Evaluation of the melting temperature of TaqMan probes as a genotyping method for IDH1, IDH2, and H3F3A in pediatric astrocytomas / Evaluación del método de temperatura de disociación de sondas TaqMan para la genotipificación de IDH1, IDH2 y H3F3A en astrocitomas pediátricos

Abstract Background: Astrocytomas are cancer tumors of the central nervous system and represent the most common type of solid tumors during human childhood. In 2016, the World Health Organization established a molecular classification system to regroup tumor entities to achieve a more accurate diagnosis and a better clinical decision-making and selection of treatment in patients with these types of tumors. Methods: We evaluated a genotyping assay for rapid and cost-effective mutation detection in astrocytomas using TaqMan probes in an asymmetric polymerase chain reaction (PCR) assay. Results: Four diffuse astrocytomas (Grade II), three anaplastic astrocytomas (Grade III), and four glioblastomas (Grade IV) were sequenced, and all of them displayed the wild-type (WT) sequence. We tried to set up this melting analysis for the genotyping of pediatric astrocytomas by identifying the specific melting temperatures of the TaqMan probes due to the presence of the WT sequences in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and H3.3 histone A genes (H3F3A). We used an IDH1-TaqMan probe to identify the WT status of IDH1 in two different WT deoxyribonucleic acid (DNA) templates (pilocytic and diffuse astrocytoma) and obtained four melting temperature values ranged from 65.6 to 92.2°C. Furthermore, only four out of 29 reactions displayed amplification of the DNA template. Sanger sequencing was faster and more reliable to detect the gene status in all the sequenced samples. Conclusions: We conclude that conventional Sanger sequencing remains the gold standard for the genotyping of pediatric astrocytomas.

Resumen Introducción: Los astrocitomas son un tipo de cáncer que afecta al sistema nervioso central y representan el tumor sólido más común durante la infancia. En el año 2016, la Organización Mundial de la Salud estableció un sistema de clasificación molecular para reagrupar tumores con identidades genéticas similares y lograr un diagnóstico más preciso, lo que lleva a tomar las decisiones clínicas idóneas al elegir el tratamiento de pacientes con este tipo de tumores. Métodos: Se evaluó un protocolo que involucra el uso de sondas TaqMan en un ensayo de reacción en cadena de la polimerasa asimétrica para la detección de mutaciones en astrocitomas. Se secuenciaron cuatro astrocitomas difusos (Grado II), tres astrocitomas anaplásicos (Grado III) y cuatro glioblastomas (Grado IV). Se intentó establecer las condiciones del análisis para la genotipificación de los astrocitomas pediátricos mediante la identificación de las temperaturas de disociación específicas de las sondas TaqMan producidas por la prescencia de las secuancias WT en los genes isocitrato deshidrogenasa 1 y 2 (IDH1, IDH2) y H3.3 histona A (H3F3A). Resultados: Los astrocitomas mostraron la secuencia wild type (WT) (silvestre) de los genes. Se utilizó una sonda TaqMan IDH1 para identificar el estado de este gen en dos templados WT de DNA (astrocitoma pilocítico y difuso) y se obtuvieron cuatro valores de temperatura de disociación (65.6-92.2 °C). Solo cuatro de las 29 reacciones mostraron amplificación de DNA. La secuenciación de Sanger fue más rápida y confiable para detectar el estado de los genes en todas las muestras. Conclusiones: La secuenciación de Sanger sigue siendo la técnica más práctica para la genotipificación de astrocitomas pediátricos.

P53 expression in glioma: an immunohistochemical study

The aim of this study was to evaluate the frequency of immunohistochemical [IHC] expression of p53 protein in different types of glioma in Mosul city, and to correlate p53 expression with the histological types and grades of gliomas, and compare the results of this study with those of others. This study was performed on 50 cases of glioma. Samples were obtained in a prospective and retrospective fashion [cross-sectional study]. The samples were collected during the period extending from October 2010 to May 2011. All cases were obtained from Al-Jamhuri Teaching Hospital in Mosul city, Northen Iraq and some private laboratories. Typing and grading of the glioma were done according to World Health Organization [WHO] classification system. P53 expression was assessed immunohistochemically. Fifty cases of gliomas were collected; they included 37 cases of astrocytoms, 8 ependymoma 4 oligodendrogliomas and 1 oligoastrocytoma. p53 expression was detected in 25 cases ofglioma [50%]. The positive cases included 59.45% ofastrocytomas, 50% of oligodendrogliomas, and one case of oligoastrocytoma which was positive also. On the other hand, all of the ependymomas were negative for p53 protein. p53 was significantly related to the grades of glioma, but not significantly related to the type ofglioma. P53 expression was expressed in 50% of gliomas in Mosul city. P53 expression is common among high grade astrocytomas and mixed gliomas, less among oligodendrogliomas, and lacking in cases of ependymomas. Statistically, p53 expression was not significantly correlated with the type of glioma. p53 index is directly correlated with the grade of glioma, and so it is of prognostic value

Current concepts in the pathology and genetics of gliomas.

