Gene expression microarray analysis reveals prognostic markers of survival in high grade astrocytomas

Objective: High grade astrocytoma (HGA) as an aggressive brain tumor, is always correlated with poor prognosis. In this paper, we aimed to explore the genetic prognostic biomarkers for HGA.

Methods: The genome-wide expression profile of 26 brain tumor samples obtained from 26 patients with HGA was downloaded from Gene Expression Omnibus. The risk genes for prognosis of HGA were identified and verified by the data in TCGA database. Protein–protein interaction (PPI) network of risk factor genes was constructed and significant module was screened. Function and pathway annotations were performed for risk genes and drug target genes were further analyzed.

Results: A total of 598 genes were identified as significant risk genes for prognosis, such as checkpoint kinase 1, potassium inwardly-rectifying channel, subfamily J, member 6, leukocyte receptor tyrosine kinase and uncharacterized LOC283887. All risk genes for prognosis of HGA were significantly enriched in cell cycle, mitotic as well as mitotic anaphase. While the genes in the network module mainly participated in functions such as cell cycle, mitotic cell cycle and cell cycle process. Moreover, the genes in the network module mainly participated in the pathways such as cell cycle and cell cycle, mitotic. Drug target analysis showed that seven genes were recorded in Drugbank database, and there were as many as 32 drug records of CHEK1.

Conclusion: The prognostic effect of CHEK1 was validated based on the expression profile data of 615 low-grade glioma and glioblastoma samples. We proposed CHEK1 as prognostic biomarker for HGA. Our work might provide the candidate target for HGA therapy.     

Expression of pERK and pAKT in pediatric high grade astrocytomas: Correlation with YKL40 and prognostic significance

The Ras signaling pathway, consisting of mitogen‐activated protein kinase (MAPK) and PI3K/AKT signaling, is a prominent oncogenic pathways in adult diffuse gliomas, but few studies have evaluated such pathways in pediatric malignant gliomas. We investigated by immunohistochemistry MAPK and AKT signaling in a series of 28 pediatric high‐grade gliomas (WHO grade III and IV). We sought a possible association of phospho‐ERK (p‐ERK) and phospho‐AKT (p‐AKT) with expression of other proteins involved in the Ras pathway, that is, YKL40, epidermal growth factor receptor (EGFR), EGFR vIII and c‐Met. Moreover we correlated the expression of p‐ERK and p‐AKT with prognosis. No cases showed expression for c‐Met and EGFR, and only one case was positive for EGFR vIII. YKL‐40 protein was expressed in 43% of cases. We detected expression of p‐ERK and p‐AKT in 61% and 57%, respectively, of pediatric high grade gliomas. Statistical analysis comparing the two groups in term of high and low p‐ERK and p‐AKT expression showed a trend toward worse overall survival in patients with high expression of p‐AKT. The activation of ERK and AKT suggest a possible role of this protein in inducing activation of the Ras signaling pathway in pediatric high‐grade gliomas. Moreover high levels of p‐AKT are associated with worse overall survival.     

Low grade astrocytomas: controversies in management

Low grade astrocytomas are common brain neoplasms that primarily affect young adults. Although these patients often have a reasonably long survival, most will ultimately succumb to their tumours. Often the tumours progress to higher grade astrocytomas. The optimal management plan for these tumours is controversial and ranges from observation to macroscopic excision, radiotherapy and chemotherapy. The evidence for each of these approaches is presented in this review and a management algorithm is presented.     

Clinico‐pathological feature of pilomyxoid astrocytomas: Three case reports

Pilomyxoid astrocytoma (PMA) is a newly identified variant of pilocytic astrocytoma (PA). We report three cases of PMA with comparison to seven cases of PA in terms of their clinicopathological features. The three cases occurred at the ages of 2, 36 and 6 years, and their tumors were located in the left basal ganglia, the pineal gland, and the cerebellum, respectively. They were diagnosed PMA by surgical specimens that showed a characteristic monomorphous architecture with an angiocentric growth pattern and myxoid background. One patient developed localized relapse at 6 months after the surgery, but the other patients remained alive without tumor progression more than 5 years after treatment. In analysis of the immunohistochemical association in PMA and PA, no specific staining was found to be useful for differential diagnosis of PMA from PA. The expression of biomarkers including O‐6‐methylguanine‐DNA methyltransferase, p53, MIB‐1, and EGF receptor neither distinguished PMA from PA nor correlated with outcome. But almost all PMA and PA that demonstrated prominent positivity for nestin showed a high MIB‐1 labelling index (LI), and four of these five patients suffered a relapse in the early phase. These results suggest that immunohistochemical expression of nestin and MIB‐1 LI may correlate with the aggressiveness of the tumor in PA and PMA.     

