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1.
Anticancer Res ; 40(11): 6229-6236, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109560

RESUMO

BACKGROUND/AIM: To compare the GeneXpert® O6-methylguanine DNA methyltransferase (MGMT) methylation prototype (GX MGMT) assay with pyrosequencing in glioblastomas. MATERIALS AND METHODS: The MGMT methylation status was retrospectively assessed in formalin-fixed paraffin embedded (FFPE) tumor blocks from 262 glioblastoma patients obtained from three independent cohorts using either a standard of care pyrosequencing laboratory developed test or the GX MGMT assay. RESULTS: The concordance rate was 92.1% (58/63) for Oregon Health and Science University (OSHU) samples, 91.7% (88/96) for Medical University of Vienna (MUV) samples, and 82.5% (85/103) for Kepler University Hospital (KUH) samples. Patients with MGMT promoter hypermethylation assessed by pyrosequencing or the GX MGMT test had a significantly longer overall survival compared to patients without hypermethylation (HR=0.43, 95%CI=0.26-0.72, p=0.001 and HR=0.51, 95%CI=0.31-0.84, p=0.008, respectively). CONCLUSION: Standardized, simplified, and on-demand testing of MGMT promoter methylation by the GX MGMT assay is feasible.


Assuntos
Bioensaio/métodos , Neoplasias Encefálicas/genética , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioblastoma/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Adulto Jovem
2.
PLoS One ; 15(8): e0238021, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841306

RESUMO

Triple-negative breast cancer (TNBC) is typically treated with chemotherapeutic agents, including carboplatin (Cb), an DNA platinating agent. The O6-methylguanine-DNA-methyltransferase gene (MGMT) encodes for the protein O6-alkylguanine-DNA-alkyltransferase (MGMT protein). MGMT protein is involved in DNA repair mechanisms to remove mutagenic and cytotoxic adducts from O6-guanine in DNA. In glioblastoma multiforme, MGMT methylation status is a predictive biomarker for increased response to temozolomide therapy. It has been suggested, that MGMT protein may have relevance for cellular adaptation and could have an influence on resistance to carboplatin therapy. We investigated the influence of MGMT promoter methylation on pathologic complete response and survival of patients with TNBC treated in the neoadjuvant GeparSixto trial. In 174 of 210 available TNBC tumors a valid MGMT promoter methylation status was determined by pyrosequencing of 5 CpG islands. In 21.8%, we detected a mean MGMT promoter methylation >10%. Overall, MGMT promoter methylation was not significantly associated with pathological complete response (pCR) rate. After stratification for the two therapy arms with and without Cb no statistically significant differences in therapy response rates between the two MGMT promoter methylation groups could be observed. Our results show that different MGMT promoter methylation status is not related to different chemotherapy response rates in the TNBC setting in GeparSixto.


Assuntos
Ensaios Clínicos como Assunto , Ensaios Clínicos Fase II como Assunto , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Neoplasias de Mama Triplo Negativas/genética , Proteínas Supressoras de Tumor/genética , Biópsia , Estudos de Coortes , Ilhas de CpG/genética , Humanos , Estudos Retrospectivos , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/patologia
3.
Nat Commun ; 11(1): 3883, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753598

RESUMO

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Rearranjo Gênico , Glioma/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Adutos de DNA/efeitos dos fármacos , Adutos de DNA/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/prevenção & controle , Regiões Promotoras Genéticas/genética , RNA-Seq , Temozolomida/uso terapêutico , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Sequenciamento Completo do Genoma , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
4.
Life Sci ; 259: 118148, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32721465

