基于高通量测序技术的骨肉瘤基因研究与个体化治疗

Study of osteosarcoma related genes and individualized treatment based on high throughput genotyping

目的探索骨肉瘤突变的新基因，探讨通过检测化疗药物相关基因来指导骨肉瘤的个体化治疗。方法采用第二代高通量测序技术对23例骨肉瘤患者的手术切除标本进行化疗相关药物靶基因检测，并对其中10例患者标本的339个基因进行突变检测。经系统分析后发现骨肉瘤相关的特异性突变以及患者对化疗药物的敏感程度。结果2009年2月至2013年4月，西京医院术前病理报告确诊为骨肉瘤的23例患者，男16例，女7例；年龄6-58岁，平均18．5岁。每个骨肉瘤标本均存在基因突变。在所有的突变中，确定了85个未报道过的基因突变位点，其中间变性淋巴瘤激酶（ana．plasticlymphoma bkinase，ALK）基因在全部样本中均出现突变，共同突变位置发生在第19和第29个外显子（第19个外显子c．A3089C：p．Hisl030Pro，第29个外显子c．A4381G：p．Iiel461V~；c．A4472G：p．Lysl491ArgIc．C4587G：p．Aspl529G1u）：NOTCH基因与患者血清碱性磷酸酶水平相关。药敏结果显示23例骨肉瘤患者均对异环磷酰胺敏感，铂类敏感率为39．1％，阿霉素敏感率为21．7％。吉西他滨、多西紫杉醇类及培美曲塞敏感率分别为36．3％、47．8％和46．5％。当术前化疗使用的药物全部对患者敏感时，化疗效果均为有效。当使用的药物不全（患者对1种或2种药物敏感）时，只有6例患者得到有效化疗，15例无效，差异有统计学意义。结论ALK基因与NOTCH基因可能均为致癌基因，在不同方面参与骨肉瘤的病理过程。骨肉瘤个体化治疗可能成为提高化疗效果并最终提高骨肉瘤生存率的关键。

Objective To identify new genes that are mutated in osteosarcomas and to guide individualized treatment of osteosarcoma by detecting genes which are associated with chemotherapy drugs. Methods Target genes associated with chemo-therapy drugs in 23 osteosarcoma samples were detected by the second generation high-throughput sequencing technology and 339 genes had undergone mutation detection in 10 of the 23 samples. Based on a systemic analysis of all the mutations, specific genes which were associated with the pathogenesis of osteosarcoma and summarized the susceptibility to chemotherapy drugs of different patients were found. Results From February 2009 to April 2013, 23 patients （7 males and 16 females, with an average age of 18.5 years） were diagnosed with osteosarcoma by preoperative pathological report. All samples had mutations. 82 new genetic mu-tations were detected which had never been reported. Notably, common mutations in 19th and 29th exons of ALK in osteosarcoma were identified. The 19th exon was c.A3089C：p.His1030Pro. The 29th exon was c.A4381G：p.Iie1461Va1, c.A4472G：p. Lys1491Arg or c.C4587G：p.Asp1529Glu. The relation between NOTCH and alkaline phosphatase were also discovered. In addi-tion, all the 23 patients were sensitive to ifosfamide. The sensitivity rate of platinum and adriamyein were 39.1% and 21.7% re-spectively. The sensitivity rate of gemcitabine, docetaxel and pemetrexed were 36.3%, 47.8% and 46.5%, respectively. Among pa- tients who were given the sensitive drugs during preoperative chemotherapy, the effective rate of chemotherapy drugs was signifi- cantly sensitive. When the drugs were not completely sensitive （one or two） to patients, only 6 cases got effective chemotherapy, and 15 cases were invalid. The changes were significantly different. Conclusion The ALK and NOTCH may participate in the pathogenesis of osteosarcoma from different aspects as oncogenes. Moreover, individualized treatment may be the key factor in im- proving the effective rate of chemotherapy drugs and the overall survival rate of osteosarcoma patients.