Oligodendroglial tumours originate from oligodendrocytes usually arising in the white matter and could be classified into grade-II oligodendrogliomas (OD) and anaplastic oligodendrogliomas (AOD, grade-III) according to the 2016 World Health Organization (WHO) grading scheme. ODs1 could be diagnosed by pathological and immunohistochemical analyses, however recent evidence suggests that they could be better diagnosed on the basis of defined genetic entities, such as the combined loss of chromosome 1p and 19q arms and IDH mutation. 1p/19q co-deletion is molecular hallmark of ODs and is clinically associated with better prognosis, response to chemo/radio-therapy and overall survival. Typical oligodendroglial histological features are strongly associated with 1p/19q loss and IDH mutation, which is critically important as diagnostic point of view. The examining of exclusive molecular signatures and transcriptome expression profiles added to histological class could compliment the classification of OD subtypes. In this regard, microRNAs (miRNAs, miRs) profiles could serve classifier signatures for tumour subsets. MiRNAs are 22nt short non-coding RNAs which are expressed endogenously and regulate diverse cellular process through negative control on gene expression at the posttranscriptional level by direct or imperfect interaction with their target mRNAs. MiRNAs are involved in regulating human tumorigenesis acting as either tumour suppressors or oncogenes. During the passage of tumorigenesis miRNA expression level is significantly increased or decreased compared to corresponding normal tissue. The same is observed with their mRNAs. Therefore, transcriptome profiling of human tumours could identify signatures associated with progression, diagnosis, prognosis and response to therapy. However, until recently the information regarding the expression of miRNAs and mRNA in oligodendroglial tumours is scarce. In this study we performed miRNA and mRNA differential expression profiling between grade II and grade III ODs using microarray based expression profiling platforms (723 transcripts and 41,000 genes, respectively). 7 cases for OD grade-II, and 7 for AOD grade-III, and 15 non neoplastic white matter (nnWM) samples were used after microdissection with no previous history of treatment. We performed a systematic evaluation of miRNAs and mRNAs expressions and determined miRNAs and putative target genes that are differentially expressed in grade III AOD, but not in grade II OD and in non-neoplastic white matter (nnWM). 1 ODs when used with ,,s\" will represent both OD and AOD. 50 miRNAs were overexpressed and 43 were down regulated in AOD-III, whereas 7 miRNAs showed significant reduction in expressions in OD-II group. 3 miRNAs were commonly down regulated in comparisons of both groups. The hsa-miR-23a was strongly upregulated and hsamiR-27a was strongly downregulated in AOD-III. The functions of hsa-miR-23a and hsa-miR- 27a were tested in human adult fibroblasts for cell proliferation assay and apoptosis detection. Cells treated with pre-miR-23a and pre-miR-27a showed 20% reduction in cell proliferation as compared with controls. Further, the functional relevance of miRNAs to their target mRNAs was validated for each group, using real time qPCR. 10 key-miRNAs from AOD were subjected to validation by qPCR. We were able to confirm 7 miRNAs (p? 0.05). Among these, 5 miRs (miR- 193a-3p, miR-24, miR-27a, miR-30a-5p and miR-30c) showed reduced expression whose target genes (CCND1, HDAC2, PDGFA and RAB-26) were upregulated. Whereas, 2 miRNAs likewise miR-301b and miR-378 were overexpressed whose target genes BCL2, FGF2, CD44 and PPP4R4 confirmed by qPCR (p? 0.05). Bioinformatics based gene ontology (GO), and networking analysis revealed that differential expression and targets are attributed to differentiation of embryonic stem cells, cell adhesion, angiogenesis and neurogenesis, resistance to apoptosis, protein-protein interactions and cell proliferation. It was possible to identify and validate miRNAs and their mRNA-targets potentially involved in the progression of oligodendrogliomas particularly in grade III-AOD. Collectively, this analysis provides new insights to malignant progression of oligodendroglial tumours and could compliment WHO-2016 diagnosis scheme and may provide predictive outcome in patients