RESUMO

Pancreatic cancer is a malignant cancer with poor prognosis. This study aimed to explore how O6-methylguanine-DNA methyltransferase (MGMT) affects the gemcitabine resistance of pancreatic cancer cells by the regulatory role of SHH/GLI signaling pathway. MGMT inhibition induced by lomeguatrib (LM) suppressed the proliferation, invasion, migration and autophagy, promoted the apoptosis of PanC-1/GEM cells and up-regulated the GEM inhibition rates for PanC-1/GEM cells. Moreover, MGMT inhibition increased the expression of Caspase-3 and Bax and decreased the expression of Bcl-2, Beclin1 and Atg5 in PanC-1/GEM cells. PVT1 silencing could also produce the similar effects of MGMT inhibition induced by LM on PanC-1/GEM cells. And, PVT1 silencing could inhibit the SHH/GLI signaling pathway in PanC-1/GEM cells by regulating the MGMT expression. miR-409 was demonstrated to be a potential target of PVT1 and SHH was demonstrated to be a potential target of miR-409. Furthermore, GLI overexpression could reverse the effects of PVT1 silencing. In the xenograft model of pancreatic cancer, nude mice were treated with GEM. MGMT inhibition suppressed the tumor growth and autophagy and promoted the apoptosis in tumor tissues. And, PVT1 silencing could inhibit the SHH/GLI signaling pathway in tumor tissues. In conclusion, MGMT inhibition could suppress the proliferation, invasion, migration and autophagy and promote the apoptosis of PanC-1/GEM cells in vitro and in vivo. PVT1 silencing may affect the PanC-1/GEM cells through changing the MGMT expression by inhibiting the SHH/GLI signaling pathway.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Metilases de Modificação do DNA/biossíntese , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/biossíntese , Enzimas Reparadoras do DNA/genética , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética , Animais , Autofagia/efeitos dos fármacos , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Metilases de Modificação do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/antagonistas & inibidores , Desoxicitidina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Nus , Invasividade Neoplásica , Neoplasias Pancreáticas/patologia , Purinas/farmacologia , Purinas/uso terapêutico , Proteínas Supressoras de Tumor/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Brain Tumor Pathol ; 37(2): 50-59, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32361941

RESUMO

Aging is a known negative prognostic factor in glioblastomas (GBM). Whether particular genetic backgrounds are a factor in poor outcomes of elderly patients with GBM warrants investigation. We aim to elucidate any differences between older and younger adult patients with IDH-wildtype GBM regarding both molecular characteristics and clinical outcomes. We collected adult cases diagnosed with IDH-wildtype GBM from the Kansai Network. Clinical and pathological characteristics were analyzed retrospectively and compared between older (≥ 70 years) and younger (≤ 50 years) cases. Included were 92 older vs. 33 younger cases. The older group included more patients with preoperative Karnofsky performance status score < 70 and had a shorter survival time than the younger group. MGMT promoter was methylated more frequently in the older group. TERT promoter mutation was more common in the older group. There were significant differences in DNA copy-number alteration profiles between age groups in PTEN deletion and CDK4 amplification/gain. In the older group, no molecular markers were identified, but surgical resection was an independent prognostic factor. Age-specific survival difference was significant in the MGMT methylated and TERT wildtype subgroup. Elderly patients have several potential factors in poor prognosis of glioblastomas. Varying molecular profiles may explain differing rates of survival between generations.


Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Isocitrato Desidrogenase/genética , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/mortalidade , Estudos de Coortes , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Feminino , Glioblastoma/mortalidade , Humanos , Japão , Masculino , Metilação , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
6.
PLoS One ; 15(4): e0231932, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324779

RESUMO

BACKGROUND: Epigenetic silencing of the O6-methylguanine-DNA methyltransferase (MGMT) DNA repair enzyme via promoter hypermethylation (hmMGMT) may increase mutations in the TP53 oncosuppressor gene and contribute to carcinogenesis. The effects of smoking, which is a risk factor for head and neck squamous cell carcinoma (HNSCC), were investigated to determine whether they up- or down-regulate hmMGMT. Additionally, the impact of hmMGMT and disruptive TP53-mutations on relapse was investigated in patients with HNSCC. METHODS: This study included 164 patients with HNSCC who were negative for both p16 protein expression and human papilloma virus infection. The association of smoking and hmMGMT was investigated using multiple logistic regression analysis. Competing risk regression was used to evaluate the effects of hmMGMT and TP53-mutations in exon 2 to 11 on relapse of HNSCC. RESULTS: hmMGMT was observed in 84% of the 164 patients. TP53-mutations, specifically, G:C>A:T transition, were more frequent in patients with hmMGMT (32%) than in those without hmMGMT (8%). The frequency of disruptive TP53-mutations was not significantly different between groups. Compared with nonsmoking, heavy smoking of 20 pack-years or more was significantly associated with decreased hmMGMT (adjusted odds ratio, 0.08; 95% CI, 0.01 to 0.56; P = 0.01). Patients who had both hmMGMT and disruptive TP53-mutations showed a significantly higher relapse rate than all other patients (subdistribution hazard ratio, 1.77; 95% CI, 1.07 to 2.92; P = 0.026). CONCLUSIONS: It was found that hmMGMT was suppressed by heavy smoking, and hmMGMT combined with disruptive TP53-mutations may indicate a poor prognosis in patients with HNSCC.