Evaluation of the prognostic potential of EGFL7 in pilocytic astrocytomas

Pilocytic astrocytoma (PA) is the most frequent solid neoplasm in childhood. It has a good 5‐year overall survival (90% in childhood and 52% in adults). However, up to 20% of patients experience residual tumor growth, recurrence, and death. Although the main genetic alteration of PAs, including KIAA1549:BRAF fusion, involves chromosome 7q34, we previously found frequent loss in chr9q34.3 locus in a small subset of these tumors. Among the genes present in this locus, EGFL7 is related to poor prognosis in several tumor types. In this study, we aimed to assess EGFL7 expression through immunohistochemistry, and to evaluate its prognostic value in a series of 64 clinically and molecularly well‐characterized pilocytic astrocytomas. We found high expression of EGFL7 in 71.9% of patients. Low EGFL7 expression was associated with older patients, the mean age mainly older than 11 years (P = 0.027). EGFL7 expression was not associated with presence of KIAA1549:BRAF fusion, BRAF mutation, FGFR1 mutation, nor FGFR1 duplication. Moreover, high EGFL7 expression was associated with high FGFR1 (P = 0.037) and 5′‐deoxy‐5′‐methyltioadenosine phosphorylase (MTAP) (P = 0.005) expression, and with unfavorable outcome of patients (P = 0.047). Multivariate analysis revealed low EGFL7 expression related to older patients and high EGFL7 expression related to retained expression of MTAP. In addition, we found a borderline significance of unfavorable outcome and high EGFL7 expression. Finally, EGFL7 expression was not associated with overall or event‐free survival of PA patients. Our findings point to EGFL7 expression as a novel candidate prognostic marker in PA, which should be further investigated.     

miR-181b在星形细胞瘤中的表达及其临床意义

目的 分析星形细胞瘤中miR-181b的表达及其与临床意义.方法 选取星形细胞瘤标本84例,以20例癌旁组织为对照,采用Real-time PCR对组织中miR-181b的含量进行测定.结果 miR-181b在星形细胞瘤组织中含量显著下调,差异有统计学意义(P =0.0087).miR-181b的表达与患者预后密切相关,低表达miR-181b的患者预后较差,而高表达miR-181b的患者预后良好(P =0.035).miR-181b的表达与患者年龄、性别和WHO分级无统计学意义(P＞0.05).结论 miR-181b在星形细胞瘤组织中表达显著降低,且其表达量与患者的存活时间密切相关,提示其可能为抑癌基因参与了星形细胞瘤的发生与发展,具有重要临床诊疗意义.     

以癫癎为首发症状的低级别星形细胞瘤病人术后癫癎预后分析

目的探讨影响以癫癎为首发症状的低级别星形细胞瘤癫癎预后的因素。方法回顾性分析以癫癎为首发症状的低级别星形细胞瘤病例,经手术切除肿瘤1年后的癫癎症状改善情况,本组研究数据应用SPSS17．0统计软件进行卡方检验和多因素Logistic回归分析。结果术后癫癎预后为改良Engel I级102例（67．5％）,Ⅱ级以上49例（32．5％）。卡方检验显示：术前病人有语言障碍（P=0．038）、术前肿瘤累及岛叶（P=0．010）、肿瘤未全切除（P=0．001）对术后癫癎预后差异有统计学意义。多因素Logistic回归分析：术前病人有语言障碍（P=0．022）、术前肿瘤累及岛叶（P=0．007）及肿瘤未全切除（P=0．002）均为影响术后癫癎发作的独立危险因素。结论术前病人有语言障碍、术前肿瘤累及岛叶及肿瘤未全切除是影响以癫癎为首发症状的低级别星形细胞瘤病人术后癫癎预后的独立危险因素。     

