中医辨证治疗晚期结直肠癌化疗失败患者循环肿瘤DNA状态与预后的相关性

Relationship Between Circulating Tumor DNA Status and Prognosis in the Advanced Colorectal Cancer Patients Treated with Traditional Chinese Medicine Syndrome Differentiation and Treatment after Failure of Chemotherapy

目的探讨中医辨证治疗晚期结直肠癌化疗失败患者循环肿瘤DNA(ctDNA)状态、突变基因及其与预后相关性。方法收集83例晚期结直肠癌化疗失败患者外周血10ml,利用定制的覆盖416个肿瘤相关基因探针的Geneseeq One NGS面板进行杂交富集。高效捕获后进行聚合酶链式反应(PCR)扩增,在HiSeq NGS平台(Illumina)上对目标富集的文库进行测序。结果在纳入的83例经中医辨证治疗的晚期结直肠癌化疗失败患者中,有54例(65.06%)血浆中检测出肿瘤相关突变基因,共152个基因。根据突变频率筛选出APC、TP53、KRAS、PIK3CA等前20位的基因;ctDNA突变频率、肿瘤突变负荷(TMB)、KRAS、PIK3CA均与预后显著相关(P<0.05);临床相关因素分析显示,肝转移与多个肿瘤基因不良突变相关(P<0.05或P<0.01),脾肾两虚证与ctDNA突变频率、TMB、TP53相关(P<0.05),八纲辨证中阴证、阳证与TMB相关(P<0.05)。结论外周血ctDNA可以较好地反映中医辨证治疗的晚期结直肠癌化疗失败患者分子生物学状态,脾肾两虚证与较低的ctDNA突变频率、较低的抑癌基因突变存在相关性;阴证患者TMB较低,阳证患者TMB较高。

Objective To explore the relationship between circulating tumor DNA(ctDNA)state,mutation gene and prognosis in patients with advanced colorectal cancer after failure of chemotherapy.Methods Collect 10 ml of peripheral blood of 83 patients with advanced colorectal cancer who failed to receive chemotherapy,and customized Geneseq one NGS panel covering 416 tumor related gene probes were used to hybridize and enrich.After highly efficient capture,polymerase chain reaction(PCR)amplification was carried out,and the target enriched library was sequenced on the hillseq ngs platform(Illumina).Results Among 83 patients with advanced colorectal cancer after chemotherapy failure,tumor related mutations were found in 54(65.06%)patients,involving a total of 152 genes.The top 20 genes including APC,TP53,KRAS,PIK3 CA,etc.were screened according to the mutation frequency,ctDNA mutation frequency,tumor mutation burden(TMB),KRAS and PIK3 CA were significantly correlated with prognosis(P<0.05).Clinical correlation analysis showed that liver metastasis was strongly correlated with multiple undesirable mutations in tumor genes(P<0.05 or P<0.01).There was a significant correlation between spleen kidney deficiency syndrome and ctDNA mutation frequency,TMB,TP53(P<0.05),and there was a significant correlation between yin syndrome,yang syndrome in eight syndrome differentiation and TMB(P<0.05).Conclusion Peripheral blood ctDNA can well reflect the molecular biological status of patients with advanced colorectal cancer after chemotherapy failure by TCM syndrome differentiation treatment.Spleen-kidney deficiency syndrome is related to lower ctDNA mutation frequency and lower tumor suppressor gene mutation.Patients with yin syndrome have lower TMB and patients with yang syndrome have higher TMB.