Assuntos
Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Mutação , Fumar , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Recidiva
7.
J Clin Neurosci ; 75: 45-51, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32229069

RESUMO

Due to the varied overall survival (OS), limited studies focus on the factors that affect the prognosis for lower grade glioma patients (LGGs) with MGMT promoter methylated. A total of 579 samples (TCGA LGGs 456; CGGA LGGs 123) were included to identify potential genes for LGGs with MGMT promoter methylated. All bioinformatics analyses were conducted using SPSS software and GraphPad Prism 6. Based on COX regression analysis, we established a four-gene signature (ALDOC, APOBEC3C, ANXA1 and ARPP21) and divided LGGs into two groups based on median risk score. The OS of LGGs in high risk group was shorter than low risk group (P < 0.0001). Furthermore, the OS in high risk group were shorter than low risk group in Grade II and III, respectively (P = 0.0003; P = 0.0104). It showed that the signature was an independent prognosis factor on multivariate Cox regression analysis (P = 0.033). Patients in high group tended to displayed high grade (GIII), IDH1 wild type and mesenchymal subtype preference. Four-gene signature was discovered for LGGs with MGMT promoter methylated. Our findings suggested that the four genes could serve as prognostic biomarkers.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , RNA Mensageiro/genética , Transcriptoma/genética , Adulto , Neoplasias Encefálicas/mortalidade , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Feminino , Glioma/mortalidade , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Prognóstico , Regiões Promotoras Genéticas , Fatores de Risco , Proteínas Supressoras de Tumor/genética
8.
PLoS Comput Biol ; 16(4): e1007195, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275652

RESUMO

DNA methylation is a heritable epigenetic modification that plays an essential role in mammalian development. Genomic methylation patterns are dynamically maintained, with DNA methyltransferases mediating inheritance of methyl marks onto nascent DNA over cycles of replication. A recently developed experimental technique employing immunoprecipitation of bromodeoxyuridine labeled nascent DNA followed by bisulfite sequencing (Repli-BS) measures post-replication temporal evolution of cytosine methylation, thus enabling genome-wide monitoring of methylation maintenance. In this work, we combine statistical analysis and stochastic mathematical modeling to analyze Repli-BS data from human embryonic stem cells. We estimate site-specific kinetic rate constants for the restoration of methyl marks on >10 million uniquely mapped cytosines within the CpG (cytosine-phosphate-guanine) dinucleotide context across the genome using Maximum Likelihood Estimation. We find that post-replication remethylation rate constants span approximately two orders of magnitude, with half-lives of per-site recovery of steady-state methylation levels ranging from shorter than ten minutes to five hours and longer. Furthermore, we find that kinetic constants of maintenance methylation are correlated among neighboring CpG sites. Stochastic mathematical modeling provides insight to the biological mechanisms underlying the inference results, suggesting that enzyme processivity and/or collaboration can produce the observed kinetic correlations. Our combined statistical/mathematical modeling approach expands the utility of genomic datasets and disentangles heterogeneity in methylation patterns arising from replication-associated temporal dynamics versus stable cell-to-cell differences.


