脑胶质瘤化疗药物敏感性的临床研究

背景与目的 :脑胶质瘤是一个需要多学科综合治疗的恶性脑部疾病,国内外学者都在积极探索更加有效的综合治疗方法。本文探讨脑胶质瘤细胞在体外对化疗药物的敏感性,为临床有效治疗胶质瘤提供实验依据。方法:应用四甲基偶氮唑盐比色法(methyl thiazolyl tetrazolium,MTT)对胶质瘤化疗的7种常用药物体外敏感性进行检测。结果:总体化疗药物敏感性BCNU﹥VM-26、TMZ﹥DDP﹥CBP﹥VP-16﹥VCR;低级别胶质瘤(WHO分级Ⅰ、Ⅱ级)化疗药物敏感性BCNU﹥TMZ﹥VM-26、DDP﹥CBP、VCR﹥VP-16;高级别胶质瘤(WHO分级Ⅲ、Ⅳ级)化疗药物敏感性BCNU、VM-26﹥TMZ、DDP﹥VP-16﹥CBP﹥VCR。结论:体外药敏试验对排除无效药物、筛选敏感药物对脑胶质瘤进行个体化的化疗,提高临床化疗效果,具有重要意义。     

长周期替莫唑胺治疗脑胶质瘤:附32例报告

背景与目的 :替莫唑胺(temozolomide,TMZ)国内外多推荐为胶质瘤的一线化疗药物,化疗周期通常为六周期。长周期(超过六周期)TMZ治疗胶质瘤国外已有多篇文献报道,但中国脑胶质瘤患者这方面信息较少。本文总结我们近年来长周期TMZ治疗32例胶质瘤的临床经验,重点探讨其安全性。方法:32例高级别胶质瘤(high-grade gliomas,HGGs)或低级别胶质瘤(low-grade gliomas,LGGs)采用了TMZ长周期治疗。TMZ化疗方案的选择基于肿瘤组织DNA甲基转移酶(O6-methylguanine-DNA methyltransferase,MGMT)的免疫组化检测结果,用甲基化特异PCR(MSP-PCR)检测了其中6例的MGMT启动子甲基化程度。MGMT阴性表达(±或-)者接受TMZ标准化疗(200mg/(m2.d),d1-5,四周方案),MGMT阳性表达(+或++)者接受TMZ剂量密度方案[75mg/(m2.d),d1-21,4周方案],或顺铂(cisplatin,DDP)联合TMZ化疗方案[DDP 75mg/(m2.d),d1-2;TMZ 200mg/(m2.d),d2-6,四周方案]。结果:32例患者共接受318周期TMZ方案化疗。患者化疗周期数为7~24(中位周期数为9.4)。最常见的严重毒性反应是Ⅲ度淋巴细胞减少症与白细胞减少症,发生率均为9.4%(3/32)。最常见的轻至中度毒性反应依次为疲乏(86.9%)、中性粒细胞减少症(46.9%)、脱发(46.9%)、血小板减少症(40.6%)、便秘(41.2%)及淋巴细胞减少症(25.0%)等。中位无进展生存(progress free survival,PFS)为28.6月。6月PFS、12月PFS分别为100%与71%。32例患者中,15例肿瘤完全切除,至今无病生存。依据意向性治疗原则(intention-to-treatprinciple,ITT),1例(3.1%)取得完全缓解(complete response,CR),14例(43.8%)微效(minor response,MR),2例(6.2%)稳定(stable disease,SD)。总反应率(overall response rate,ORR)为81.5%(95%CI,50%~96%),疾病控制率(disease control rate,DCR)为89.2%(95%CI,64%~98%)。结论:长周期TMZ化疗治疗胶质瘤是安全的。长周期TMZ化疗具有较高的反应率(ORR)与无进展生存(PFS)。     

