Molecular mechanisms of drug resistance in cancer.-GIST resistant to imatinib as a model-

对抗癌药物的耐药性是晚期癌症患者预后和生活质量的决定因素。目前已有多种耐药机制，但对新出现的靶向药物耐药的分子机制尚未阐明。在本项目中，我们以GIST对伊马替尼和舒尼替尼的耐药性为模型，研究了靶向药物的分子机制。虽然伊马替尼在晚期GIST中表现出较高的活性，并改善了GIST患者的预后，但有一半的患者在治疗2年后出现了伊马替尼耐药的GIST。我们重点分析了KIT和PDGFRA基因的分子机制，并分析了42例耐伊马替尼的GIST患者中的25例(48个耐药灶)，其中5例为原发耐药，37例为继发性耐药。难治性…的临床特征：CT显示15个病灶呈肿块增大，10个呈肿块状结节，仅3个病灶显示为新的GIST更多的损伤。部分患者表现为混合型。手术的安全性和疗效：22例病灶抵抗或症状性全身抵抗的患者接受了手术和/或RFA。耐药皮损的手术通常是安全和可接受的，有三个轻微的并发症。虽然全身阻力手术的结果令人失望，但与没有手术的患者相比，预后没有延长，但局部进展的手术获得了额外的6个月的PFS。完全切除耐药灶的患者预后较好。KIT和PDGFRA蛋白的表达及下游激酶的激活：所有耐药的皮损均显示有活性的梭形和/或上皮样肿瘤细胞，免疫组织化学显示KIT蛋白的表达。包括AKT和MAPK在内的下游激酶也被重新激活。KIT和/或PDGFRA基因的获得性突变：20名患者被赋予KIT基因激动域的获得性突变(除原始突变外，11例在EX13中，1例在EX14中，10例在EX17中，1例在EX16+17中)。这些突变位于相同的等位基因中，耐药皮损呈克隆性发展。其余5例患者均为原发突变。突变和对舒尼替尼的耐药性：6名对伊马替尼耐药的GIST患者接受了舒尼替尼作为二线治疗。5例患者对舒尼替尼表现出原发耐药，包括3例Ex 11+Ex 17突变、Ex 9+无额外突变和Ex 11+Ex 13突变。最后一组患者的血中舒尼替尼水平没有因为扩大的肠切除而增加。1例Ex11+Ex13突变的患者对Sunitinib表现出PR，然而，Ex17的第三个突变允许在Sunitinib下发生另一种情况。因此，KIT基因Ex17的第二个或第三个突变赋予了对舒尼替尼的抗性。综上所述，靶向耐药加上KIT基因的额外突变可能是对伊马替尼和舒尼替尼的分子靶标制剂产生继发性耐药的主要原因。较少

Resistance to the anticancer drugs is a determinant of the prognosis and QOL of patients with advanced cancer. There have been several mechanisms for drug resistance, however, molecular mechanisms of resistance to newly emerged target agents have not yet been elucidated. In this project, we have investigated molecular mechanisms of target agents using GIST resistant to imatinib and sunitinib as a model. Although imatinib has shown high activities on advanced GIST and improves the prognosis of GIST patients, half of patients suffered from appearance of imatinib-resistant GIST after 2 years treatment. We have focused analysis of a molecular mechanism on the KIT and PDGFRA genes and analyzed 25 patients (48 resistant lesions) of 42 patients who have imatinib-resistant GIST including 5 primary resistance and 37 secondary.(1). Clinical features of resistant GIST :CT revealed that 15 lesions showed a mass enlargement, 10 a nodule in a mass appearance, and only three lesions appeared as a new … More lesion. Some patients showed mixed type.(2). Safety and therapeutic effects of surgery :Twenty two patients with focal resistance or symptomatic systemic resistance underwent surgery and/or RFA. Surgery for resistant lesions is generally safe and acceptable with three minor complications. Although surgery for systemic resistance showed disappointed results without no prolongation of the prognosis compared to patients without surgery, surgery for focal progression conferred additional 6 months PFS. Patients with complete resection of resistant lesions had much better prognosis.(3). Expression of KIT ＆ PDGFRA proteins and activations of downstream kinases :All resistant lesions show viable spindle and/or epithelioid tumor cells which express KIT proteins in immunohistochemistory. Downstream kinases including AKT and MAPK are also re-activated.(4). Acquired mutations in the KIT and/or PDGFRA genes :twenty patients are endowed with acquired mutations in kinase domains of the KIT gene (11 in Ex13, 1 in Ex14, 10 in Ex17, 1 in Ex16+17 in addition to primary mutations). These mutations are in the same allele and resistant lesions showed clonal development. The other 5 patients had only primary mutations.(5). Mutations and sunitinib resistance :Six patients with imatinib-resistant GIST received sunitinib as a second-line therapy. Five patients showed primary resistance to sunitinib including three Ex 11+ Ex 17 mutations, Ex 9 +no additional mutation, and Ex 11+ Ex 13 mutation. The last patients had no increase in blood levels of sunitinib because of extended bowel resection. One patient with Ex 11+Ex 13 mutation showed PR to sunitinib, however, third mutation in Ex 17 allowed another development under sunitinib. Thus, second or third mutation in Ex 17 of the KIT gene conferred resistance to sunitinib.In summary, target resistance with additional mutations in the KIT gene may be a main cause of secondary resistance to molecular targets agent of imatinib and sunitinib. Less