Assuntos
Biologia Computacional/métodos , Metilação de DNA/fisiologia , Animais , Bromodesoxiuridina/química , Ilhas de CpG , Citosina/metabolismo , DNA/metabolismo , Metilases de Modificação do DNA/genética , Células-Tronco Embrionárias/metabolismo , Epigênese Genética/genética , Epigênese Genética/fisiologia , Epigenômica/métodos , Genoma , Genômica , Humanos , Cinética , Modelos Estatísticos , Modelos Teóricos , Processos Estocásticos
9.
Sci Rep ; 10(1): 3711, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111869

RESUMO

Diffuse low-grade gliomas (LGG) have been reclassified based on molecular mutations, which require invasive tumor tissue sampling. Tissue sampling by biopsy may be limited by sampling error, whereas non-invasive imaging can evaluate the entirety of a tumor. This study presents a non-invasive analysis of low-grade gliomas using imaging features based on the updated classification. We introduce molecular (MGMT methylation, IDH mutation, 1p/19q co-deletion, ATRX mutation, and TERT mutations) prediction methods of low-grade gliomas with imaging. Imaging features are extracted from magnetic resonance imaging data and include texture features, fractal and multi-resolution fractal texture features, and volumetric features. Training models include nested leave-one-out cross-validation to select features, train the model, and estimate model performance. The prediction models of MGMT methylation, IDH mutations, 1p/19q co-deletion, ATRX mutation, and TERT mutations achieve a test performance AUC of 0.83 ± 0.04, 0.84 ± 0.03, 0.80 ± 0.04, 0.70 ± 0.09, and 0.82 ± 0.04, respectively. Furthermore, our analysis shows that the fractal features have a significant effect on the predictive performance of MGMT methylation IDH mutations, 1p/19q co-deletion, and ATRX mutations. The performance of our prediction methods indicates the potential of correlating computed imaging features with LGG molecular mutations types and identifies candidates that may be considered potential predictive biomarkers of LGG molecular classification.


Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/genética , Glioma/diagnóstico por imagem , Glioma/genética , Imagem por Ressonância Magnética/métodos , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Humanos , Mutação , Telomerase/genética , Proteínas Supressoras de Tumor/genética , Proteína Nuclear Ligada ao X/genética
10.
Cancer Res ; 80(11): 2101-2113, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32213541

RESUMO

Colorectal cancer initiation and progression result from the accumulation of genetic and epigenetic alterations. Although aberrant gene expression and DNA methylation profiles are considered hallmarks of colorectal cancer development, the precise timing at which these are produced during tumor establishment remains elusive. Here we investigated the early transcriptional and epigenetic changes induced by adenomatous polyposis coli (Apc) inactivation in intestinal crypts. Hyperactivation of the Wnt pathway via Apc inactivation in crypt base columnar intestinal stem cells (ISC) led to their rapid accumulation driven by an impaired molecular commitment to differentiation, which was associated with discrete alterations in DNA methylation. Importantly, inhibiting the enzymes responsible for de novo DNA methylation restored the responsiveness of Apc-deficient intestinal organoids to stimuli regulating the proliferation-to-differentiation transition in ISC. This work reveals that early DNA methylation changes play critical roles in the establishment of the impaired fate decision program consecutive to Apc loss of function. SIGNIFICANCE: This study demonstrates the functional impact of changes in DNA methylation to determine the colorectal cancer cell phenotype following loss of Apc function.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Metilação de DNA , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Receptores Acoplados a Proteínas-G/biossíntese , Células-Tronco/patologia , Proteína da Polipose Adenomatosa do Colo/deficiência , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Inativação Gênica , Intestino Delgado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas-G/genética , Células-Tronco/metabolismo , Via de Sinalização Wnt
11.
Int J Mol Sci ; 21(3)2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32023925