低场强术中磁共振BOLD导航在脑功能区胶质瘤术中的应用

目的 探讨低场强术中磁共振(iMRI)融合术前采集血液氧饱和水平检测功能成像(BOLD)功能导航的可行性和应用价值.方法 48例脑皮层功能区的胶质瘤患者随机分组,试验组用术前高场强的BOLD影像与术中iMRI解剖影像融合,对照组将高场强的BOLD与术前高场强MRI解剖影像融合,分别导航手术.两组均采用术中电生理来验证BOLD功能区定位的准确性并随访.结果 试验组BOLD敏感性73.3%,特异性83.3%;对照组BOLD敏感性75.5%,特异性81.2%,差异无统计学意义.试验组肿瘤全切率为85.1%,对照组77.8%,差异有统计学意义(P:0.012);试验组术后功能改善或保持率为80.8%,对照组83.3%,差异无统计学意义.结论 低场强iMRI与术前BOLD功能导航手术可实时提供病灶与功能区可视化解剖信息,有助于提高肿瘤切除率,降低术后致残率.     

高场强术中磁共振成像对脑胶质瘤全切率的影响及其评估

目的 探讨高场强术中磁共振成像(iMRI)对脑胶质瘤手术全切率的影响及其意义.方法 自2009年2月至6月应用高场强iMRI施行脑胶质瘤切除术40例.运用术中影像数据对胶质瘤体积及全切率做回顾性分析.结果 术中第1次行iMRI扫描仅10例胶质瘤完全切除,30例肿瘤仍有残留,23例行进一步切除,其中21例胶质瘤最终全切除.最终肿瘤的伞切率从25%提高到78%,残存肿瘤的体积也明显下降.结论 高场强iMRI的应用显著提高脑胶质瘤手术的全切率.     

Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression

Although rodent glioblastoma (GBM) models have been used for over 30 years, the extent to which they recapitulate the characteristics encountered in human GBMs remains controversial. We studied the histopathological features of dog GBM and human xenograft GBM models in immune-deficient mice (U251 and U87 GBM in nude Balb/c), and syngeneic GBMs in immune-competent rodents (GL26 cells in C57BL/6 mice, CNS-1 cells in Lewis rats). All GBMs studied exhibited neovascularization, pleomorphism, vimentin immunoreactivity, and infiltration of T-cells and macrophages. All the tumors showed necrosis and hemorrhages, except the U87 human xenograft, in which the most salient feature was its profuse neovascularization. The tumors differed in the expression of astrocytic intermediate filaments: human and dog GBMs, as well as U251 xenografts expressed glial fibrillary acidic protein (GFAP) and vimentin, while the U87 xenograft and the syngeneic rodent GBMs were GFAP⁻ and vimentin⁺. Also, only dog GBMs exhibited endothelial proliferation, a key feature that was absent in the murine models. In all spontaneous and implanted GBMs we found histopathological features compatible with tumor invasion into the non-neoplastic brain parenchyma. Our data indicate that murine models of GBM appear to recapitulate several of the human GBM histopathological features and, considering their reproducibility and availability, they constitute a valuable in vivo system for preclinical studies. Importantly, our results indicate that dog GBM emerges as an attractive animal model for testing novel therapies in a spontaneous tumor in the context of a larger brain.     

