Aberrant MGMT (O6-methylguanine-DNA methyltransferase) promoter methylation in choroid plexus tumors

Aberrant methylation of the MGMT (O6-methylguanine-DNA methyltransferase) DNA-repair gene is a predictive marker for the response to chemotherapy with alkylating agents (e.g., temozolomide) in malignant gliomas. Since temozolomide is considered for the treatment of choroid plexus tumors, MGMT promoter methylation status was retrospectively assessed in 36 choroid plexus tumors using methylation specific PCR, combined bisulfite restriction analysis (COBRA), and clone sequencing. By methylation specific PCR, all samples demonstrated a signal for MGMT methylation. COBRA confirmed >10% methylation of CpGs 17 and 31 in 58% of tumors. Clone sequencing of six cases methylated by COBRA confirmed aberrant methylation including a previously recognized enhancer element. In conclusion, MGMT promoter methylation is frequent in choroid plexus tumors and can be quantified using COBRA. Determination of MGMT promoter methylation status might be useful for the stratification of patients for alkylator-based treatments in future clinical trials. The authors M. Hasselblatt and J. Mühlisch have contributed equally.     

Trial of a 5-day dosing regimen of temozolomide in patients with relapsed small cell lung cancers with assessment of methylguanine-DNA methyltransferase

Objectives

Small cell lung cancers (SCLCs) are characterized by aberrantly methylated O⁶-methyl-guanine-DNA methyltransferase (MGMT). Epigenetic silencing of MGMT is associated with loss of MGMT activity and improved sensitivity to alkylating agents in glioblastomas. We have reported the activity of temozolomide, a non-classical alkylating agent, in patients with relapsed sensitive or refractory SCLCs, given at 75 mg/m²/day for 21 of 28 days. However, prolonged myelosuppression was noted. We therefore evaluated a 5-day dosing schedule of temozolomide and examined MGMT as a predictive biomarker for temozolomide treatment in SCLC.

Materials and methods

Patients with sensitive or refractory SCLCs and progression after one or two prior chemotherapy regimens received temozolomide 200 mg/m²/day for 5 consecutive days in 28-day cycles. The primary endpoint was tolerability. We also assessed MGMT promoter methylation status by PCR and MGMT expression by immunohistochemistry in tumor specimens.

Results

Of 25 patients enrolled, 5 experienced grade 3 or 4 toxicity (anemia, thrombocytopenia, neutropenia, and constipation). The partial response rate was 12% [95% CI: 3–31%], with partial responses in 2 refractory patients. We were able to obtain tumor samples for more than half of patients for MGMT testing.

Conclusion

Temozolomide 200 mg/m²/day for 5 days in 28-day cycles is tolerable and active in patients with relapsed SCLCs. No treatment-limiting prolonged cytopenias were observed, making this our preferred schedule for further studies. Acquisition of archived biospecimens is feasible and necessary in order to continue evaluating the role of MGMT as a predictive biomarker in SCLCs.     

FANCD2 re-expression is associated with glioma grade and chemical inhibition of the Fanconi Anaemia pathway sensitises gliomas to chemotherapeutic agents

Brain tumours kill more children and adults under 40 than any other cancer. Around half of primary brain tumours are glioblastoma multiforme (GBMs) where treatment remains a significant challenge. GBM survival rates have improved little over the last 40 years, thus highlighting an unmet need for the identification/development of novel therapeutic targets and agents to improve GBM treatment. Using archived and fresh glioma tissue, we show that in contrast to normal brain or benign schwannomas GBMs exhibit re-expression of FANCD2, a key protein of the Fanconi Anaemia (FA) DNA repair pathway, and possess an active FA pathway. Importantly, FANCD2 expression levels are strongly associated with tumour grade, revealing a potential exploitable therapeutic window to allow inhibition of the FA pathway in tumour cells, whilst sparing normal brain tissue. Using several small molecule inhibitors of the FA pathway in combination with isogenic FA-proficient/deficient glioma cell lines as well as primary GBM cultures, we demonstrate that inhibition of the FA pathway sensitises gliomas to the chemotherapeutic agents Temozolomide and Carmustine. Our findings therefore provide a strong rationale for the development of novel and potent inhibitors of the FA pathway to improve the treatment of GBMs, which may ultimately impact on patient outcome.     