RESUMO

DNA methylation is a process through which methyl groups are added to the DNA molecule, thereby modifying the activity of a DNA segment without changing the sequence. Increasing evidence has shown that DNA methylation is involved in various aspects of plant growth and development via a number of key processes including genomic imprinting and repression of transposable elements. DNA methylase and demethylase are two crucial enzymes that play significant roles in dynamically maintaining genome DNA methylation status in plants. In this work, 22 DNA methylase genes and six DNA demethylase genes were identified in rapeseed (Brassica napus L.) genome. These DNA methylase and DNA demethylase genes can be classified into four (BnaCMTs, BnaMET1s, BnaDRMs and BnaDNMT2s) and three (BnaDMEs, BnaDML3s and BnaROS1s) subfamilies, respectively. Further analysis of gene structure and conserved domains showed that each sub-class is highly conserved between rapeseed and Arabidopsis. Expression analysis conducted by RNA-seq as well as qRT-PCR suggested that these DNA methylation/demethylation-related genes may be involved in the heat/salt stress responses in rapeseed. Taken together, our findings may provide valuable information for future functional characterization of these two types of epigenetic regulatory enzymes in polyploid species such as rapeseed, as well as for analyzing their evolutionary relationships within the plant kingdom.


Assuntos
Brassica napus/crescimento & desenvolvimento , Metilação de DNA , Perfilação da Expressão Gênica/métodos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Brassica napus/genética , Brassica napus/metabolismo , Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico , Família Multigênica , Filogenia , Proteínas de Plantas/química , Domínios Proteicos , Estresse Salino , Análise de Sequência de RNA , Distribuição Tecidual
12.
BMC Cancer ; 20(1): 79, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005184

RESUMO

BACKGROUND: Gliomas consist of a heterogeneous group of tumors. This study aimed to report the incidences of O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation, 1p19q co-deletion, isocitrate dehydrogenase (IDH) gene mutations, and inactivating mutations of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) in high-grade gliomas in an ethnically diverse population. METHODS: Records of patients who underwent surgery for high-grade gliomas from January 2013 to March 2017 at our institution were obtained. The patients' age, gender, ethnicity, Karnofsky Performance Scale (KPS) score, ability to perform activities of daily living (ADLs), tumor location and biomarkers status were recorded. Data were analyzed using chi-square and Mann-Whitney U tests, Kaplan-Meier estimates and log-rank test. RESULTS: 181 patients were selected (56 with grade III gliomas, 125 with grade IV gliomas). In the grade III group, 55% had MGMT promoter methylation, 41% had 1p19q co-deletion, 35% had IDH1 mutation and none had ATRX loss. In the grade IV group, 30% had MGMT promoter methylation, 2% had 1p19q co-deletion, 15% had IDH1 mutation and 8% had ATRX loss. After adjusting for effects of age, surgery and pre-operative ADL statuses, only MGMT promoter methylation was found to be significantly associated with longer overall survival time in grade III (p = 0.024) and IV patients (p = 0.006). CONCLUSIONS: The incidences of MGMT promoter methylation and IDH1 mutation were found to be comparable to globally reported rates, but those of 1p19q co-deletion and ATRX loss seemed to be lower in our cohort. MGMT promoter methylation was associated with increased overall survival in our cohort and might serve as favorable prognostic factor.


Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioma/cirurgia , Isocitrato Desidrogenase/genética , Proteínas Supressoras de Tumor/genética , Proteína Nuclear Ligada ao X/genética , Atividades Cotidianas , Adulto , Ásia Sudeste/etnologia , Cromossomos Humanos Par 1/genética , Epigênese Genética , Feminino , Glioma/genética , Glioma/mortalidade , Glioma/patologia , Humanos , Incidência , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Prognóstico , Regiões Promotoras Genéticas , Estudos Retrospectivos , Deleção de Sequência , Análise de Sobrevida
13.
PLoS One ; 15(2): e0229534, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101575

