17, of 120 glioma patients and 133 controls via TaqMan® genotyping assay. To assess the associations between GM genotypes and the risk of glioma, we applied an unconditional multivariate logistic regression analysis adjusted for potential confounding variables. In comparison to subjects who were homozygous for the GM 17 allele, the GM 3 homozygotes were over twice as likely, and the GM 3/17 heterozygotes were over three times as likely, to develop glioma. Similar results were achieved when analyzed by combining the data corresponding to alleles GM 3 and GM 3/17 in a dominant model. The GM 3/17 genotype and the combination of GM 3 and GM 3/17 were found to be further associated with over 3 times increased risk for high-grade astrocytoma (grades III-IV). Allele frequency analyses also showed an increased risk for gliomas and high-grade astrocytoma in association with GM 3. Our findings support the premise that the GM 3 allele may present risk for the development of glioma, possibly by modulating immunity to cytomegalovirus., This work was supported in part by the US. National Institute of Neurological Disorders and Stroke and by Fundacao a Ciencia e Tecnologia, Portugal (PTDC/SAU-ONC/115513/2009). The authors thank the Immunochemotherapy Department of Hospital S. Marcos, and Clinica Laboratorial Dr. Edgar Botelho Moniz, S. Tirso, Portugal, for their helpful assistance in the management of controls, and to Monica Ferreira from ICVS for her valuable assistance in the management of cases and controls.

Microsatellite instability (MSI) frequency in pediatric high-grade glioma remains a controversial research topic, and there is lack of clarity in the literature. Overall, it has been shown that MSI-positivity in adult high-grade glioma is a rare event, whereas in pediatric tumors reported frequencies are highly variable, probably due to the lack of consistency in the screening strategies. Furthermore, in contrast to colorectal MSI-positive tumors that harbor Type B MSI, high-grade gliomas have been reported as presenting Type A MSI, increasing the difficulty of an accurate analysis. In this chapter, we will review the type of MSI mostly found in pediatric high-grade gliomas and the distinct approaches to detect it, the role of the mismatch repair system (MMR) in these tumors, as well as the relation of MSI with other genomic abnormalities and the frequency of MSI target genes mutations.

1.1 Brain tumours in children and adults Primary Central Nervous System (CNS) tumours are considered to be those that originate in the CNS and usually remain there. Primary CNS tumours, despite not leading the cancer frequency rates, rank first among cancer types for the average years of life lost (Burnet et al., 2005). By contrast with other malignancies, where research has lead to the establishment of more successful treatment options, the diagnosis of a CNS tumour, particularly the more malignant histological types, still has devastating effects on the patients and their relatives, not only due to the dismal prognosis of these tumours with extremely high rates of mortality, but also to the great morbidity that actual treatment options cause (Laughton et al., 2008; Mulhern et al., 2004; Silber et al., 1992). The estimated annual worldwide age-standardized incidence of malignant primary CNS tumours is 3.5 per 100,000 people, which represents more than 200,000 cases. The rate is slightly higher in males (3.9 per 100,000 people per year) than in females (3.1 per 100,000 people per year) (Ferlay et al., 2008). The frequency of brain tumours is also higher in more developed countries (5.2 per 100,000 people per year) compared to less developed countries (3.0 per 100,000 people per year), probably due to diagnostic advances, as well as access to adequate health care in the developed regions (Ferlay et al., 2008; Wrensch et al., 2005). Similarly, estimated annual global age-standardized mortality of malignant primary CNS tumours is higher in males (3.0 per 100,000 people) than in females (2.2 per 100,000 people), with higher rates also in more developed countries (3.2 per 100,000 people) than in less developed regions (2.3 per 100,000 people) (Ferlay et al., 2008). The median age of diagnosis for all primary CNS tumours is 57 years, however the histology-specific median age ranges from 9 to 70 years ( Central Brain Tumor Registry of the United States [CBTRUS], 2010).