In recent years, there has been a marked improvement in our understanding of molecular genetics of gliomas. These advancements offer hope for development of tailored therapies targeting a tumor's unique molecular profile, and may also translate into improved classification and identification of newer prognostic markers. This review focuses on the neuropathological features of different types of glial neoplasms according to the World Health Organization classification, and the recent advances in their molecular biology with emphasis on the genetic mechanisms underlying tumor progression, diagnostic and prognostic markers and potential therapeutic targets.

Current concepts and newer developments in the treatment of malignant gliomas.

Primary malignant brain tumors account for only 2% of all adult cancers but they cause a disproportionately high cancer-related disability and death. Survival of malignant glioma patients has changed only modestly over the past three decades despite the emergence of new treatment strategies for these tumors. In this review, we describe the standard treatment modalities for malignant glioma, which include surgery, radiation therapy and chemotherapy, as well as the status of novel therapies that have been developed to target various aspects of glioma cell biology. We also address this issue of drug delivery as a factor limiting the efficacy of systemic administration of therapeutics and attempts to overcome this barrier. Further progress towards a cure for malignant gliomas will require a greater understanding of the underlying mechanisms driving the growth, and resistance to therapy, of these challenging tumors.

Epidermal growth factor receptor and proliferating cell nuclear antigen in astrocytomas.

AIMS: The involvement of various growth factors, growth factor receptors and proliferative markers in the molecular pathogenesis of astrocytic neoplasms are being studied extensively. Epidermal Growth Factor Receptor (EGFR) gene overexpression occurs in nearly 50% of cases of glioblastoma. Since EGFR and proliferating cell nuclear antigen (PCNA) are involved in mitogenic signal transduction and cellular proliferation pathway, we have studied the correlation between the expression of EGFR and PCNA labeling index in astrocytic tumors. MATERIALS AND METHODS: We investigated the immunohistochemical expression of EGFR and PCNA using the appropriate monoclonal antibodies in 40 cases of astrocytic tumors of which 21 cases were glioblastoma, eight cases were Grade III or anaplastic astrocytomas and six cases were Grade II or diffuse astrocytomas and five cases were Grade I or pilocytic astrocytomas. RESULTS: Both the EGFR expression and PCNA labeling index increase with increasing grades of astrocytomas with a significantly high percentage of cells showing positive staining for both EGFR and PCNA in GBM and Grade III astrocytomas compared to Grade II astrocytomas. The expression levels of both EGFR and PCNA were low in Grade I or pilocytic astrocytomas. CONCLUSIONS: A significant correlation was found between EGFR overexpression and PCNA labeling index in Grade III and Grade II astrocytomas and glioblastoma. These suggest that the tumor proliferation, at least in higher grades of astrocytomas is dependent in some measure on EGF and EGFR-related signaling pathways.

Identification of COL6A1 as a differentially expressed gene in human astrocytomas

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.

Immunoexpression of tumor suppressor genes p53, p21WAF1/CIP1 and p27KIP1 in humam astrocystic tumors

The aim of the present study was to evaluate the tumor suppressor genes p53, p21WAF1/CIP1 and p27KIP1 expression in astrocytic tumors, correlating the findings with the histopathological grade (WHO). An immunohistochemical study of the p53, p21 and p27 proteins using the streptavidin-biotin-peroxidase method was performed in fifty-five astrocytomas (13 grade I, 14 grade II, 7 grade III and 21 grade IV) and five samples of non-tumor brain tissue (negative control). p53 positive indices (PI) and labeling indices (LI) showed tendency to increase according to malignant progression. The nuclear expression of p27 presented similar inclination, except for the PI reduction verified in grade IV tumors. Otherwise, the cytoplasmic p27 staining was more evident between high-grade tumors (III and IV). p53 and nuclear p27 expression was correlated with the histological classification (p<0.01; test H). On the other hand, p21 indices revealed a propensity to reduction in agreement with malignant evolution of the astrocytic tumors, except for high scores observed in grade IV tumors. The non-tumor samples did not show any expression of these proteins. These results indicated the p53 mutation as an initial, relevant and potentially predictor of tumor progression event in astrocytomas, with the detection of p21 protein as an important resource for the deduction of functional situation of this gene. Moreover, the activation of p27KIP1 was preserved in the astrocytic tumors and its cytoplasmic manifestation seems to be resultant of its nuclear expression, not demonstrating a direct impact in astrocytomas tumorigenesis.