RESUMO

Temozolomide (TMZ) is a drug of choice in glioblastoma treatment. Its therapeutic applications expand also beyond high grade gliomas. However, a significant number of recurrences and resistance to the drug is observed. The key factor in each chemotherapy is to achieve the therapeutic doses of a drug at the pathologic site. Nonetheless, the rate of temozolomide penetration from blood to cerebrospinal fluid is only 20-30%, and even smaller into brain intestinum. That makes a challenge for the therapeutic regimens to obtain effective drug concentrations with minimal toxicity and minor side effects. The aim of our research was to explore a novel epigenetic mechanism of temozolomide action in therapeutic conditions. We analyzed the epigenetic effects of TMZ influence on different glioblastoma cell lines in therapeutically achieved TMZ concentrations through total changes of the level of 5-methylcytosine in DNA, the main epigenetic marker. That was done with classical approach of radioactive nucleotide post-labelling and separation on thin-layer chromatography. In the range of therapeutically achieved temozolomide concentrations we observed total DNA hypomethylation. The significant hypermethylating effect was visible after reaching TMZ concentrations of 10-50 µM (depending on the cell line). Longer exposure time promoted DNA hypomethylation. The demethylated state of the glioblastoma cell lines was overcome by repeated TMZ applications, where dose-dependent increase in DNA 5-methylcytosine contents was observed. Those effects were not seen in non-cancerous cell line. The increase of DNA methylation resulting in global gene silencing and consecutive down regulation of gene expression after TMZ treatment may explain better glioblastoma patients' survival.


Assuntos
Glioblastoma/genética , Glioblastoma/metabolismo , Temozolomida/farmacologia , 5-Metilcitosina , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Epigenômica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioma/patologia , Humanos , Recidiva Local de Neoplasia/genética , Temozolomida/metabolismo
14.
J Clin Neurosci ; 73: 231-236, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32070670

RESUMO

Although promoter methylation status is known to correlate with response to alkylating agents, immunohistochemistry (IHC) is the only available method for MGMT status in many institutions. However, the clinical utility of MGMT IHC is controversial. A hundred and twenty four cases of primary glioblastoma diagnosed by morphology-based criteria over 2 decades were re-appraised based on WHO 2016 classification. Tumor MGMT status was evaluated by IHC and methylation-specific PCR (MSP) to see if any correlation between the results of the 2 methods. The association with patients' prognoses was also investigated. Among 124 cases, 116 were confirmed to be glioblastoma by definition of WHO2016, and median overall survival (OS) of glioblastoma, IDH-wildtype and epithelioid glioblastoma were 18 and 27 months, respectively. MGMT promoter methylation status significantly correlated with progression-free survival (PFS) (p = 0.014) but not with OS (p = 0.364) in patients with glioblastoma by the integrated diagnosis. With the cut-off value of 24.5% of positive cell ratio as determined by receiver operating characteristic (ROC) analysis, there was a good correlation between the results of IHC and MSP (p = 0.08 × 10-24). Accordingly, there was a marginal association between the results of IHC and patients' PFS (p = 0.063), but not OS (p = 0.563). When the patients were divided into pre and post temozolomide era, the association of MGMT promoter methylation status with PFS was only noted in the patients of temozolomide era (pre, p = 0.432; post, p = 0.015).


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/diagnóstico , Metilação de DNA , Metilases de Modificação do DNA/genética , Glioblastoma/patologia , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Metilases de Modificação do DNA/metabolismo , Feminino , Glioblastoma/genética , Humanos , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Regiões Promotoras Genéticas , Curva ROC
15.
World Neurosurg ; 137: e213-e220, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32001415

RESUMO

BACKGROUND: Patients of lower socioeconomic status (SES) may experience barriers to their oncologic care, but current data conflict over whether SES affects the prognosis of patients with glioblastoma (GB). OBJECTIVE: We sought to determine whether SES disparities impaired delivery of neuro-oncologic care and affected the prognosis of GB patients. METHODS: The records of GB patients treated from 2010 to 2014 at a safety-net hospital (SNH) or private hospital (PH), both served by 1 academic medical institution, were retrospectively reviewed and compared. Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method. RESULTS: A total of 55 SNH and 39 PH GB patients were analyzed with median 11-month follow-up. SNH patients were predominantly Hispanic, low income, enrolled in Medicaid, were less likely to receive radiation (89% vs. 100%), took longer to start radiation (41 vs. 29 days), and were less likely to complete radiation treatment (80% vs. 95%). Concurrent and adjuvant temozolomide use were also lower (85% vs. 94% and 60% vs. 71%, respectively). OS and PFS were not significantly different (15 vs. 16 months and 8 vs. 11 months, respectively). On multivariate analysis, adjuvant chemotherapy and RT completion predicted for better OS, whereas hospital type, income, and insurance did not. CONCLUSION: Although GB patients at our SNH received less adjuvant treatment compared with PH, outcomes were similar. Access to multidisciplinary care staffed by academic physicians may play an important role in overcoming socioeconomic barriers to treatment availability and quality at SNHs.


Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Disparidades em Assistência à Saúde/estatística & dados numéricos , Hospitais Privados , Procedimentos Neurocirúrgicos , Provedores de Redes de Segurança , Temozolomida/uso terapêutico , Tempo para o Tratamento/estatística & dados numéricos , Idoso , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Quimiorradioterapia Adjuvante/estatística & dados numéricos , Quimioterapia Adjuvante/estatística & dados numéricos , Estudos de Coortes , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Grupos Étnicos/estatística & dados numéricos , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Isocitrato Desidrogenase/genética , Estimativa de Kaplan-Meier , Masculino , Medicaid , Pessoa de Meia-Idade , Pobreza/estatística & dados numéricos , Intervalo Livre de Progressão , Radioterapia Adjuvante/estatística & dados numéricos , Estudos Retrospectivos , Classe Social , Padrão de Cuidado , Taxa de Sobrevida , Carga Tumoral , Proteínas Supressoras de Tumor/genética , Estados Unidos
16.
Cell ; 180(2): 263-277.e20, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31955845

RESUMO

Cytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles. In the yeast Cryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 mya. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 million years through a process analogous to Darwinian evolution of the genome.


Assuntos
Cryptococcus neoformans/genética , Metilação de DNA/genética , Metiltransferases/genética , Evolução Biológica , Cryptococcus neoformans/metabolismo , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/fisiologia , Metilases de Modificação do DNA/genética , Elementos de DNA Transponíveis/genética , Epigenômica/métodos , Evolução Molecular , Genoma/genética , Metiltransferases/metabolismo , Filogenia
18.
Talanta ; 210: 120609, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31987176

RESUMO

DNA methylation is involved in the oncogenesis of head and neck squamous cell carcinoma and could be used for early detection of cancer to increase the chances of cure, but unfortunately diagnosis is usually made at late stages of the disease. In this work we developed genosensors to detect DNA methylation of the MGMT gene in head and neck cancer cell lines. The probe for MGMT promoter methylation was immobilized on gold electrodes modified with 11-mercaptoundecanoic acid (11-MUA) self-assembled monolayers (SAM). Detection was performed with electrochemical impedance spectroscopy, with clear distinction between methylated and non-methylated DNA from head and neck cell lines. The genosensor is sensitive with a low detection limit of 0.24 × 10-12 mol L-1. In addition, the cell lines FaDu, JHU28 and SCC25 for the MGMT gene, could be distinguished from the HN13 cell line which has a high degree of MGMT methylation (97%), thus confirming the selectivity. Samples with different percentages of MGMT DNA methylation could be separated in multidimensional projections using the visualization technique interactive document mapping (IDMAP). The genosensor matrix and the immobilization procedures are generic, and can be extended to other DNA methylation biomarkers.


Assuntos
Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Técnicas Eletroquímicas , Ácidos Graxos/química , Neoplasias de Cabeça e Pescoço/genética , Compostos de Sulfidrila/química , Tioglicolatos/química , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Eletrodos , Ouro/química , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Metilação , Regiões Promotoras Genéticas/genética , Espectrofotometria Infravermelho , Proteínas Supressoras de Tumor/metabolismo
19.
Acad Radiol ; 27(12): e263-e271, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-31983532

