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 occurrence and development of CRC, and can be used as genetic markers to generate reliable maps of intratumor heterogeneity in large numbers of patients with minimal time and cost expenditure.
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。.
Keywords: Colorectal neoplasms; Intra-tumor heterogeneity; Mutation; Phylogenetic tree; Poly-guanine.