as well as decision to therapy. / Oligodendrogliomas originários de oligodendrócitos que geralmente surgem na substância branca podendo ser classificados em grau oligodendroglioma (II-OD), e anaplastic oligodendrogliomas (grau III-AOD). Os ODs2 podem ser diagnosticados por análises patológicas e imuno-histoquímicas, porém evidências recentes sugerem que poderiam ser melhor diagnosticados com base em assinaturas moleculares, como a deleção combinada dos cromossomas 1p e 19q - marcadores moleculares dos OD associados clinicamente a um melhor prognóstico, resposta à terapia e melhor sobrevida. As características histológicas típicas dos oligodendrogliomas também estão fortemente associadas à deleção de 1p/19q, que é criticamente importante como ponto de vista diagnóstico. Assim, os subtipos de gliomas podem ser fortemente diferenciados não somente em relação ao seu perfil histológico mas também com base em seu perfil de expressão genica e suas assinaturas moleculares exclusivas. Os microRNAs (miRNAs, miRs) emergiram como assinaturas moleculares para os diferentes graus. Os miRNAs são RNAs não codificantes, contendo em torno de 22 nucleótidos. São expressos endogenamente e regulam diversos processos celulares através do controle negativo da expressão gênica em nivel pós-transcricional e por interacção directa ou imperfeita com o RNAm-alvo. Os miRNAs estão envolvidos na regulação da tumorigenese humana atuando como supressores de tumour ou oncogenes. Durante o processo da tumorigenese o nível de expressão dos miRNAs é aumentado ou diminuído significativamente em comparação com tecido normal correspondente. O perfil de expressão de miRNA de tumores humanos poderia identificar assinaturas associadas com progressão, diagnóstico, prognóstico e resposta à terapia. Contudo, até recentemente a informação sobre a expressão de miRNAs em oligodendrogliomas é escassa. Neste estudo, avaliamos o perfil de expressão diferencial de miRNA e RNAm em ODs graus II e III usando plataformas de perfis de expressão baseadas em microarray (723 transcritos e 41.000 genes, respectivamente). Foram utilizados 14 casos de ODs microdissecados, sendo 7 OD grau II, e 7 AOD grau III (anaplasicos) sem histórico prévio de tratamento, além de 15 amostras de substancia branca não neoplásica (nnSB). Por meio de avaliações sistemáticas foram determinados miRNAs e mRNAs expressos em AOD grau III, mas não em OD grau II e em substancias brancas não neoplásicas (nnSB). 2 ODs when used with ,,s\" will represent both OD and AOD. Assim, foram encontrados 50 miRNAs com alta expressão e 43 miRNAs com baixa expressão em AOD-III, enquanto que 7 miRNAs apresentaram expressões reduzidas no grupo OD-II. Na comparação entre os dois grupos, 3 miRNAs apresentaram baixa expressão. A hsa-miR-23a mostrou alta expressão e a hsa-miR-27a apresentou uma diminuição de expressão importante em AOD III. A atividade dos hsa-miR-23a e hsa-miR-27a foram testadas em células de fibroblastos adultos humanos usando ensaios de proliferação celular e detecção de apoptose. As células tratadas com pre-miR-23a e pre-miR-27a mostraram 20% redução de proliferação celular em comparação com os controles. Para cada grupo, a relevância funcional dos miRNAs e seus mRNAs alvos foi validada utilizando qPCR. Dos 10 miRNAs submetidos a validação em grau III, foi possivel confirmar 7 miRNA(p<0,05). Entre esses, 5 miRs (miR-193a-3p, miR-24, miR- 27a, miR-30a-5p e miR-30c) mostraram expressão reduzida, cujos genes alvos (CCND1, HDAC2, PDGFA e RAB-26) apresentavam alta expressão. Enquanto que, 2 miRNAs como miR-301b e miR-378 apresentaram alta expressão cujos genes alvo BCL2, FGF2, CD44 e PPP4R4 foram confirmados por qPCR (p<0,05). Ferramentas de bioinformática (Gene Ontology) e a análises em rede revelaram que a expressão diferencial e os alvos são atribuídos à diferenciação de células-tronco embrionárias, adesão de celular, angiogênese e neurogênese, resistência à apoptose, interações proteína-proteína e proliferação celular. Foi possível identificar e validar miRNAs e RNAm-alvos potencialmente envolvidos na progressão de oligodendrogliomas. Coletivamente, esta análise fornece novos achados relacionados a progressão maligna de tumores oligodendrogliais e poderia facilitar o diagnóstico preciso e mais restritivo, o desfecho preditivo em pacientes, bem como auxiliar na decisão da terapia.