替莫唑胺对胶质瘤干细胞抗凋亡与多重耐药基因及细胞周期的影响

目的 分析替莫唑胺(TMZ)对胶质瘤U251细胞及其干细胞livin、MRP基因表达及细胞周期的影响.方法 分离培养U251细胞及其干细胞后,不同浓度的TMZ(0、25、50、100、200、400 μmol/L)干预72 h,CCK-8检测细胞增殖变化趋势,流式细胞术(FCM)检测细胞周期变化,RTPCR技术检测livin、MRP1、MRP3 mRNA表达.结果 本研究成功从U251细胞中分离出U251干细胞;与未干预组相比,25 μmol/L的TMZ干预U251细胞及其干细胞后其增殖率分别为0.952±0.011(t=-8.219,P=0.001)、0.937±0.029(t=-3.822,P=0.019)和100 μmol/L的TMZ干预U251细胞及其干细胞后其增殖率分别为0.750±0.018、0.887±0.039(t=5.480,P=0.005);正常情况下,livin基因在U251细胞及其干细胞中的表达量分别为(0.411 ±0.025)×10-3、(0.571 ±0.040)×10-3(t=-3.348,P=0.004),MRP1基因的表达量分别为(0.295 ±0.018)×-3、(0.481 ±0.034)×10-3(t=-8.439,P=0.001),MRP3基因的表达量分别为(1.128±0.117)×10-3、(0.963±0.059)×10-3(=2.176,P=0.095);200 μmol/L的TMZ干预U251细胞及其干细胞后livin mRNA表达量在分别为(0.020±0.002)×10-3(t=27.123,P =0)、(0.066 ±0.007)×10-3(￡=21.345,P =0);200 μmol/L干预后MRP1 mRNA表达量为(0.556±0.041)×10-3 (t=-10.214,P=0.001)、(0.609 ±0.044)×10-3(t=-3.98,P=0.016);200 μmol/L干预MRP3 mRNA表达量为(1.397±0.192)×10-3(t=-2.073,P=0.107)、(1.496 ±0.302)×10-3(t=-2.993,P=0.040);100 μmol/L干预后细胞周期U251细胞G2/M期百分比为11.353 ±0.516、39.1±1.2(t=-37.1,P=0)和U251干细胞S期百分比为21.1±1.0、30.2±1.7(t=-8.07,P=0.001).结论 替莫唑胺能有效降低U251及其干细胞中livin基因的表达,并使细胞分裂周期停滞,降低细胞增殖率,促进细胞凋亡.MRP1和MRP3可能分别是原发胶质瘤与复发胶质瘤耐药的主要原因之一.     

调强放疗联合替莫唑胺治疗丘脑胶质瘤

目的探讨调强放疗(IMRT)联合替莫唑胺(TMZ)化疗治疗丘脑胶质瘤的疗效,分析影响预后的因素。方法回顾性分析2005年1月~2010年12月联合放化疗治疗的25例丘脑胶质瘤。所有病例治疗前均行立体定向活检明确病理诊断,其中低级别胶质瘤(WHOⅡ级)11例,高级别胶质瘤(WHOⅢ~Ⅴ级)14例。记录治疗反应,计算总体生存率(OS)和无疾病进展生存率(PFS),并应用COX回归模型进行多因素预后分析。结果本组病例急性治疗反应多为Ⅰ~Ⅱ级,没有Ⅲ级以上的反应。同期放化疗结束时,中位KPS上升20。6个月、1年和2年的OS分别为84.0%、52.0%和22.2%,PFS分别为76.0%、33.0%和19.8%,中位生存时间为13.0个月(95%CI 9.5~16.5),中位无进展生存时间9.0个月(95%CI6.9~11.1)。多因素分析显示,性别、年龄、病程以及放疗前KPS与预后无显著关系,病理级别与预后显著相关。结论调强放疗联合TMZ化疗治疗丘脑胶质瘤的急性反应小,患者能够耐受,近期疗效有改善,但长期疗效仍不理想。     

地塞米松拮抗替莫唑胺抑制胶质瘤细胞生长的体外试验

目的:探讨替莫唑胺(TMZ)联合地塞米松(DXM)对体外培养的人胶质瘤细胞系U251细胞的增殖影响。方法:U251细胞分为TMZ组(分别加入10、25、50、100、200、400μmol·L~(-1)浓度的TMZ)、TMZ+DXM组(在各浓度TMZ的基础上加入40μmol·L~(-1)的DXM)和对照组(不加任何药物)。各组细胞加入相应药物72 h后,观察细胞形态学改变,检测细胞增殖抑制率及细胞周期和凋亡率。结果:TMZ浓度大于100μmol·L~(-1)时,TMZ组抑制率大于TMZ+DXM组(P<0.01)。与TMZ组比较, TMZ+DXM组细胞G_(0-1)期比例增加、G_2～M期比例减少,细胞的凋亡率进一步降低。结论:DXM可能通过改变细胞周期分布而减弱TMZ抑制胶质瘤细胞生长作用,2药联用具有负协调作用。     