O presente estudo objetivou avaliar a expressão dos supressores tumorais p53, p21WAF1/CIP1 e p27KIP1 em tumores astrocíticos humanos, correlacionando os achados com a graduação histopatológica (OMS). Procedeu-se o estudo imuno-histoquímico para as proteínas p53, p21 e p27 utilizando o método da estreptavidina-biotina-peroxidase em 55 astrocitomas (13 do grau I, 14 do grau II, 7 do grau III e 21 do grau IV) e 5 amostras de tecido cerebral não-tumoral (controle negativo). Os índices de positividade (PI) e de marcação (LI) para p53 demonstraram tendência de aumento conforme a progressão maligna. A expressão nuclear do p27 apresentou semelhante inclinação, exceto pela redução do PI verificada nos tumores do grau IV. Já a marcação citoplasmática do p27 foi mais evidente entre tumores de alto grau (III e IV). As expressões de p53 e p27 nuclear demonstraram correlação com a classificação histológica (p<0,01; teste H). Por outro lado, os índices para p21 manifestaram propensão à redução conforme a evolução maligna dos tumores astrocíticos, salvo significante aumento observado nos tumores do grau IV. Não houve expressão dessas proteínas nas amostras não-tumorais. Tais resultados indicaram a mutação do p53 como um evento inicial, relevante e potencialmente indicador de progressão maligna nos astrocitomas, sendo a detecção da proteína p21 um importante recurso para a dedução da situação funcional desse gene. Além disso, a ativação do p27KIP1 mostrou-se preservada nos tumores astrocíticos e sua manifestação citoplasmática parece ser reflexo de sua expressão nuclear, não demonstrando impacto direto na tumorigênese dos astrocitomas.

Transcription analysis of TIMP-1 and NM23-h1 genes in glioma cell invasion / Análise transcricional dos genes TIMP-1 e NM23-H1 na invasão celular em astrocitoma difuso e glioblastoma multiforme

PURPOSE: To evaluate using transcription analysis the presence and importance of two genes: NM23-H1 and TIMP-1 on control of tumor cell invasion in diffuse astrocytomas (WHO II) and glioblastoma multiforme (WHO IV). METHOD: Northern blot analysis of NM23-H1 and TIMP-1 was performed. Eight diffuse astrocytomas and 19 glioblastomas (WHO IV) were analyzed to determine if TIMP-1 and NM23-H1 were candidates to inhibition of tumor cell invasion quantitated RNA levels. The samples were collected directly from operating room. Total cellular RNA was extracted from frozen tissue samples using guanidinium-isothiocyanate and cesium chloride gradients. Total RNA (10 mg per sample) from tumor tissue were size fractionated through 1 percent agarose-formaldehyde gel and transferred to nylon filters and then hybridized to 32P-labeled DNA probes and placed for autoradiography. Levels of specific RNAs were determined by computer-assisted laser densitometry. Blot filters were sequentially hybridized to nm23 and TIMP-1 probes in addition to GAPDH, as a control. Statistical analyses were carried out according to t-test for equality of means. RESULTS: NM23-H1 were detected in each sample, however it did not correlate with malignancy and invasiveness. On the other side TIMP-1 gene expression showed a clear correlation between low expression and invasiveness. CONCLUSION: The data suggest that TIMP-1 is an inhibitor of high grade gliomas invasion. NM23-H1 was present in the entire gliomas sample, but it did not vary in diffuse astrocytomas and glioblastomas.

OBJETIVO: Comparar através da análise da expressão dos níveis de RNA, a presença e a relevância dos genes NM23-H1 e TIMP-1 no controle da invasão celular tumoral dentro do tecido cerebral normal em: astrocitoma difuso (OMS II) e glioblastoma multiforme (OMS:IV). MÉTODO: Análise em "Northern blot" dos genes NM23-H1 e TIMP-1. Oito astrocitomas fibrilares difusos (OMS II) e 19 glioblastomas multiformes foram analisados para determinar se TIMP-1 e NM23-H1 estavam relacinados à inibição da invasão tumoral nas neoplasias do sistema nervoso central, quantificando os níveis de RNA dos respectivos genes extraídos diretamente dos tumores. 10 mg por amostra de RNA total foram fracionados de gel de formaldeído e transferidos para os filmes de hibridação. Níveis específicos de RNAs foram determinados na espectrofotometria. Valores das razões entre NM23-H1/GAPDH e TIMP-1/GAPDH foram submetidos à análise de variabilidade das médias. RESULTADOS: A análise da expressão do gene TIMP-1 mostrou supressão em tumores gliais malignos. CONCLUSÃO: Os resultados indicam que existe relação direta entre níveis baixos de TIMP-1 e malignidade dos gliomas. O gene NM23-H1 foi detectado em todas as amostras, mas não foi possível relacionar sua subexpressão ou superexpressão com algum fenótipo de invasividade.