Oligodendroglial tumours originate from oligodendrocytes usually arising in the white matter and could be classified into grade-II oligodendrogliomas (OD) and anaplastic oligodendrogliomas (AOD, grade-III) according to the 2016 World Health Organization (WHO) grading scheme. ODs1 could be diagnosed by pathological and immunohistochemical analyses, however recent evidence suggests that they could be better diagnosed on the basis of defined genetic entities, such as the combined loss of chromosome 1p and 19q arms and IDH mutation. 1p/19q co-deletion is molecular hallmark of ODs and is clinically associated with better prognosis, response to chemo/radio-therapy and overall survival. Typical oligodendroglial histological features are strongly associated with 1p/19q loss and IDH mutation, which is critically important as diagnostic point of view. The examining of exclusive molecular signatures and transcriptome expression profiles added to histological class could compliment the classification of OD subtypes. In this regard, microRNAs (miRNAs, miRs) profiles could serve classifier signatures for tumour subsets. MiRNAs are 22nt short non-coding RNAs which are expressed endogenously and regulate diverse cellular process through negative control on gene expression at the posttranscriptional level by direct or imperfect interaction with their target mRNAs. MiRNAs are involved in regulating human tumorigenesis acting as either tumour suppressors or oncogenes. During the passage of tumorigenesis miRNA expression level is significantly increased or decreased compared to corresponding normal tissue. The same is observed with their mRNAs. Therefore, transcriptome profiling of human tumours could identify signatures associated with progression, diagnosis, prognosis and response to therapy. However, until recently the information regarding the expression of miRNAs and mRNA in oligodendroglial tumours is scarce. In this study we performed miRNA and mRNA differential expression profiling between grade II and grade III ODs using microarray based expression profiling platforms (723 transcripts and 41,000 genes, respectively). 7 cases for OD grade-II, and 7 for AOD grade-III, and 15 non neoplastic white matter (nnWM) samples were used after microdissection with no previous history of treatment. We performed a systematic evaluation of miRNAs and mRNAs expressions and determined miRNAs and putative target genes that are differentially expressed in grade III AOD, but not in grade II OD and in non-neoplastic white matter (nnWM). 1 ODs when used with ,,s\" will represent both OD and AOD. 50 miRNAs were overexpressed and 43 were down regulated in AOD-III, whereas 7 miRNAs showed significant reduction in expressions in OD-II group. 3 miRNAs were commonly down regulated in comparisons of both groups. The hsa-miR-23a was strongly upregulated and hsamiR-27a was strongly downregulated in AOD-III. The functions of hsa-miR-23a and hsa-miR- 27a were tested in human adult fibroblasts for cell proliferation assay and apoptosis detection. Cells treated with pre-miR-23a and pre-miR-27a showed 20% reduction in cell proliferation as compared with controls. Further, the functional relevance of miRNAs to their target mRNAs was validated for each group, using real time qPCR. 10 key-miRNAs from AOD were subjected to validation by qPCR. We were able to confirm 7 miRNAs (p? 0.05). Among these, 5 miRs (miR- 193a-3p, miR-24, miR-27a, miR-30a-5p and miR-30c) showed reduced expression whose target genes (CCND1, HDAC2, PDGFA and RAB-26) were upregulated. Whereas, 2 miRNAs likewise miR-301b and miR-378 were overexpressed whose target genes BCL2, FGF2, CD44 and PPP4R4 confirmed by qPCR (p? 0.05). Bioinformatics based gene ontology (GO), and networking analysis revealed that differential expression and targets are attributed to differentiation of embryonic stem cells, cell adhesion, angiogenesis and neurogenesis, resistance to apoptosis, protein-protein interactions and cell proliferation. It was possible to identify and validate miRNAs and their mRNA-targets potentially involved in the progression of oligodendrogliomas particularly in grade III-AOD. Collectively, this analysis provides new insights to malignant progression of oligodendroglial tumours and could compliment WHO-2016 diagnosis scheme and may provide predictive outcome in patients