基于配对样本间的聚鸟嘌呤基因型差异构建结直肠癌系统进化树  

Poly-G for tumor matched samples chronicles the evolution of human colorectal cancer

作　　者：高鑫[1] 于涛[2] 张琦[1] 张师垚 黄迪 赵信禹 刘刚[1] Gao Xin;Yu Tao;Zhang Qi;Zhang Shiyao;Huang Di;Zhao Xinyu;Liu Gang(Department of General Surgery,Tianjin Medical University General Hospital,Tianjin 300052,China;Department of Oncology,Tianjin Medical University General Hospital,Tianjin 300052,China)

机构地区：[1]天津医科大学总医院普通外科,天津300052 [2]天津医科大学总医院肿瘤内科,天津300052

出　　处：《中华肿瘤杂志》2023年第5期382-388,共7页Chinese Journal of Oncology

摘　　要：目的探讨结直肠癌(CRC)原发灶和淋巴结转移灶的肿瘤内异质性空间分布及其与转移的关系。方法2017年1—12月在天津医科大学总医院普通外科接受手术切除并伴有区域淋巴结转移的原发性CRC患者20例。对患者手术切除组织的蜡块进行连续切片,经Histogene染色后行显微切割,获取原发肿瘤组织样本、淋巴结转移组织样本和正常组织样本。提取DNA,采用多重PCR扩增及毛细管电泳检测配对样本的聚鸟嘌呤(Poly-G)基因型。分析临床病理特征与Poly-G突变频率的关系。基于配对样本间的Poly-G基因型差异计算距离矩阵,构建CRC系统进化树。结果共检测20例患者的237个样本,其中134个原发肿瘤组织样本,66个淋巴结转移组织样本,37个正常组织样本,每例患者均检测出Poly-G突变。肿瘤低、未分化患者的Poly-G突变频率[(74.10±23.11)%]高于高、中分化患者[(31.36±12.04)%,P<0.001],微卫星不稳定患者的Poly-G突变频率[(68.19±24.80)%]高于微卫星稳定患者[(32.40±14.90)%,P=0.003)],Poly-G突变频率与患者的性别、年龄、病理分期无关(均P>0.05)。基于配对样本的Poly-G基因型差异构建了20例患者的系统进化树,能明确显示患者肿瘤的进化过程和淋巴结转移的亚克隆来源。结论CRC原发灶和淋巴结转移灶的肿瘤内异质性普遍存在。Poly-G突变积累于CRC的发生发展过程中,并广泛存在于CRC的原发灶和淋巴结转移灶,可作为遗传标志物构建系统进化树,高效、便捷地展示CRC肿瘤内异质性的空间分布和转移途径。Objective To analyze poly-guanine(poly-G)genotypes and construct the phylogenetic tree of colorectal cancer(CRC)and provide an efficient and convenient method for the study of intra-tumor heterogeneity and tumor metastasis pathway.Methods The clinicopathological information of patients with primary colorectal cancer resection with regional lymph node metastases were retrospectively collected in the Department of General Surgery,General Hospital of Tianjin Medical University from January 2017 to December 2017.The paraffin sections of the paired tumor samples were performed consecutively,and multi-region microdissection was performed after histogene staining.The phenol-chloroform extraction and ethanol precipitation scheme was used to obtain DNA,and Poly-G multiplex PCR amplification and capillary electrophoresis detection were performed.The correlation between Poly-G mutation frequency and clinicopathological parameters was analyzed.Based on the difference of Poly-G genotypes between paired samples,the distance matrix was calculated,and the phylogenetic tree was constructed to clarify the tumor metastasis pathway.Results A total of 237 paired samples were collected from 20 patients including 134 primary lesions,66 lymph node metastases,37 normal tissues,and Poly-G mutation was detected in 20 patients(100%).The mutation frequency of Poly-G in low and undifferentiated patients was(74.10±23.11)%,higher than that in high and medium differentiated patients[(31.36±12.04)%,P<0.001].In microsatellite instability patients,the mutation frequency of Poly-G was(68.19±24.80)%,which was higher than that in microsatellite stable patients[(32.40±14.90)%,P=0.003].The Poly-G mutation frequency was not correlated with age,gender,and pathological staging(all P>0.05).Based on Poly-G genotype difference of the paired samples,the phylogenetic trees of 20 patients were constructed,showing the evolution process of the tumor,especially the subclonal origins of lymph node metastasis.Conclusion Poly-G mutations accumulate in the occurren

关 键 词：结直肠肿瘤 肿瘤内异质性 聚鸟嘌呤 突变 系统进化树 

分 类 号：R735.34[医药卫生—肿瘤]