替莫唑胺治疗恶性脑胶质瘤97例多中心随机对照研究

目的 评价国产替莫唑胺(TMZ)胶囊治疗恶性脑胶质瘤的临床疗效和安全性.方法 采用多中心、随机、双盲、阳性药平行对照研究,将恶性脑胶质瘤患者随机分为试验组(TMZ组)和环已亚硝脲对照组(CCNU)组.试验组于第1～5天给予TMZ胶囊150 mg/m2口服,第1天需同时口服CCNU安慰剂130 mg/m2;对照组于第1天口服CCNU胶囊130 mg/m2,第1～5天口服TMZ安慰剂150 mg/m2.两组均每28天为1个周期,连续治疗3个周期.结果 共入组97例,可评价疗效者86例:TMZ组42例,CCNU组44例.治疗12周后,TMZ组:完全缓解(CR)1例,部分缓解(PR)14例,稳定(SD)23例,进展(PD)4例;CCNU组:CR 3例,PR 1例,SD 29例,PD 11例.TMZ组和CCNU组的有效率(CR+PR)分别为35.71%和9.09%(P<0.01),临床受益率(CR+PR+SD)分别为90.48%和75.00%(P<0.05).TMZ常见的不良反应为恶心呕吐,多为轻、中度.结论 国产TMZ胶囊治疗恶性脑胶质瘤的疗效优于CCNU,耐受性良好,是一种安全有效的治疗恶性脑胶质瘤的化疗药.     

p53上调凋亡调控因子增强胶质瘤干细胞对替莫唑胺化疗敏感性的研究

 目的　探讨不同CD133免疫原型的胶质瘤细胞对替莫唑胺（TMZ）耐药性的差异以及p53上调凋亡调控因子（PUMA）改变胶质瘤细胞对TMZ敏感性的影响。方法　利用免疫磁珠细胞分选法筛选出CD+133和CD-133的胶质瘤细胞U87MG,分别传代培养,转染Ad-PUMA,并用TMZ干预。四甲基偶氮唑蓝比色法（MTT法）检测细胞增殖情况及对TMZ的半数抑制浓度（IC50）,流式细胞术观察外源性PUMA、TMZ干预前后CD+133和CD-133细胞的凋亡情况。结果　CD+133免疫磁珠分选出胶质瘤干细胞,CD+133胶质瘤细胞IC50较CD-133胶质瘤细胞高[（200±10）μmol／L比（80±11）μmol／L],转染Ad-PUMA后,两组细胞IC50均降低[（100±8）μmol／L比 （20±7）μmol／L],两组转染前后IC50差异均有统计学意义（P < 0.05）。TMZ联合PUMA组CD+133细胞凋亡率为68.05 %,对照组、TMZ组及PUMA组分别为6.05 %、32.91 %和40.61 %,联合组与其他组比较差异均有统计学意义（均P＜0.05）。结论　PUMA联合TMZ可以促进耐药的胶质瘤干细胞凋亡,从而增强胶质瘤对化疗的敏感性。     

Treatment of elderly patients with glioblastoma: From clinical evidence to molecular highlights

Elderly patients with glioblastoma are characterized by a high rate of associated morbidities, and a poor prognosis. Therefore, they have been excluded from most prospective clinical trials. However, the poorer outcome retrospectively reported in these patients might be also related to that those are less likely to receive the appropriate treatment than their younger counterparts. We reviewed the literature with regard to the optimal therapeutic management of this particular population, with focus on molecular perspectives for improving patients' selection. Clinical data have demonstrated that open craniotomy with resection of the tumor was superior to biopsy only in elderly patients with good Karnofsky Performance Status (KPS) score. Then, postoperative radiotherapy (RT) improves survival without impairing functional status or neurocognitive functions, compared with best supportive care only following resection. Despite promising preliminary data, the addition of concomitant temozolomide to RT has not been validated in patients more than 70-years old. In case of additional poor prognostic factors or after biopsy only, there is no definitive demonstration that RT, chemotherapy, or both could improve outcome. Incorporation of more sensitive predictive and/or prognostic molecular factors could help physicians in patients' selection. Further prospective trials should incorporate age-dependent molecular specificities in their design, and better focus on particular subgroup of patients exhibiting specific molecular alterations.