The authors would like to thank Dr. Steve Jackson's Laboratory at the University of Cambridge for the MRE11 antibody and Dr. Sandra Costa from ICVS, University of Minho, for the disease-free controls DNA, High grade gliomas (HGG) are one of the leading causes of cancer-related deaths in children, and there is increasing evidence that pediatric HGG may harbor distinct molecular characteristics compared to adult tumors. We have sought to clarify the role of microsatellite instability (MSI) in pediatric versus adult HGG. MSI status was determined in 144 patients (71 pediatric and 73 adults) using a well established panel of five quasimonomorphic mononucleotide repeat markers. Expression of MLH1, MSH2, MSH6 and PMS2 was determined by immunohistochemistry, MLH1 was assessed for mutations by direct sequencing and promoter methylation using MS-PCR. DNA copy number profiles were derived using array CGH, and mutations in eighteen MSI target genes studied by multiplex PCR and genotyping. MSI was found in 14/71 (19.7%) pediatric cases, significantly more than observed in adults (5/73, 6.8%; p=0.02, Chi-square test). MLH1 expression was downregulated in 10/13 cases, however no mutations or promoter methylation were found. MSH6 was absent in one pediatric MSI-High tumor, consistent with an inherited mismatch repair deficiency associated with germline MSH6 mutation. MSI was classed as Type A, and associated with a remarkably stable genomic profile. Of the eighteen classic MSI target genes, we identified mutations only in MSH6 and DNAPKcs and described a polymorphism in MRE11 without apparent functional consequences in DNA double strand break detection and repair. This study thus provides evidence for a potential novel molecular pathway in a proportion of gliomas associated with the presence of MSI., British Council and the Portuguese Council of Rectors, Treaty of Windsor Anglo-Portuguese Joint Research Programme (R.M. Reis, C. Jones). M. Viana-Pereira is funded by a PhD fellowship ‘[SFRH/BD/29145/2006]’ of Fundação para a Ciência e Tecnologia, Portugal. (FCT).

B.M. Costa and M. Viana-Pereira contributed equally to this work; The authors thank the Immunochemotherapy Department of Hospital S. Marcos, and Clinica Laboratorial Dr. Edgar Botelho Moniz, S. Tirso, Portugal, for their helpful assistance in the management of controls, BACKGROUND: The epidermal growth factor receptor (EGFR) regulates important cellular processes and is frequently implicated in human tumors. Three EGFR polymorphisms have been described as having a transcriptional regulatory function: two single-nucleotide polymorphisms in the essential promoter region, -216G/T and -191C/A, and a polymorphic (CA)(n) microsatellite sequence in intron 1. We aimed to elucidate the roles of these EGFR polymorphisms in glioma susceptibility and prognosis. METHODS: We conducted a case-control study with 196 patients with glioma and 168 cancer-free controls. Unconditional multivariate logistic regression models were used to calculate ORs and 95% confidence intervals. A Cox regression model was used to evaluate associations with patient survival. False-positive report probabilities were also assessed. RESULTS: None of the EGFR -216G/T variants was significantly associated with glioma risk. The -191C/A genotype was associated with higher risk for glioma when the (CA)(n) alleles were classified as short for ≤16 or ≤17 repeats. Independently of the (CA)(n) repeat cutoff point used, shorter (CA)(n) repeat variants were significantly associated with increased risk for glioma, particularly glioblastoma and oligodendroglioma. In all tested models with different (CA)(n) cutoff points, only -191C/A genotype was consistently associated with improved survival of patients with glioblastoma. CONCLUSIONS: Our findings implicate EGFR -191C/A and the (CA)(n) repeat polymorphisms as risk factors for gliomas, and suggest -191C/A as a prognostic marker in glioblastoma. Impact: Our data support a role of these EGFR polymorphisms in determining glioma susceptibility, with potential relevance for molecularly based stratification of patients with glioblastoma for individualized therapies., Schering-Plough Farma, Portugal, Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BPD/33612/2009; SFRH/BD/29145/2006

As genome-scale technologies begin to unravel the complexity of the equivalent tumors in adults, we can attempt detailed characterization of high-grade gliomas in children, that have until recently been lacking. Toward this end, we sought to validate and extend investigations of the differences between pediatric and adult tumors. Purpose: As genome-scale technologies begin to unravel the complexity of the equivalent tumors in adults, we can attempt detailed characterization of high-grade gliomas in children, that have until recently been lacking. Toward this end, we sought to validate and extend investigations of the differences between pediatric and adult tumors. Experimental Design: We carried out copy number profiling by array comparative genomic hybridization using a 32K bacterial artificial chromosome platform on 63 formalin-fixed paraffin-embedded cases of high-grade glioma arising in children and young people (, National Health Service funding to the NIHR Biomedical Research Centre. This work was supported by The Royal Marsden Children's Department Fund, Fundação para a Ciência e Tecnologia, Portugal, and Breakthrough Breast Cancer

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.