as well as decision to therapy. / Oligodendrogliomas originários de oligodendrócitos que geralmente surgem na substância branca podendo ser classificados em grau oligodendroglioma (II-OD), e anaplastic oligodendrogliomas (grau III-AOD). Os ODs2 podem ser diagnosticados por análises patológicas e imuno-histoquímicas, porém evidências recentes sugerem que poderiam ser melhor diagnosticados com base em assinaturas moleculares, como a deleção combinada dos cromossomas 1p e 19q - marcadores moleculares dos OD associados clinicamente a um melhor prognóstico, resposta à terapia e melhor sobrevida. As características histológicas típicas dos oligodendrogliomas também estão fortemente associadas à deleção de 1p/19q, que é criticamente importante como ponto de vista diagnóstico. Assim, os subtipos de gliomas podem ser fortemente diferenciados não somente em relação ao seu perfil histológico mas também com base em seu perfil de expressão genica e suas assinaturas moleculares exclusivas. Os microRNAs (miRNAs, miRs) emergiram como assinaturas moleculares para os diferentes graus. Os miRNAs são RNAs não codificantes, contendo em torno de 22 nucleótidos. São expressos endogenamente e regulam diversos processos celulares através do controle negativo da expressão gênica em nivel pós-transcricional e por interacção directa ou imperfeita com o RNAm-alvo. Os miRNAs estão envolvidos na regulação da tumorigenese humana atuando como supressores de tumour ou oncogenes. Durante o processo da tumorigenese o nível de expressão dos miRNAs é aumentado ou diminuído significativamente em comparação com tecido normal correspondente. O perfil de expressão de miRNA de tumores humanos poderia identificar assinaturas associadas com progressão, diagnóstico, prognóstico e resposta à terapia. Contudo, até recentemente a informação sobre a expressão de miRNAs em oligodendrogliomas é escassa. Neste estudo, avaliamos o perfil de expressão diferencial de miRNA e RNAm em ODs graus II e III usando plataformas de perfis de expressão baseadas em microarray (723 transcritos e 41.000 genes, respectivamente). Foram utilizados 14 casos de ODs microdissecados, sendo 7 OD grau II, e 7 AOD grau III (anaplasicos) sem histórico prévio de tratamento, além de 15 amostras de substancia branca não neoplásica (nnSB). Por meio de avaliações sistemáticas foram determinados miRNAs e mRNAs expressos em AOD grau III, mas não em OD grau II e em substancias brancas não neoplásicas (nnSB). 2 ODs when used with ,,s\" will represent both OD and AOD. Assim, foram encontrados 50 miRNAs com alta expressão e 43 miRNAs com baixa expressão em AOD-III, enquanto que 7 miRNAs apresentaram expressões reduzidas no grupo OD-II. Na comparação entre os dois grupos, 3 miRNAs apresentaram baixa expressão. A hsa-miR-23a mostrou alta expressão e a hsa-miR-27a apresentou uma diminuição de expressão importante em AOD III. A atividade dos hsa-miR-23a e hsa-miR-27a foram testadas em células de fibroblastos adultos humanos usando ensaios de proliferação celular e detecção de apoptose. As células tratadas com pre-miR-23a e pre-miR-27a mostraram 20% redução de proliferação celular em comparação com os controles. Para cada grupo, a relevância funcional dos miRNAs e seus mRNAs alvos foi validada utilizando qPCR. Dos 10 miRNAs submetidos a validação em grau III, foi possivel confirmar 7 miRNA(p<0,05). Entre esses, 5 miRs (miR-193a-3p, miR-24, miR- 27a, miR-30a-5p e miR-30c) mostraram expressão reduzida, cujos genes alvos (CCND1, HDAC2, PDGFA e RAB-26) apresentavam alta expressão. Enquanto que, 2 miRNAs como miR-301b e miR-378 apresentaram alta expressão cujos genes alvo BCL2, FGF2, CD44 e PPP4R4 foram confirmados por qPCR (p<0,05). Ferramentas de bioinformática (Gene Ontology) e a análises em rede revelaram que a expressão diferencial e os alvos são atribuídos à diferenciação de células-tronco embrionárias, adesão de celular, angiogênese e neurogênese, resistência à apoptose, interações proteína-proteína e proliferação celular. Foi possível identificar e validar miRNAs e RNAm-alvos potencialmente envolvidos na progressão de oligodendrogliomas. Coletivamente, esta análise fornece novos achados relacionados a progressão maligna de tumores oligodendrogliais e poderia facilitar o diagnóstico preciso e mais restritivo, o desfecho preditivo em pacientes, bem como auxiliar na decisão da terapia.

