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.

Replication of GWAS identifies RTEL1, CDKN2A/B, and PHLDB1 SNPs as risk factors in Portuguese gliomas 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.

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.

Effects of the functional HOTAIR rs920778 and rs12826786 genetic variants in glioma susceptibility and patient prognosis.

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.

Study of hTERT and Histone 3 Mutations in Medulloblastoma.

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 <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 medulloblastoma.

The prognostic impact of TERT promoter mutations in glioblastomas is modified by the rs2853669 single nucleotide polymorphism.

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.

Significance of glycolytic metabolism-related protein expression in colorectal cancer, lymph node and hepatic metastasis.

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.

Impact of TGF-ß1 -509C/T and 869T/C polymorphisms on glioma risk and patient 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.

Impact of Suppressor of Mothers Against Decapentaplegic (SMAD) 7 Gene Single Nucleotide Polymorphisms on Colorectal Cancer Risk and Prognosis.

Colorectal cancer (CRC) is a prevalent diagnosis worldwide with significant associated mortality. Single nucleotide polymorphic (SNP) variants have been identified as being associated with CRC risk. Although SMAD7 SNPs have been associated with the risk of developing CRC, their prognostic effect is still unclear. We carried out a case-control study to establish an association between genotypes of the suppressor of mothers against decapentaplegic (SMAD) 7 SNP rs4464148, rs4939827, and rs12953717 and CRC risk. Furthermore, we retrospectively assessed whether these SNPs had prognostic implications in CRC patients by evaluating survival with Kaplan-Meier curves and Cox regression. Only the CT genotype of the rs4939827 variant showed an association with CRC risk, and no genotype (CC, CT, or TT) of any of the three SNPs was shown to have prognostic implications in overall survival. Our study failed to show an association between certain SNP genotypes and the risk of CRC, which has already been well documented in two meta-analyses. Furthermore, it showed no prognostic relevance for these SNPs. More studies are needed to understand whether there are population variations or haplotype effects that could disturb the evaluation of these results.

Heart Failure: From Typical Clinical Manifestations to the Surprising Final Diagnosis.

The authors report a case of an 80-year-old woman with multiple cardiovascular risk factors, with exuberant acute congestive heart failure at admission. Fever, anemia, and an increase in inflammatory parameters were present, with imaging suggesting a respiratory infection as the main reason for decompensation. Empirical antibiotic therapy was instituted, with no clinical improvement even after escalation to broad-spectrum antibiotics and non-invasive ventilation with high support pressures, with no possibility of weaning. Due to maintenance of symptoms, a transthoracic echocardiogram was performed, revealing a large left atrial myxoma, obstructing the mitral valve in diastole. This case illustrates the potential severity of these benign tumors and their ability to mimic symptoms that are often evaluated in the daily life of an internist. The high clinical suspicion led to a diagnosis that was surprising due to its rarity and severity, with the patient being urgently referred for cardiac surgery.

Seronegative Celiac Disease - A Challenging Case.

Celiac disease is an inflammatory disorder of the small intestine caused by sensitivity to gluten. This enteropathy results from the interaction between genetics, autoimmunity, and an environmental trigger (gluten). It can manifest at all ages. We present a case of a 76-year-old woman with nausea and vomiting for six months. She reported asthenia, weight loss, and a brief period of diarrhea without blood or mucus. The search for evidence of infection, tumours, and endocrinopathies was negative, as well as the immunological study, including antibodies for celiac disease. Upper endoscopy with biopsies revealed villous atrophy. Capsule endoscopy showed macroscopic features suggestive of celiac/Whipple's disease. Duodenal biopsies were reviewed, and Whipple's disease was considered unlikely. The genetic analysis was positive for HLA DR7-DQ2. After one year on a gluten-free diet, there was a clinical and histological improvement. The diagnosis of seronegative celiac disease is complex and requires the exclusion of other causes of villous atrophy, as well as a histological improvement after one year of treatment. The genetic test has a high negative predictive value.

Microsatellite Instability Analysis in Gastric Carcinomas of Moroccan Patients.

Aim: To investigate correlations between microsatellite instability (MSI) and the phenotype, clinicopathological features, and overall survival (OS) in Moroccan gastric cancer (GC) patients. We evaluated the mutation frequency of 22 MSI-target genes in MSI-positive tumors. Materials and Methods: MSI evaluation were performed for 97 gastric tumors by multiplex polymerase chain reaction (PCR) using a panel of five quasimonomorphic mononucleotide repeat markers (NR27, NR21, NR24, BAT25, and BAT26). The mutation profiles of 22 MSI-target genes were assessed by multiplex PCR and genotyping. Kaplan-Meier curves, the log-rank test, and the Cox proportional hazard regression model were used to conduct survival analyses. Results: Microsatellite stable (MSS) status was observed in 77/97 (79.4%) gastric cancer samples, MSI-Low in 7 (7.2%) samples, and MSI-High (MSI-H) in 13 (13.4%) cases. The MSI-H phenotype was significantly associated with older age (p = 0.004), tumor location (p < 0.001), and intestinal-type of Lauren classification (p < 0.001). Among the 22 MSI target genes analyzed, the most frequently altered genes were HSP110 (84.6%), EGFR (30.8%), BRCA2 (23.1%), MRE11 (23.1%), and MSH3 (23.1%). Multivariate analysis revealed the MSS phenotype (Hazard ratio, 0.23; 95% confidence interval, 0.7-7.4; p = 0.014) as an independent indicator of poor prognosis in our population. Conclusions: This study is the first analysis of MSI in Moroccan GC patients. MSI-H GCs have distinct clinicopathological features and an improved OS. We have identified candidate target genes altered in MSI-positive tumors with potential clinical implications. These findings can guide immunotherapy designed for Moroccan GC patients.

Reproduction of the Cancer Genome Atlas (TCGA) and Asian Cancer Research Group (ACRG) Gastric Cancer Molecular Classifications and Their Association with Clinicopathological Characteristics and Overall Survival in Moroccan Patients.

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.

Loss of SPINT2 expression frequently occurs in glioma, leading to increased growth and invasion via MMP2.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

Low frequency of MAP kinase pathway alterations in KIT and PDGFRA wild-type GISTs.

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.

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.

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 non-neoplastic 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.

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.

Molecular Profiling of a Rare Rosette-Forming Glioneuronal Tumor Arising in the Spinal Cord.

Rosette-forming glioneuronal tumor (RGNT) of the IV ventricle is a rare and recently recognized brain tumor entity. It is histologically composed by two distinct features: a glial component, resembling pilocytic astrocytoma, and a component forming neurocytic rosettes and/or perivascular rosettes. Herein, we describe a 33-year-old man with RGNT arising in the spinal cord. Following an immunohistochemistry validation, we further performed an extensive genomic analysis, using array-CGH (aCGH), whole exome and cancer-related hotspot sequencing, in order to better understand its underlying biology. We observed the loss of 1p and gain of 1q, as well as gain of the whole chromosomes 7, 9 and 16. Local amplifications in 9q34.2 and 19p13.3 (encompassing the gene SBNO2) were identified. Moreover, we observed focal gains/losses in several chromosomes. Additionally, on chromosome 7, we identified the presence of the KIAA1549:BRAF gene fusion, which was further validated by RT-PCR and FISH. Across all mutational analyses, we detected and validated the somatic mutations of the genes MLL2, CNNM3, PCDHGC4 and SCN1A. Our comprehensive molecular profiling of this RGNT suggests that MAPK pathway and methylome changes, driven by KIAA1549:BRAF fusion and MLL2 mutation, respectively, could be associated with the development of this rare tumor entity.