【作者】 王春萌；

【导师】 师英强； 傅红； 周晓燕； 周烨；

【作者基本信息】 复旦大学 ， 肿瘤学， 2009， 博士

【摘要】 研究目的:1.分析及评估伊马替尼治疗胃肠道间质瘤(Gastrointestinal stromal tumor,GIST)患者的临床疗效和副反应,总结伊马替尼治疗经验。2.分析原发GIST患者基因突变和伊马替尼继发耐药GIST患者基因突变的发生规律,分析基因突变与临床特征之间的关系,为防止伊马替尼耐药以及新靶向药物治疗提供依据。了解基因突变与伊马替尼继发耐药的关系,更好地阐明伊马替尼耐药机制。3.目的是发现GIST KIT癌基因信号转导通路的特征,探测哪条通路与靶向治疗干预潜在相关。研究方法:1.胃肠道间质瘤的靶向治疗分析我院2003年11月至2008年8月使用分子靶向药物——伊马替尼治疗32例GIST患者的临床资料,总结伊马替尼治疗经验。2.基因突变与伊马替尼耐药的相关研究用PCR扩增和基因测序的方法检测32例胃肠道间质瘤KIT基因第9,11,13,17外显子和PDGFRA基因第12,18外显子序列,总结胃肠道间质瘤基因突变的发生规律,并且结合临床随访资料,探索GIST患者发生伊马替尼继发耐药的某些规律。分析基因突变与临床特征之间的关系,为防止伊马替尼耐药以及新靶向药物治疗提供依据。3.信号通路与伊马替尼耐药的相关研究我们通过Western blotting检测信号通路中介物的表达,评价KIT突变类型与不同信号通路激活的相关性。发现GIST KIT癌基因信号转导机制的特征,探测哪条通路与靶向治疗干预潜在相关。研究结果:1.在接受治疗的32例患者中,28例(87.5%)患者CD117阳性,23例(71.9%)患者CD34阳性。获得最佳疗效时评估,有10例(31.3%)获得PR,9例(28.1%)获得SD,4例(12.5%)出现疾病进展(Progression disease,PD),1例(3.1%)获得疾病完全缓解(Complete response,CR),8例(25%)患者为术后伊马替尼辅助治疗,随访时仍然有疗效,表现为无瘤生存。9例(28.1%)患者无明显副反应,23例(71.9%)出现药物副反应,包括17例(53.1%)出现2种或以上副反应,均为Ⅰ级或Ⅱ级副反应,3例(9.4%)患者出现Ⅲ级副反应,没有1例出现Ⅳ级副反应。2.32例患者中22例病例存在KIT基因激活性突变,其中2例发生KIT基因第9外显子突变,17例发生第11外显子编码的跨膜区突变,1例发生第13外显子突变。2例发生PDGFRA基因第18外显子突变,8例未发现KIT基因和PDGFRA基因突变,表现为野生基因型。有13例伊马替尼耐药患者,其中包括4例原发耐药患者和9例继发耐药患者。9例继发患者中有5例没有发生二次突变,4例发生二次突变。而4例患者发生二次突变有两种表现类型。其中3例(病例8,12,18)突变为同一种类型,表现为KIT基因第17外显子同一处碱基发生替换突变,即密码子第2467位点T为G所替换(T2467G),可导致823密码子编码氨基酸由酪氨酸转变为天冬氨酸(Y823D)。1例患者为KIT基因第13外显子第1924位点A为G所替换(A1924G),导致642密码子编码氨基酸由赖氨酸改变为谷氨酸(K642E)。3.所有GIST均见KIT强烈表达,伊马替尼治疗有效后,PKIT表达下降,其中获得完全缓解GIST PKIT表达水平最低,如果出现继发耐药,则PKIT表达再次上升。所有GIST中MAPK及PMAPK均见强烈表达,表达水平相似。未经伊马替尼治疗GIST和继发耐药GIST PAKT表达强烈,伊马替尼治疗有效GIST PAKT表达水平下降。所有GIST PMTOR表达水平相当。GIST中PCNA和BCL2表达相似,未经伊马替尼治疗GIST和伊马替尼耐药GIST第一次手术标本,高于伊马替尼有效GIST和完全缓解GIST表达,但同一患者不同时段,不同部位标本表达却有差异。结论:1.伊马替尼治疗GIST疗效肯定,不良反应较轻,且毒性能够耐受。具有高危因素的GIST患者,术后推荐辅助治疗。2.伊马替尼继发耐药可能同KIT基因第17外显子密码子第2467位点T为G所替换(T2467G)和第13外显子第1924位点A为G所替换(A19246)相关。3.RAS/RAF/MAPK和PI3-K/AKT/MTOR两条信号通路在GIST形成过程中起着非常重要的作用,均为KIT依赖性的,在伊马替尼继发耐药GIST中,PI3-K/AKT/MTOR通路比RAS/RAF/MAPK通路更为关键更多还原