Purpose: The epidermal growth factor receptor (EGFR) is amplified and overexpressed in adult glioblastoma, with response to targeted inhibition dependent on the underlying biology of the disease. EGFR has thus far been considered to play a less important role in pediatric glioma, although extensive data are lacking. We have sought to clarify the role of EGFR in pediatric high-grade glioma (HGG). Experimental Design: We retrospectively studied a total of 90 archival pediatric HGG specimens for EGFR protein overexpression, gene amplification, and mutation and assessed the in vitro sensitivity of pediatric glioma cell line models to the small-molecule EGFR inhibitor erlotinib. Results: Amplification was detected in 11% of cases, with corresponding overexpression of the receptor. No kinase or extracellular domain mutations were observed; however, 6 of 35 (17%) cases harbored the EGFRvIII deletion, including two anaplastic oligodendrogliomas and a gliosarcoma overexpressing EGFRvIII in the absence of gene amplification and coexpressing platelet-derived growth factor receptor α. Pediatric glioblastoma cells transduced with wild-type or deletion mutant EGFRvIII were not rendered more sensitive to erlotinib despite expressing wild-type PTEN. Phosphorylated receptor tyrosine kinase profiling showed a specific activation of platelet-derived growth factor receptor α/β in EGFRvIII-transduced pediatric glioblastoma cells, and targeted coinhibition with erlotinib and imatinib leads to enhanced efficacy in this model. Conclusions: These data identify an elevated frequency of EGFR gene amplification and EGFRvIII mutation in pediatric HGG than previously recognized and show the likely necessity of targeting multiple genetic alterations in the tumors of these children., Cancer Research UK grants C1178/A10294, C309/A2187, and C309/A8274; Oak Foundation (L. Marshall); La Fondation de France (N. Gaspar); and Breakthrough Breast Cancer (J.S. Reis-Filho). We acknowledge NHS funding to the National Institute for Health Research Biomedical Research Centre.

Background: Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines. Principal Findings: All lines proliferate as adherent monolayers and express glial markers. Copy number profiling revealed complex genomes including amplification and deletions of genes known to be pivotal in core glioblastoma signalling pathways. Expression profiling identified 93 differentially expressed genes which were able to distinguish between the adult and paediatric high grade cell lines, including a number of kinases and co-ordinated sets of genes associated with DNA integrity and the immune response. Significance: These data demonstrate that glioma cell lines derived from paediatric patients show key molecular differences to those from adults, some of which are well known, whilst others may provide novel targets for evaluation in primary tumours. We thus provide the rationale and demonstrate the practicability of using paediatric glioma cell lines for preclinical and mechanistic studies., (undefined)

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.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).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.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.

Background: The smads are intracellular proteins critical to the transmission of signals from the transforming growth factor-β (TGF- β) pathway to the nucleus. SMAD7 is a potent antagonist of TGF- β signalling pathways and genome-wide association studies (GWAS) linked three single nucleotide polymorphisms (SNPs) in SMAD7 gene (rs4464148, rs4939827 and rs12953717) to a modest increase in the susceptibility to colorectal cancer (CRC) development. Some studies even hypothesized that SMAD7 correlates with the development of metastasis and prognosis of CRC. The main aim of this study is to investigate the association between these three SNPs and the risk of CRC in an independent Portuguese population. The authors also accessed the eventual impact on the prognosis of this disease. Methods: This was a population-based case-control retrospective study that included 202 patients diagnosed with CRC and 197 healthy controls, from northern Portugal. The DNA (obtained from nucleated blood cells) was genotyped for a panel of 3 SNPs (rs4464148, rs4939827 and rs12953717) using a Sequenom® MassARRAY platform. We performed a multivariate logistic regression analysis (which included age and sex as covariates) to calculate Odds Ratio (OR) and 95% confidence intervals (CI) of association between SMAD7 SNPs and risk of CRC. ACox regression model was used to calculate Hazard ratios (HR), adjusted to age and sex, in the survival analysis. Results: The median age of patients at diagnosis was 66 years old and the majority of the tumors were located in the colon (61%). The median age of controls was 50 years old. Median time offollow up was 40 months. We replicated the association of the SNP rs4939827 with the risk of CRC. Compared with the TT genotype, the carriers of the CT genotype had an adjusted OR of 2.38 (95% CI: 1.03-5.46; p = .04). The analysis of the SNP rs12953717 revealed a strong tendency for association of the genotype TT with increased risk of CRC (OR = 3.87, 95% CI: 0.99-15.2; p = .052). The rs4464148 did not show any association with cancer risk. None of the SNPs depicted an association with patient’s survival. Conclusion: Replications offindings in GWAS are important to secure that the evidence of association with disease risk is robust. This is the first study that analyzes the SMAD7 SNPs in the Portuguese population. We found an association between the genotype CT in the SNP rs4939827 and increased risk of CRC. The other two SNPs studied did not correlate with CRC risk in our population. Overall survival of the patients also did not relate to the variants of these three SMAD7 polymorphisms.