血浆DNA全外显子测序研究分化型甲状腺癌获得性碘抵抗的发生机理

Thyroid cancer is the most common endocrine malignancy, with differentiated thyroid carcinoma (DTC) composing approximately 93% of all thyroid cancer. While most cases of DTC are curable with the use of surgery and radioactive iodine (RAI) ablation of the remaining thyroid remnant, prognosis is dire and treatment options limited when DTC becomes RAI refractory(RAI-R).The molecular mechanisms of RAI-RDTC are still unclear,which is key problem that unattainable molecular profile of acquired resistance for RAI therapy in thyroid carcinoma. Cancers acquire resistance to systemic treatment as a result of clonal evolution and selection. Repeat biopsies to study genomic evolution as a result of therapy are difficult, invasive and may be confounded by intra-tumour heterogeneity. Recent studies have shown that genomic alterations in solid cancers can be characterized by massively parallel sequencing of circulating cell-free tumour DNA released from cancer cells into plasma, representing a noninvasive liquid biopsy. We performed whole exome depth sequencing of plasma DNA in one patient with advanced DTC,which acquired RAI resistance during the course of RAI therapy. Several mutations selected by RAI therapy were identified. After a preliminary validation and bioinformatics analysis,we speculate that expression of mutation gene RasGRP3c.848A﹥G may modulate expression of thyroid-specific genes to acquiring RAI resistance by activating MAPK and PI3K-AKT signaling pathways in DTC cells. In this project,using Sanger sequencing and digital PCR, gene transfection and silence, nude mice subcutaneous transplantation and micro-PET,we will further investigate the role and mechanism of RasGRP3 c.848A﹥G in acquired RAI resistance of DTC by expanding the samples validation and function characterization in vivo and in vitro from clinical samples,cell,animal levels. In this project,we will identify a novel activating mutation and its mechanism,which will provide a novel molecular target and noninvasive,dynamic and warning marker for therapy and monitoring of RAI-RDTC patients and improve their prognosis.

放射碘难治性分化型甲状腺癌（RAI-RDTC）病人预后差，其分子机理不清楚，关键在于难以获得肿瘤放射碘治疗获得性抵抗的分子谱。现有证据支持循环肿瘤DNA能动态反映肿瘤基因组全貌。我们对一分化型甲状腺癌出现放射碘抵抗病例进行治疗前后血浆DNA全外显子深度测序，显示存在继发性基因突变，经初步验证与信息学分析推测RasGRP3c.848A﹥G可能激活MAPK与PI3K-AKT途径调节甲状腺特异基因表达而获得放射碘抵抗。拟进一步采用Sanger测序与数字PCR、基因转染与沉默、裸鼠皮下移植及micro-PET等手段，通过扩大样本验证与体内外功能鉴定，从临床样本、细胞、动物解析RasGRP3c.848A﹥G在分化型甲状腺癌放射碘抵抗中的作用及机理。本项目研究将识别新的基因突变体及其作用机制，为RAI-RDTC病人的治疗、监测提供新的分子靶点与非侵袭性动态预警标志物，进而改善RAI-RDTC病人预后。

部分分化型甲状腺癌(WDTC)在诊断、治疗过程中出现放射碘抵抗，进而降低了其预后。理解获得性放射碘抵抗的分子遗传学机理就成为改善分化型放射碘抵抗的甲状腺癌（RAIR-WDTC)预后的关键。本项目对RAIR-WDTC外周血ctDNA及对转移性RAIR-WDTC组织全外显子深度测序；分析碘抵抗甲状腺癌的分子遗传突变谱；调查野生型RasGRP3在WDTC中的表达及其细胞内分子机制；扩大样本检测RasGRP3突变体在多种甲状腺癌类型中的表达频率；解析RasGRP3突变体在RAIR-WDTC中的PI3K/AKT信号通路激活路径；探讨有放射碘抵抗潜能的侵袭性乳头状甲状腺微癌的分子遗传谱。结果显示RAIR-DTC外周血ctDNA体细胞突变谱与原发肿瘤有部分重叠，同时也新出现了不同的体细胞突变基因。相比正常甲状腺，野生型RasGRP3基因在乳头状癌中高表达，且通过调节AKT/MDM2信号通路促进细胞增殖与迁移。突变型RasGRP3基因在转移性RAIR-WDTC中突变频率较放射碘抵抗的原发灶、放射碘亲和性的甲状腺癌中突变频率明显高；癌细胞的突变型RasGRP3基因通过激活AKT信号通路降低Na+/I-转运泵基因表达，进而出现放射碘抵抗表型。细胞粘附相关基因突变组合明显富集在有放射碘抵抗潜能的侵袭性甲状腺乳头状微癌。本项目研究的关键数据包括RAIR-WDTC循环ctDNA与组织有共同体细胞突变基因，占癌组织体细胞突变基因的31.3%，明显比癌组织体细胞突变基因多。突变型RasGRP3基因在转移性RAIR-WDTC中突变频率高达20%，在原发灶6.67%，而在放射碘亲和性甲状腺癌中小于3.5%；RasGRP3突变体大幅度抑制NIS基因的表达，明显降低细胞摄碘率。13个粘附相关体细胞突变基因组合在有放射碘抵抗潜能的侵袭性甲状腺乳头状微癌表达频率高达74%，而在非侵袭组仅10%。通过本项目研究，癌组织及循环中检测RasGRP3c.1924G＞T 基因可能用于RAIR-WDTC的诊断、预测及实时监测标志，也可能用于开发RAIR-WDTC病人治疗的药物靶点；13个粘附相关体细胞突变基因组合可用于有放射碘抵抗潜能的甲状腺乳头状微癌侵袭性的预测标志，进而有利于制定有放射碘抵抗潜能的甲状腺乳头状微癌的精准治疗决策。