| BackgroundHepatocellular carcinoma(HCC),as the main type of primary liver cancer,has the characteristics of hidden onset and rapid progression,and the recurrence rate and mortality remain higher after treatment.The construction of clinical prognostic model can effectively guide clinical decision-making,improve treatment measures and prolong the survival time of patients.However,the existing prognostic evaluation system is constructed in view of clinicopathological and serological characteristics,and does not take into account the factors of inherent genetic variation of tumors,resulting in inaccurate prognostic evaluation and cannot be carried out dynamically in real time.Based on the development and application of liquid biopsy technology and next-generation sequencing technology,new tumor markers represented by circulating tumor DNA(ct-DNA),which can reflect the genomic variation information of tumors,have been gradually discovered and proved to have better clinical diagnosis and prognosis evaluation efficacy.Novel tumor markers are being discovered and have been shown to be effective in clinical diagnosis and prognosis assessment.Mitochondrial DNA(mt-DNA)mutation is closely associated with the occurrence and development of tumors,and mt-DNA has the unique advantages of high copy number,higher mutation rate and easier detection in tumors than nuclear DNA.Previous studies have captured and analyzed the variation of plasma cell free-mitochondrial DNA(cf-mt-DNA)to determine the disease progression of cancer patients and confirmed that mitochondrial genomic variation is closely related to the occurrence and development of HCC.Therefore,the construction of a prognostic model by integrating the characteristics of plasma free mtDNA variation with the clinical imagery of HCC patients is helpful to improve the efficacy of clinical prediction and guide clinical decision-making.Purpose1.Variation characteristics of plasma free mt-DNA of HCC patients were found through targeted capture sequencing analysis;2.To construct a prognosis assessment model based on plasma free mt-DNA characteristics and clinical characteristics of HCC patients;3.Evaluate the predictive efficacy of the prognostic model.MethodsPart Ⅰ: Plasma samples of 300 HCC patients were obtained and targeted to capture plasma free mt-DNA.Illumina Hiseq ×10 platform was used for sample sequencing,mtDNA sequencing data were analyzed,and mt-DNA copy number,fragment distribution,mutation and terminal base motif characteristics were obtained.Part Two: A total of 222 HCC patients with a history of hepatitis B virus(HBV)infection who received radical surgical treatment in two affiliated hospitals of our university from June 2016 to September2020 were selected as the training set.Clinical data and follow-up information of these patients were collected and sorted.Using R software,independent risk factors were obtained by univariate and multivariate COX analysis,and prognostic models of overall survival and recurrence rate of HCC patients were constructed.ROC(AUC)curve,calibration curve and DCA curve were used to evaluate the predictive efficacy of the prognostic model.Part III:Based on a total of 65 HCC patients(history of HBV infection)who received radical surgical treatment in Shanghai Zhongshan Hospital from June 2017 to June 2019,as an external validation set,R software was used to further evaluate the predictive efficacy of the prognostic model by differentiating,calibrating and clinical effectiveness.ResultsPart Ⅰ: Through sequencing analysis of 300 HCC patients’ plasma free mt-DNA,it was found that the copy number,fragment distribution,low frequency mutation and terminal base motif of mt-DNA had certain rules.The results showed that the copy number of genes in the regulatory region of mt-DNA was significantly higher than that in the coding region,and the copy number of protein coding region was significantly higher than that in other regions.The size of plasma free mt-DNA fragments mainly distributed around 120 bp,and the proportion decreased with the increase of the length of mt-DNA fragments.The mutation of plasma free mt-DNA was mainly low frequency mutation.The end bases of plasma free mt-DNA fragments are mainly CCCC-end and CCCT-end,and the fragment truncation mode is favorable.These results suggest that the variation of plasma free mtDNA is specific and non-random,which may be related to the disease status of patients.Part Ⅱ: A prognostic model based on clinical indicators and mt-DNA characteristic indicators of HCC patients was constructed and efficacy was evaluated.According to the above screening methods,the results showed that the copy number of plasma free mt-DNA coding region,the proportion of low-frequency mutations,the fragment size distribution ratio,TNM stage and cirrhosis were independent risk factors affecting the overall survival of patients with hepatocellular carcinoma.Plasma free mt-DNA coding region copy number,low frequency mutation proportion,fragment size distribution ratio,maximum tumor tissue diameter,vascular invasion,TNM stage and cirrhosis were independent risk factors affecting disease-free survival in patients with hepatocellular carcinoma.The prognostic model diagram of overall survival rate and recurrence rate of HCC patients after surgery was constructed by using the selected independent risk factors,and the differentiation,calibration and effectiveness evaluation were conducted.The results showed that the prognostic model had good predictive efficacy,which could provide reference for clinical decision-making.Part Ⅲ: The external cohort validation of the prognostic model was completed based on the external validation set of 65 HBV-related HCC patients to further verify the predictive efficacy of the prognostic model.Through the differentiation,calibration and effectiveness evaluation,the results showed that the prognostic model had good predictive efficacy,suggesting that the prediction model has certain reference value for the overall postoperative survival and recurrence rate of HBV infected HCC patients,and can provide a reference for clinical selection of appropriate treatment methods and postoperative intervention.ConclusionIn this study,for the first time,the characteristics of plasma free mt-DNA in HCC patients and tumor clinical phenotype were used to construct a prognosis model for HBVrelated HCC patients after surgery,and the predictive efficacy of the model was tested in the training set and validation set.The results showed that the characteristic indexes of plasma free mt-DNA in HCC patients were correlated with the survival period and diseasefree survival period of patients after HCC surgery,and could be used as an independent risk factor to evaluate the prognosis of HCC patients.The prognosis model can better predict the disease status and evaluate prognosis of HCC patients,which can provide valuable reference for clinical treatment decision-making. |
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