药物相关性分子靶标检测在指导Ⅲ-Ⅳ期儿童神经母细胞瘤治疗中的初步经验

Primary identification of genomic markers of drug sensitivity for individualized therapy in children with neuroblastoma of Ⅲ-Ⅳ stage

目的探讨药物相关性分子靶标检测在儿童神经母细胞瘤个体化治疗中的作用和意义。方法2011年4月至2013年4月间在上海交通大学医学院附属新华医院诊治的13例儿童神经母细胞瘤患儿，取其手术肿瘤及外周血标本共31份（手术/二次手术标本共15份，外周血标本16份）。通过免疫组化，酶联免疫吸附试验，荧光原位杂交，PCR扩增测序等方法检测常见肿瘤药物的相关分子靶标。共检测12种常用化疗药物和1种靶向药物的相关性基因的表达或突变。结果 共检测肿瘤标本分子靶点7个，包括： DNA拓扑异构酶ⅡA（TOPOⅡA），微管蛋白β3（Tubulinβ3），人切除修复交叉互补基因（ERRC 1），DNA拓扑异构酶Ⅰ（TOPOⅠ），DNA修复蛋白-O6甲基鸟嘌呤-DNA-甲基转移酶（MGMT），二氢叶酸还原酶（DHFR），胸苷酸合成酶（TS）；外周血标本分子靶点2个，包括：CYP2C9＊3和二氢叶酸还原酶DHFR（C829T）基因多态性测序。根据靶标对应的相关药物进行总结分析，研究结果提示：氟尿嘧啶类，长春碱类，环磷酰胺和甲氨蝶呤四类化疗药物敏感性较高；而紫杉醇类、拓扑替康/伊立替康、替莫唑胺/卡莫斯汀/司莫斯汀、蒽环类/依托泊苷和铂类的药物敏感性较低。研究共检测肿瘤标本和外周血标本涉及的靶向药物基因2个：血管内皮生长因子受体（VEGFR-2）和细胞间粘附分子1（ICAM-1）。结果提示：贝伐单抗敏感性高。依据上述检测结果，我们及时调整治疗策略，优化化疗方案，取得了良好的效果，并为部分患儿创造了手术机会。结论本研究在儿童神经母细胞瘤的多学科治疗过程中引入药物相关性分子靶标的检测，对于开展规范化治疗基础上的个体化治疗方案的选择，提供了依据和经验。

Objective To explore the significance of genomic markers of drug sensitivity for individualized therapy in children with neuroblastoma. Methods The surgical specimens and peripheral blood samples were collected from 13 children with neuroblastoma between April 2011 and April 2013. The genomic markers of anti-neoplastic drug sensitivity were examined, including gene expression or mutation of 12 chemotherapeutic agents and Ⅰ targeted drug by methods ol immunohistocherfdcal staining, enzyme-linked immunosorbent assay, fluorescence in situ hybridization, polymerase chain reaction amplification and sequencing. Results The tumor tissue of targets for such chemotherapeutic agents as topoisomerase Ⅱ A （TOPO Ⅱ A）, tubulinβ3, excision repair cross-complementing Ⅰ （ ERRCI ）, topoisomerase Ⅰ（ TOPO Ⅰ ）, 06-methylguanine-DNA methytransferase （MGMT）, dihydrofolate reductase （DHFR） and thyrmdylate synthase （TS） were examined. Furthermore, the peripheral blood of targets for such chemotheraprutic agents as CYP2C9 ＊ 3 genotypes and dihydro{olate reductase （DHFR C829T） genotypes were detected. It showed that tumor/blood samples were highly sensitive to fluorouracil （5-Fu）, vincristine （VCR）, cyclophosphamide （CTX） and methotrexate （MTX） while poorly sensitive to paclitaxel, temozolondde/carmustine/semustine, anthracycline/etoposide, topotecan/irinotecan and platinum. Additionally, tumor tissues or blood samples of genomic markers for such targeted drugs as vascularendothelial growth factor receptor-2 （VEGFR-2） and intercellular cell adhesion molecule-1 （ICAM-1） were examined. The results showed samples were highly sensitive to bevacizumab （Avastin）. Based upon the above test results, therapeutic strategies for poor responders were promptly adjusted for optimized outcomes. Conclusions The genomic markers of drug sensitivity in children with neuroblastoma may guide clinical decision-making for individualized treatment.