RESUMO

RATIONALE AND OBJECTIVES: The World Health Organization 2016 classification of central nervous system tumors added the molecular classification of gliomas and has guiding significance for the operation and prognosis of glioma patients. At present, the perfusion technique plays an important role in judging the malignant degree of glioma. To evaluate the performance of dynamic susceptibility contrast (DSC)- and dynamic contrast-enhanced (DCE)-magnetic resonance imaging (MRI) histogram analyses in discriminating the states of molecular biomarkers and survival in glioma patients. MATERIALS AND METHODS: Forty-three glioma patients who underwent DCE- and DSC-MRI were enrolled. Relevant molecular test results, including those on isocitrate dehydrogenase (IDH), O6-methylguanine-DNA methyltransferase (MGMT) and telomere reverse transcriptase (TERT), were collected. The mean relative cerebral blood volume of DSC-MRI and histogram parameters derived from DCE-MRI (volume transfer coefficient (Ktrans), fractional volume of the extravascular extracellular space (Ve), fractional blood plasma volume (Vp), rate constant between the extravascular extracellular space and blood plasma (Kep) and area under the curve (AUC)) were calculated. Differences in each parameter between gliomas with different expression states (IDH, MGMT, and TERT) were evaluated. The diagnostic efficiency of each parameter was analyzed. The overall survival of all patients was assessed. RESULTS: The 10th percentile AUC (AUC = 0.830, sensitivity = 0.78, specificity = 0.80), the 90th percentile Ve (AUC = 0.816, sensitivity = 0.84, specificity = 0.79), and the mean Kep (AUC = 0.818, sensitivity = 0.76, specificity = 0.78) provided the highest differential efficiency for IDH, MGMT, and TERT, respectively. Kaplan-Meier curves showed a significant difference between subjects with a 10th percentile AUC higher or lower than 0.028 (log-rank = 7.535; p = 0.006) for IDH and between subjects with different 90th percentile Ve values (log-rank = 6.532; p = 0.011) for MGMT. CONCLUSION: Histogram DCE-MRI demonstrates good diagnostic performance in identifying different molecular types and for the prognostic assessment of glioma.


Assuntos
Neoplasias Encefálicas , Glioma , Telomerase , Biomarcadores , Neoplasias Encefálicas/diagnóstico por imagem , Meios de Contraste , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioma/diagnóstico por imagem , Humanos , Isocitrato Desidrogenase/genética , Imagem por Ressonância Magnética , Gradação de Tumores , Proteínas Supressoras de Tumor
20.
Cancer Genet ; 241: 42-50, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31653608

RESUMO

Testicular germ cell tumor (TGCT) development may involve a series of modification at epigenetic and genetic level which may act synergistically and transform the primordial gonocyte. This study evaluated the frequency and distribution pattern of RASSF1A/MGMT gene methylation and KRAS, BRAF and cKIT gene mutation in Indian TGCT patient, and their correlation with clinicopathological features. Forty-one TGCT tumors were used to investigate hypermethylation of RASSF1A and MGMT gene and mutations of KRAS codon 12/13, BRAF V600E and cKIT exon 17 mutations. RASSF1A and MGMT methylation was noted in 58.5% and 10% of the TGCTs. A higher frequency of RASSF1A methylation was noted in seminomas (71%, 17/24), while MGMT methylation was frequent in mixed tumors (23%, 3/13). Interestingly, 3 of 41 cases showed concurrent methylation of both the genes. The absence of tumor necrosis was significantly associated with increased frequency of MGMT hypermethylation (30% vs. 3%, p = 0.03). KRAS mutation was identified in 17% (7/41), while none showed BRAF and cKIT mutation. KRAS mutations were predominantly found in codon 12 with G12V as the most recurrent mutations. Mixed germ tumors tends to show increased frequency of KRAS mutation (31%, 4/13), followed by pure seminomas (4%, 1/24). Interestingly, KRAS mutation rate was significantly higher in metastatic tumors in comparison to primary tumors (100% vs. 13%, p = 0.02). No other association of RASSF1A/MGMT/KRAS alterations with other clinicopathological features was noted. In conclusion, these data support the notion that the cancer-associated alterations in the RASSF1, MGMT and KRAS gene may significantly contribute to TGCT pathogenesis.


Assuntos
Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Embrionárias de Células Germinativas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Seminoma/genética , Neoplasias Testiculares/genética , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Ilhas de CpG/genética , Metilação de DNA , Análise Mutacional de DNA , Epigênese Genética , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Mutação , Taxa de Mutação , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-kit/genética , Seminoma/patologia , Testículo/patologia , Adulto Jovem
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