Estudo das alterações de TP53 em astrocitomas difusos / Analysis of TP53 alterations in diffuse astrocytomas

Alterações de TP53, estão associadas com a maioria dos cânceres, incluindo tumores do sitema nervoso central(SNC). Astrocitomas difusos são as neoplasias intracranianas mais frequentes perfazendo cerca de 60 por cento de todos os tumores primários do SNC. As mutações de TP53 são observadas em aproximadamente 30 a 40 por cento de todos os graus de astrocitomas difusos / TP53 alterations, are associated to a large number of human cancers, including central nervous system (CNS) tumors. Diffuse astrocytomas are the most frequent intracranial neoplasms and account for approximately 30 a 40 per cent of all three grades of diffuse astrocytoma, sugggesting that inactivation...

Ploidia de DNA em astrocitomas: estudo em 66 pacientes brasileiros / DNA ploidy in glioma: study in 66 Brazilian patients

A determinação do conteúdo de DNA nuclear (fração de fase S e ploidia de DNA) foi realizada por meio de análise de imagem em 66 astrocitomas, a partir de material fixado em formalinas e seccionado em cortes de 5 micrômetros corados pela técnica de Feulgen. Nossos resultados mostraram forte relação entre a idade do paciente, grau histológico e sobrevida, com a ploidia de DNA e o percentual de células em fase de síntese. A análise da atividade proliferativa de astrocitomas intracranianos é a nosso ver muito útil no entendimento do comportamento biológico, do prognóstico e para o planejamento terapêutico dessas lesões.

Alterations in brain tumor DNA detected by a fingerprinting probe.

We used a novel DNA fingerprinting probe O-chi-1 (ref. 1) to detect differences in the hybridization pattern of brain tumor DNA and paired normal tissue of a given individual. Representatives of meningiomas (two), glioblastoma multeforme (three) and astrocytoma (one) were studied. Alterations, which included amplification as well as the loss of a normal band in tumor DNA, were observed in four of the six tumours. While the increased intensity of a band can be taken to imply increased copy number, the disappearance of bands could either be due to loss of DNA sequence or rearrangement resulting in different sized bands.

Loss of heterozygosity on chromosome 10, 13q(Rb), 17p, and p53 gene mutations in human brain gliomas

Using the methods of restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) analyses, we have examined 33 cases of human gliomas with various malignant grades to detect the deletions of putative tumor suppressor gene loci, chromosome 10, 13q(retinoblastoma gene, Rb), 17p, and p53 mutation. We observed loss of heterozygosity (LOH) at loci on chromosome 10 (36%), 13q(Rb) (54%), and 17p(50%) in malignant gliomas. There, however was no allelic loss on chromosome 10 and 17p in low-grade gliomas. Rb gene deletions were seen in low-grade gliomas, including oligodendroglioma and ependymoma. This finding suggests that Rb inactivation may be an early genetic event in the development and progression of gliomas. We correlated the results of LOH on chromosome 17p and p53 mutation. Among the 8 cases which showed LOH on chromosome 17p, only three cases (38%) revealed p53 mutations. Low incidence of p53 mutations in cases with chromosome 17p deletions suggests that some other tumor suppressor genes may be located on chromosome 17p.

Cytogenetic investigation of four low grade gliomas

Four low-grade gliomas - two oligodendrogliomas and two astrocytomas - were analyzed cytogenetically. All cases exhibited monosomies of chromosomes 10 and 11. The astrocytomas shared monosoniies of chromosomes 8, 9, 1O, 11, 12, 18 and 20. Losses of chromosomes 3, 5, 6, 10 and I I were present in both oligodendrogliomas, and except for monosomy of chromosome 6, were also identified in the pilocytic astrocytoma.

Estudios citogenéticos en tumores del sistema nervioso / Cytogenetic studies in tumors of the nervous system

Se presenta el estudio citogenético efectuado en 3 pacientes con tumores del sistema nervioso: 2 astrocitomas grado III y un neuroblastoma estadio I. Los dos primeros mostraron una distribución bimodal en tanto que el neuroblatoma presentó un número modal diploide. En los 3 casos se encontraron alteraciones cromosómicas clonales, observándose los marcadores i(lq), 9p+, 3p+, dicéntricos y un metacéntrico pequeño de origen desconocido en gliomas. En el neuroblastoma se observó el marcador 11p+. Los dos astrocitomas presentaron cromosomas dobles diminutos (DM), fenómeno indicador de amplificación génica que correlaciona con el estadio avanzado de la enfermedad. Las anormalías numéricas clonales fueron trisomías 16 y 18 en los gliomas y monosomía 15 en el neuroblastoma