脑星形细胞瘤癌基因表达

52例星形细胞瘤应用免疫组化方法,检测癌基因p~(53)、C-erbB-2及增殖细胞核抗原(PCNA)的表达,结果发现①p53异常表达率为41.2%(24/52),C-erbB-2过度表达率为39%(20/52),PCNA(PI>0.05)增殖指数为77%(40/52),与对照组正常脑组织对比有显著差异(P<0.001).②p53,C-erbB-2,PCNA异常表达与病理级别有明显相关性.病理Ⅲ,Ⅳ级的阳性率分别为80%(16/20),40%(8/20),100%(20/20),(P0.05);C-erbB-2阳性组,PCNA指数;0.361±0.27,阴性组PCNA指数;0.399±0.39,两组间亦无差异(P>0.05).④11例胶质增生组织有1例(9%)显示p53表达,C-erbB-2,PCNA无表达.随访3年,病变复发,病理证实为星形细胞瘤Ⅰ～Ⅱ级,C-erbB-2,PCNA表达.结果提示:①星形细胞瘤p53、C-erbB-2及PCNA的异常表达可作为星形细胞瘤恶性程度及病人预后的指标,以p53过度表达尤为重要;②3种抗体的联合应用对星形细胞瘤发病机理研究及预测早癌发生有一定价值,p53异常表达主要是影响星形细胞瘤的分化,而C-erbB-2对肿瘤进展早期起一定作用.③胶质增生的胶质细胞具有恶性表型.     

Immunohistochemical estimation of cell cycle entry and phase distribution in astrocytomas: applications in diagnostic neuropathology

An immunohistochemical method for assessing cell cycle phase distribution in neurosurgical biopsies would enable such data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in glial neoplasms, without the requirement for flow cytometric analysis. Paraffin-embedded sections of intracerebral gliomas (n = 48), consisting of diffuse astrocytoma (n = 9), anaplastic astrocytoma (n = 8) and glioblastoma (n = 31), were analysed by immunohistochemistry using markers of cell cycle entry, Mcm-2 and Ki67, and putative markers of cell cycle phase, cyclins D1 (G1-phase), cyclin A (S-phase), cyclin B1 (G2-phase) and phosphohistone H3 (Mitosis). Double labelling confocal microscopy confirmed that the phase markers were infrequently coexpressed. Cell cycle estimations by immunohistochemistry were corroborated by flow cytometric analysis. There was a significant increase in Mcm-2 (P < 0.0001), Ki67 (P < 0.0001), cyclin A (P < 0.0001) and cyclin B1 (P = 0.002) expression with increasing grade from diffuse astrocytoma through anaplastic astrocytoma to glioblastoma, suggesting that any of these four markers has potential as a marker of tumour grade. In a subset of glioblastomas (n = 16) for which accurate clinical follow-up data were available, there was a suggestion that the cyclin A:Mcm-2 labelling fraction might predict a relatively favourable response to radical radiotherapy. These provisional findings, however, require confirmation by a larger study. We conclude that it is feasible to obtain detailed cell cycle data by immunohistochemical analysis of tissue biopsies. Such information may facilitate tumour grading and may enable information of prognostic value to be obtained in the routine diagnostic laboratory.     

Clinicopathological correlation of cell proliferation, apoptosis and p53 in cerebellar pilocytic astrocytomas

We have analysed 78 cerebellar pilocytic astrocytomas to assess whether histopathology, cell proliferation, apoptosis rate, p53 immunoreactivity, or flow cytometry could predict their long-term behaviour. Classic pilocytic/microcystic pattern was seen in 62 patients and 16 patients had mixed pattern with an additional non-pilocytic glial component. The overall 5-year survival was 93%, complete resection providing 100% survival. The four patients who died during the follow-up were more than 14 years of age, their primary operation had been incomplete and three of them were mixed variants. In 15 cases the tumour recurred giving a recurrence-free 5-year survival of 77%. The proliferation indices were low: Ki-67_MIB-1 (median 2.0%), PCNA (1.2%) and S-phase fraction (4.4%). The Ki-67_MIB-1-labelling index was significantly higher in young patients, but did not differ between the classic and mixed variants. Twenty-two per cent of the tumours were aneuploid with a significantly higher S-phase fraction than in diploid tumours. p53 seems to act as ardian of the genome' in pilocytic astrocytomas, because aberrant/increased expression of p53 and aneuploidy associated with enhanced apoptosis. Only patient age ( P =0.01), radicality of the primary operation ( P =0.0001) and histology (classic vs mixed, P =0.008) significantly correlated with survival. The poorer prognosis of the mixed variant suggests that this may represent a distinct entity. Although none of the novel parameters significantly predicted recurrence or survival, they indicate substantial biological variation among cerebellar pilocytic astrocytomas.     