Background Molecular data of histologically classified oligodendrogliomas are available offering the possibility to stratify these human brain tumors into clinically relevant molecular subtypes. Methods Gene copy number, mutation, and expression data of 193 histologically classified oligodendrogliomas from The Cancer Genome Atlas (TCGA) were analyzed by well-established computational approaches (unsupervised clustering, statistical testing, network inference). Results We applied hierarchical clustering to tumor gene copy number profiles and revealed three molecular subgroups within histologically classified oligodendrogliomas. We further screened these subgroups for molecular glioma markers (1p/19q co-deletion, IDH mutation, gain of chromosome 7 and loss of chromosome 10) and found that our subgroups largely resemble known molecular glioma subtypes. We excluded glioblastoma-like tumors (7a10d subgroup) and derived a gene expression signature distinguishing histologically classified oligodendrogliomas with concurrent 1p/19q co-deletion and IDH mutation (1p/19q subgroup) from those with predominant IDH mutation alone (IDHme subgroup). Interestingly, many signature genes were part of signaling pathways involved in the regulation of cell proliferation, differentiation, migration, and cell-cell contacts. We further learned a gene regulatory network associated with the gene expression signature revealing novel putative major regulators with functions in cytoskeleton remodeling (e.g. APBB1IP, VAV1, ARPC1B), apoptosis (CCNL2, CREB3L1), and neural development (e.g. MYTIL, SCRT1, MEF2C) potentially contributing to the manifestation of differences between both subgroups. Moreover, we revealed characteristic expression differences of several HOX and SOX transcription factors suggesting the activity of different glioma stemness programs in both subgroups. Conclusions We show that gene copy number profiles alone are sufficient to derive molecular subgroups of histologically classified oligodendrogliomas that are well-embedded into general glioma classification schemes. Moreover, our revealed novel putative major regulators and characteristic stemness signatures indicate that different developmental programs might be active in these subgroups, providing a basis for future studies.

Background Molecular data of histologically classified oligodendrogliomas are available offering the possibility to stratify these human brain tumors into clinically relevant molecular subtypes. Methods Gene copy number, mutation, and expression data of 193 histologically classified oligodendrogliomas from The Cancer Genome Atlas (TCGA) were analyzed by well-established computational approaches (unsupervised clustering, statistical testing, network inference). Results We applied hierarchical clustering to tumor gene copy number profiles and revealed three molecular subgroups within histologically classified oligodendrogliomas. We further screened these subgroups for molecular glioma markers (1p/19q co-deletion, IDH mutation, gain of chromosome 7 and loss of chromosome 10) and found that our subgroups largely resemble known molecular glioma subtypes. We excluded glioblastoma-like tumors (7a10d subgroup) and derived a gene expression signature distinguishing histologically classified oligodendrogliomas with concurrent 1p/19q co-deletion and IDH mutation (1p/19q subgroup) from those with predominant IDH mutation alone (IDHme subgroup). Interestingly, many signature genes were part of signaling pathways involved in the regulation of cell proliferation, differentiation, migration, and cell-cell contacts. We further learned a gene regulatory network associated with the gene expression signature revealing novel putative major regulators with functions in cytoskeleton remodeling (e.g. APBB1IP, VAV1, ARPC1B), apoptosis (CCNL2, CREB3L1), and neural development (e.g. MYTIL, SCRT1, MEF2C) potentially contributing to the manifestation of differences between both subgroups. Moreover, we revealed characteristic expression differences of several HOX and SOX transcription factors suggesting the activity of different glioma stemness programs in both subgroups. Conclusions We show that gene copy number profiles alone are sufficient to derive molecular subgroups of histologically classified oligodendrogliomas that are well-embedded into general glioma classification schemes. Moreover, our revealed novel putative major regulators and characteristic stemness signatures indicate that different developmental programs might be active in these subgroups, providing a basis for future studies.