【Abstract】 【Objective】1.To assess the efficacy and safety of imatinib mesylate in patients with unresectable and/or metastatic GIST or in adjuvant treatment.2.To explore the mechanisms responsible for the acquired resistance to imatinib.3.To characterize oncogenic KIT signaling mechanisms in GISTs,and to determine which signaling pathways might be of potential relevance to therapeutic interventions.【Methods】1.32 patients with GIST were analyzed for efficacy and safety retrospectively.Front-line imatinib treatment consisted of 400 mg once daily.Dose escalation to 600mg or 800 mg a day was allowed if the tumor progressed and the patients tolerated the initial dose.2.With the bidirectional DNA sequencing and the analysis of an ABI PRISM 310 capillary electrophoresis system,we sequenced the KIT and PDGFRA gene in the 32 GIST patients before or after imatinib treatment.3.Detected by Western blottying,ctivation of signaling intermediates was evaluated in GISTs with different KIT mutational mechanisms to evaluate the relevance of the type of KIT mutation for differential activation of signaling pathways.【Results】1.Of 32 patients with GIST,CD117-positive was confirmed in 28 cases(87.5%),and 23 patients(71.9%) were judged to be positive in expressing CD34.1 patient had CR(3.1%),10 patients(31.3%) achieved PR,9 patients(28.1%)had SD,and 4 patients(12.5%)had PD.8 patients(25%) who obtained neoadjuvant therapy after radical resection were alive without disease.9(28.1%) out of 32 patients had no adverse effects related to the drug.23 cases (71.9%)appeared mild adverse effects which were of gradeⅠorⅡ, including 17 patients had two or more side effects.3 patients(9.4%) displayed gradeⅢadverse reaction,and all cases had no adverse effects of gradeⅣ.2.In 22 of 32 cases carried activating mutations in KIT,17 cases were found mutations in exon 11 encoding the juxtamembrane domain,2 patients were found in exon 9,1 case was found mutation in exon 13. Two cases were found mutation in PDGFRA gene exon 18,and 8 patients were found no mutation in KIT and PDGFRA gene(Wild type).In 13 patients,including 9 patients who were secondary resistance to imatinib and 4 cases who were primary resistance to imatinib,secondary mutation were only identified in patients with acquired resistance to imatinib.Secondary KIT mutations were identified in 4 out of 9 imatinib-resistant patients.In 3 patients,we found an identical novel exon-17 missense mutation,T2467G, resulting in a substitution of Tyr by Asp at codon 823(Y823 D)in tyrosine kinase domain of KIT.We found a novel exon 13 missense mutation,A1924G,in the other one patient.3.KIT expressed strongly in all GIST patients.The level of p-KIT expression decreased dramatically in patients with GIST after imatinib therapy,especially in patients who obtained CR during the treatment,nonetheless,p-KIT expression increased when the GIST patients acquired resistance.The level of MAPK,p-MAPK,and p-MTOR expression in all GIST patients were strong,and the deference were no significace.P-AKT expressed strongly in untreated GIST patients and in GIST patients who showed secondary resistance to imatinib,by contrast,the level of p-AKT expression in GIST patients who responsed to imatinib were lower.Of all GIST patients,the level of PCNA and BCL2 expression were similar. PAKT expression in untreated GIST patients and in GIST patients who showed secondary resistance to imatinib were stronger than other GIST patients who responsed to imatinib.【Conclusion】1.Imatinib is generally safe and has significant activity in the treatment of GIST patients.Most of the adverse effects were mild and manageable.2.The exon-17 missense mutation,T2467G,and exon 13 missense mutation,A1924G,in tyrosine kinase domain of KIT are correlated with imatinib resistance.3.Signal transduction pathways such as RAS/RAF/MAPK and PI3-K/AKT/MTOR pathways are constitutively activated in a KIT-dependent manner.They are essential to GIST pathogenesis,and the PI3-K/AKT/MTOR pathway is particularly relevant for therapeutic targeting in imatinib-resistant GIST.更多还原