儿童与成人脑干星形细胞瘤临床特点及手术疗效比较

目的比较病理级别相同的儿童和成人脑干星形细胞瘤病人的临床特点和手术疗效的差异。方法回顾性分析40例病理级别相同(WHO分级Ⅱ级)的脑干星形细胞瘤病人的临床资料,包括儿童组10例,成人组30例。比较两组的一般资料和手术疗效差异,并采用Kaplan-Meier法分析两组术后1年生存率。结果肿瘤侵犯脑神经数目,在儿童组和成人组组间的差异具有统计学意义(P=0.017);病人性别、MRI显示肿瘤有无强化、肿瘤部位、大小、切除程度,入院时的日常生活质量评分(KPS)及出现症状至手术时间等方面,两组间差异均无统计学意义。生存分析显示:儿童组平均生存16.5个月,术后1年生存率为55.6%,成人组平均生存35.2个月,术后1年生存率为83.3%;成人组脑干星形细胞瘤预后优于儿童组(P=0.005)。结论病理级别相同的成人和儿童脑干星形细胞瘤,成人预后好于儿童;与成人比较,儿童脑干星形细胞瘤浸润生长明显。     

星形细胞瘤中错配修复基因HMSH2蛋白表达的改变

目的探讨HMSH2与星形细胞瘤发生、发展及恶性程度的关系。方法应用免疫组化技术检测50例星形细胞瘤组织和10例正常脑组织标本中HMSH2蛋白的表达情况。结果正常脑组织、低级别星形细胞瘤、高级别星形细胞瘤中HMSH2蛋白缺失率分别为0、33.3%、65.2%。结论HMSH2蛋白缺失率与星形细胞瘤的病理分级和恶性程度有密切相关,可能在星形细胞瘤的发生、发展过程中起重要作用。     

Mipu1在人脑星形细胞瘤中的表达及临床意义初步探讨

目的研究心肌缺血预适应上调蛋白1(Mipu1)在人脑星形细胞瘤中mRNA及蛋白水平的表达情况,并探讨其临床意义。方法收集24例临床上病理级别分别为Ⅰ、Ⅱ、Ⅲ、Ⅳ级的脑星形细胞瘤组织标本,应用逆转录酶-聚合酶链式反应(RT-PCR)和蛋白印迹技术(Western blot)分别检测Mipu1的mRNA和蛋白质表达水平。结果 RT-PCR结果显示Mipu1的mRNA的表达水平随着星形细胞瘤病理级别的升高而上调;相似,Western blot分析结果显示Mipu1蛋白表达水平也随着星形细胞瘤病理级别的升高而上调,差异有显著性(P<0.05)。结论 Mipu1在脑星形细胞瘤中表达上调,其表达水平与星形细胞瘤病理级别成正相关,可作为判别星形细胞瘤分化程度的指标之一。     

Cell proliferation patterns in the diagnosis of astrocytomas, anaplastic astrocytomas and glioblastoma multiforme: a Ki-67 study

Although astrocytomas, anaplastic astrocytomas and glioblastoma multiforme differ in their clinical courses, histological distinction between these three tumours and between astrocytomas and anaplastic astrocytomas in particular may be unclear on histology alone especially in small biopsies. In the present study, a more objective way of distinguishing between the three types of tumour is sought. Frozen sections from 26 astrocytomas, 26 anaplastic astrocytomas and 38 glioblastomas were stained by an indirect immunoperoxidase technique using the monoclonal antibody Ki-67 which binds to nuclear proteins in the G1, S, G2 and M phases of the cell cycle. The Ki-67 staining was assessed quantitatively through direct observation of the stained sections by the use of a drawing tube attached to a microscope. A minimum of 1000 cells was counted in each case and labelled cells were expressed as a percentage of the total number of cells. These results, when correlated in each case with the histology of the tumour in paraffin sections, showed Ki-67 labelling indices ranging from 0 to 1.9% (mean 0.5% SD +/- 0.54) in astrocytomas, 0.6 to 10.9% (mean 4.1% SD +/- 2.8) in anaplastic astrocytomas and 0.9 to 16.2% (mean 6.4% SD +/- 3.34) in glioblastoma multiforme. The differences in the three types of tumour were statistically significant. The results suggest that Ki-67 staining is a useful addition to the panel of techniques for distinguishing between astrocytomas, anaplastic astrocytomas and glioblastoma multiforme.     