PAX6 increases glioma cell susceptibility to detachment and oxidative stress

Purpose Glioblastoma multiforme (GBM) is an incurable malignant glioma which is very resistant to radiation and alkylating agent-based chemotherapy. Necrosis is a hallmark for GBM and the layer surrounding necrotic area is packed with cells which are now believed to be those migrating out of the necrotic area. Oxidative stress is a condition that GBM cells encounter in the necrotic zone, which is one of the stressful conditions that GBM cells need to resist in order to survive. Our previous studies revealed that low PAX6 expression is a favorable molecular trait for survival acquired by GBM cells because PAX6 could suppress cell invasion and tumorigenicity. Since detachment of cells from GBM is an early event for cell migration and subsequent invasion, we examined whether PAX6 is involved in cell survival after detachment. Experimental Design PAX6 over-expression was achieved in glioma cells transiently (by adenoviral-mediated transient over-expression) or stably (by the establishment of stable cell lines after transfection). The effect of PAX6 over-expression on the survival and growth of glioma cells after detachment from the culture was determined. Result Our data revealed that GBM cells (with their low PAX6 levels) survived the detachment procedure well. However, PAX6 over-expression attenuated GBM cell recovery of growth after detachment-induced stress. Importantly, intracellular reactive oxygen species (ROS) levels increased following cell detachment and that PAX6 over-expressing cells retained higher level of ROS than control cells. This may be partially responsible for the impaird growth rate after cell detachment. Addition of anti-oxidant improved the cell viability of PAX6 over-expressing cells, but did not restore their ability to proliferate. Conclusion To survive, GBM cells must resist oxidative stress in the necrotic zone as well as the intracellular ROS generated during detachment. Since PAX6 over-expression in low PAX6-expressing glioma cells attenuated cell survival and growth after detachment, these results suggest that a reduced PAX6 expression may be a molecular trait that gives glioma cells a real selection advantage over other cell types to survive in stressful conditions, thus resulting in expansion of their population.     

Continuous intracranial administration of suberoylanilide hydroxamic acid (SAHA) inhibits tumor growth in an orthotopic glioma model

Object Current treatments for malignant gliomas produce only a modest increase in survival time. New therapeutic approaches are desperately needed. Suberoylanilide hydroxamic acid (SAHA) is an effective inhibitor of the growth of many solid and hematological malignancies. Nevertheless, very few studies have investigated the effects of SAHA on glial tumors. The present study was designed to investigate the therapeutic effects of the intracranial local delivery of SAHA in an orthotopic glioma model. Methods The antiproliferative effect of SAHA was examined in six glioblastoma and one endothelial cell lines in vitro. In addition, one glioblastoma cell line (U87MG) used in in vivo short term (14 days) and survival studies in an orthotopic human glioma athymic mice model. Tumor volume, apoptosis rate, microvessel density, and proliferation index were determined by immunohistochemistry. Results SAHA treatment inhibited the growth of all cell lines in concentrations ranging from 1 μM to 30 μM. For short-term studies, histological analysis showed an 80% reduction of tumor volume in the treatment group (P < 0.001). This reduction in tumor volume was associated with a significant increase in the apoptosis rate (31.9%, P < 0.001), a significant decrease in the proliferation (36.8%, P < 0.001) and angiogenesis rates (30%, P < 0.05). For survival studies, the mean survival time was 22 days in the control group, whereas it was 42 days in the treatment group. Conclusions These results suggest that local delivery with SAHA inhibits intracranial glioma growth in vitro and in vivo. SAHA is a promising candidate for further preclinical and clinical studies on glial tumors.     

Expression of VEGFR3 in glioma endothelium correlates with tumor grade

Angiogenic processes are regulated by vascular endothelial growth factors (VEGFs) and their receptors VEGFR1 (Flt-1), 2 (Flk-1) and 3 (Flt-4). While VEGFR2 is thought to play a central role in tumor angiogenesis, anti-angiogenic therapies targeting VEGFR2 in glioma models can show escape phenomena with secondary onset of angiogenesis. The purpose of this study was to find explanations for these processes by searching for alternative pathways regulating glioma angiogenesis and reveal a correlation with tumor grade. Thus, VEGFR3, which is not expressed in normal brain, and its ligands VEGF-C and -D, were assessed in high grade (WHO°IV, glioblastomas, GBM) and low grade gliomas [WHO°II astrocytomas (AII)]. In all GBM, a strong protein expression of VEGFR3 was found on tumor endothelium, VEGF-C and -D expression was found on numerous cells in areas of high vascularization. On RNA level, a significant up-regulation of VEGFR3 was detected in GBM compared to AII and non-neoplastic brain. In AII, only very moderate VEGFR3, VEGF-C and -D expression was found on protein and RNA level indicating a correlation of VEGFR3 expression with tumor grade. VEGFR3 signal in both grades was found predominantly on endothelial cells, confirmed by VEGFR3 expression on isolated CD31 positive cells and the expression of various endothelial markers on VEGFR3-positive cells isolated from GBM. The demonstration of a complete angiogenic signaling system that is dependent on tumor grade may influence the traditional paradigm of glioma angiogenesis and may provide a basis for more effective anti-angiogenic treatment strategies.     

