| Background:Circulating tumor cell(CTC)is a precursor of tumor metastasis,and is occasionally discovered when it gathers together with immune cells(such as white blood cells(WBC))in the blood.CTC-associated WBC(CTC-WBC)clusters can promote CTC proliferation and metastasis,which indicates that patients with CTC-WBC clusters found in peripheral blood may have a poor prognosis.However,the relationship between the CTC-WBC cluster and Hepatocellular carcinoma(HCC)is unclear.At the same time,liquid biopsy,as a method for real-time monitoring of tumor spatio-temporal heterogeneity,has attracted people’s attention and has good clinical application prospects.In this study,we explored a new CTC enrichment technology that can enrich CTC-WBC clusters in peripheral blood,and analyzed the relationship between CTC-WBC clusters and the prognosis of HCC(Chapter 2),and at the same time with next-generation sequencing(NGS)In combination,the correlation between CTC-DNA genomic changes and CTC-WBC cluster counts in HCC patients was analyzed(Chapter 3).Methods:The CanPatrolTM platform was used to separate and count peripheral blood CTC and CTC-WBC clusters,and a customized panel was used for DNA sequencing.In the second chapter of the study,samples of 214 HCC patients diagnosed and treated in Southern Medical University Zhujiang Hospital from January 2014 to December 2016 were collected.Chi-square analysis is used to calculate the correlation between CTC-WBC clusters and clinicopathological characteristics.Kaplan-Meier survival analysis and Cox regression analysis evaluate the prognosis of patients.The third chapter collected samples of 29 HCC patients diagnosed and treated by Southern Medical University Zhujiang Hospital from January 2016 to December 2019.Mann-Whitney U test analyzes the correlation between mesenchymal CTC,CTC-WBC clusters and specific genome changes.Results:The results in the chapter 2 of the study showed that CTC-WBC clusters and tumor size(P=0.001),tumor number(P=0.005),portal vein tumor thrombus(P=0.026),BCLC staging(P<0.001),AFP(P=0.002),and the total number of CTCs(P<0.001)are correlated.The CTC-WBC cluster is an independent prognostic factor of DFS(HR=1.951,95%CI;1.348-2.824,P<0.001)and OS(HR=3.026,95%CI:1.906-4.802,P<0.001).Kaplan-Meier analysis found that the DFS and OS of the CTC-WBC cluster-positive group were significantly shorter than those of the CTC-WBC cluster-negative group(P<0.001 and P<0.001).In the chapter 3 of the 29 sequencing patients,at least one somatic hotspot mutation was detected in each patient.42 individual cell hotspot mutations were detected in tumor tissues,39 mutations were detected in CTC-DNA,including PTEN,MET,EGFR,RET and FGFR3.The number of CTC-WBC clusters was positively correlated with RET genome changes(P=0.01,Mann-Whitney U test),and negatively correlated with FGFR3 genome changes(P=0.039,Mann-Whitney U test).Conclusion:1.CTC-WBC clusters in peripheral blood are independent predictors of HCC,and their existence indicates that the prognosis of HCC patients is poor.2.We report a new method of CTC enrichment platform combined with NGS to analyze genetic variation,and found that the number of CTC-WBC clusters of HCC patients is related to a specific genome profile. |