Characterization of Numb expression in astrocytomas

During early neurodevelopment, asymmetric segregation of Numb in mitotic progenitor cells influences the fate of daughter cells, whereby one daughter retains the progenitor phenotype while the other proceeds along a differentiation pathway. Numb has also been reported to function as a tumor suppressor in breast cancers and medulloblastomas. Given its role in maintaining neural progenitor pools in animal models and its reported role as a tumor suppressor, Numb could potentially contribute to astrocytoma oncogenesis. We characterized Numb expression in both human astrocytoma tissue samples and glioblastoma cell lines. We found that Numb is expressed in all grades of astrocytomas, being predominantly cytoplasmic in higher‐grade astrocytomas but nuclear in pilocytic astrocytomas. Numb is also present in normal neurons, but not in normal astrocytes. In cultured glioblastoma cells, Numb concentrates in the perinuclear region and process tips. Numb expression in astrocytomas recapitulates that of progenitor cells during neurodevelopment, and suggests a role for Numb in astrocytoma oncogenesis.     

Vascular endothelial growth factor receptor 2 (VEGFR-2) signalling activity in paediatric pilocytic astrocytoma is restricted to tumour endothelial cells

A. H. Sikkema, E. S. J. M. de Bont, G. Molema, A. Dimberg, P. J. Zwiers, S. H. Diks, E. W. Hoving, W. A. Kamps, M. P. Peppelenbosch and W. F. A. den Dunnen (2011) Neuropathology and Applied Neurobiology 37, 538–548 Vascular endothelial growth factor receptor 2 (VEGFR‐2) signalling activity in paediatric pilocytic astrocytoma is restricted to tumour endothelial cells Aims: Tumours depend on angiogenesis for enhanced tumour cell survival and progression. Vascular endothelial growth factor receptor (VEGFR) signalling plays a major part in this process. Previously, we evaluated tyrosine kinase activity in paediatric brain tumour tissue lysates using a peptide microarray containing 144 different tyrosine kinase peptide substrates. When applied to paediatric pilocytic astrocytoma tissue, this analysis revealed extensive phosphorylation of VEGFR‐derived peptides. The aim of the current study was to validate this result and determine the presence of VEGFR‐2 activity in paediatric pilocytic astrocytoma as the main VEGFR in terms of mitogenic signalling. In addition, the localization of VEGFR1–3 mRNA expression was assessed. Methods: VEGFR‐2 phosphorylation was determined by adopting a proximity ligation assay approach. Enrichment of endothelial markers and VEGFRs in tumour endothelium was determined by quantitative polymerase chain reaction (qPCR) analysis of laser‐microdissected blood vessels. Results: Proximity ligation assays on tumour cryosections showed the presence of phosphorylation of VEGFR‐2, which primarily localized to vascular endothelium. qPCR analysis of endothelial markers and VEGFRs showed a 13.6‐fold average enrichment of VEGFR‐2 expression in the laser‐microdissected endothelium compared to whole tumour. Also the expression of VEGFR‐1 and ‐3 was highly enriched in the endothelium fraction with an average fold‐enrichment of 16.5 and 50.8 respectively. Conclusions: Phosphorylated VEGFR‐2 is detected on endothelial cells in paediatric pilocytic astrocytoma. Furthermore, endothelial cells are the main source of VEGFR1–3 mRNA expression. This suggests a crucial role for VEGF/VEGFR‐induced angiogenesis in the progression and maintenance of these tumours.     

Neuropeptide Y,somatostatin, and cholecystokinin neurone preservation in anaplastic astrocytomas

Summary Using immunohistochemistry, well-preserved neuronal cell bodies and fibres containing neuropeptide Y, somatostatin, and cholecystokinin immunoreactivity have been identified in all seven supratentorial anaplastic astrocytomas studied. These neurones have been shown not only on the edge but also in the depth of the neoplastic tissue. These neuropeptides were not present in 18 other intracranial tumours (3 astrocytomas, 1 subependymoma, 8 glioblastoma multiformes, 1 meningioma, and 5 metastases). In all 25 intracranial tumours studied, no immunoreactivity was found for vasoactive intestinal polypeptide, substance P, methionine-enkephalin, leucine-enkephalin, synenkephalin, neurophysin I-II, and corticotropin releasing factor.Supported in part by the Belgian Fund for Medical Scientific Research (FRSM 3.4523.86), the Queen Elisabeth Medical Fundation (FMRE-Neurobiology 1986–87) and the Belgian National Lotery (1986).