Multivoxel 3D proton MR spectroscopy in the distinction of recurrent glioma from radiation injury

Objective To explore the usefulness of multivoxel 3D proton MR spectroscopy (¹H-MRS) in assessing the recurrent contrast-enhancing areas at the site of the previously treated gliomas. Materials and methods In 28 patients who had new contrast-enhancing lesions in the vicinity of the previously resected and irradiated high-grade glioma, 3D ¹H-MRS examinations were performed on a 3.0T MR scanner. Spectral data for N-acetylaspartate (NAA), choline (Cho), and creatine (Cr) were analyzed in all patients. Receiver operating characteristic analysis was performed, and the threshold value for tumor differentiation was determined. Diagnosis of these lesions was assigned by means of histopathology and follow-up. Results Diagnostic-quality 3D ¹H-MRS with quantifiable Cho, Cr, and NAA peaks was obtained in 92.9% of the cases. The Cho/NAA and Cho/Cr ratios were significantly higher in recurrent tumor than in radiation injury (P < 0.01), whereas the NAA/Cr ratios were lower in recurrent tumor than in radiation injury (P = 0.02). The Cho/Cr and Cho/NAA ratios were significantly higher in radiation injury than in normal-appearing white matter (P < 0.01), however, the NAA/Cr ratios were lower in radiation injury than in normal-appearing white matter (P = 0.01). Using receiver operating characteristic analysis, the resulting sensitivity, specificity and diagnostic accuracy of 3D ¹H-MRS were 94.1%, 100%, and 96.2%, respectively, based on the cut-off values of 1.71 for Cho/Cr or 1.71 for Cho/NAA or both as tumor criterion. Conclusion 3D ¹H-MRS could differentiate recurrent tumor from radiation injury in patients with recurrent contrast-enhancing lesions in the vicinity of the previously treated gliomas.     

LRIG3特异性RNA干扰真核表达载体的构建及稳定株筛选

目的构建LRIG3（leucine-rich repeats and immunoglobulin-like domains 3,LRIG3）基因特异的RNA干扰质粒,为探讨抑制LRIG3基因表达对脑胶质瘤细胞生物学行为调控的研究奠定基础。方法根据GenBank数据库提供的LRIG3基因核苷酸序列,选择设计2条能转录短发卡RNA（shRNA）的DNA序列,命名为LRIG3-shRNA1、LRIG3-shRNA2,同时设计1条非特异性序列作为阴性对照,命名为negative-shRNA。并与pGenesil2质粒载体连接,转化感受态大肠杆菌,挑选阳性克隆,抽取重组质粒,使用限制性内切酶SalⅠ酶切电泳,DNA测序鉴定。3种重组表达载体转染胶质瘤细胞系GL15细胞,用G418筛选后挑选单克隆并扩增获得稳定株。逆转录酶-聚合酶链反应（RT-PCR）和Western blot分别在mRNA和蛋白水平上检测LRIG3的表达。结果重组质粒成功转化感受态大肠杆菌,经SalⅠ酶切琼脂糖凝胶电泳分析,结果表明寡核苷酸成功插入到预计位点,经测序鉴定,序列完全正确。G418筛选出稳定转染三种质粒的GL15细胞,转染pGenesil2-LRIG3-shRNA组细胞LRIG3 mRNA和蛋白表达明显低于转染pGenesil2-negative-shRNA组。结论成功构建针对LRIG3基因的特异性shRNA真核表达载体,转染细胞后可抑制LRIG3基因表达,为进一步研究其基因功能奠定了基础。