Urinary Cell-free MtDNA Based Application Of Liquid Biopsy In Renal Cell Carcinoma

Background: Renal cell carcinoma is a malignant tumor of urinary system.Due to the lack of reliable methods of survival prediction and tumor biomarkers of renal cell carcinoma,its prognosis evaluation and disease monitoring are facing great problems.With the development of sequencing technology,molecular subtyping of renal cell carcinoma and the application of liquid biopsy provide a new scheme for the prognosis evaluation and disease monitoring of renal cell carcinoma.At present,the study of tumor molecular subtyping has found that the complex variation of some specific genes or multiple genes is related to the prognosis of tumor.At the same time,by detecting circulating tumor cells(CTC),circulating tumor DNA(ct DNA)and exosomes in human body fluids in terms of different indicators or combining multiple indicators,and studying the relationship between them and tumor,the purpose of non-invasive tumor monitoring was achieved,that is,to explore new tumor biomarkers by using liquid biopsy technology.There are few studies on molecular subtyping and prognosis of renal cancer based on the new generation sequencing technology,and the research on biological markers(mt DNA variation)of renal cell carcinoma by sequencing and liquid biopsy technology has not been reported.Objective: To establish a reliable bioinformative method and model to evaluate the prognosis of RCC;To preliminarily explore the mechanism of ct DNA metabolism in renal cancer patients;To establish a stable mt DNA sequencing and analysis process to explore a reliable biomarker of renal cell carcinoma(mt DNA variation).Methods: By analyzing and integrating TCGA and geo databases,the differentially expressed genes(DEG)of renal cell carcinoma were screened,and the prognosis prediction nomogram was established by combining with clinical indicators;The mt DNA of 10 normal human urine samples and 16 renal cell carcinoma patients’ tissue,plasma and urine samples were sequenced,and the mechanism of ct DNA metabolism kinetics and biomarkers of renal cancer were explored by combining with animal experiments.Results: 380 DEGs were screened out by GEO,28 overlapping genes were identified in TCGA database,Sl C22A8 and TNFAIP6 were related to OS of RCC(P < 0.05).The AUC of 3-year and 5-year OS predicted by the prediction model is 0.793 and 0.740 respectively.The calibration chart shows that the 3-year and 5-year OS predicted by the nomogram are in good agreement with the actual 3-year and 5-year OS respectively.Using our patent mt DNA probe to detect the mt DNA copy number and mt DNA point mutation in urine at the same time,the mt DNA copy number obtained was highly correlated with the mt DNA copy number detected by the whole genome sequencing(gold standard)(r=0.954,p<0.01),and there was no significant difference between the length distribution and terminal preference of the analyzed fragment and the whole genome sequencing(gold standard)(p>0.05).There was a significant difference in mt DNA copy number between renal cell carcinoma patients and normal people(p<0.05).There was no significant difference in the length distribution of mt DNA fragments in plasma and urine between renal cancer patients and normal people(p>0.05).The mt DNA in urine samples of normal people has terminal preference(315 site),but there is no terminal preference in urine of renal cell carcinoma patients.Moreover,the heteroplasmy mutation of mt DNA 72: T>C in urine of RCC patients was significantly higher than that of normal people(p<0.01).Animal experimental data showed that the tumor-derived mt DNA copy number at 0h,6h,12 h was 18.61±1.15,2.51±0.58,1.45±0.47 in plasma and 3.71±0.66,1.30±0.16,1.08±0.02 in urine respectively.Conclusion: Based on high-throughput sequencing data,we have established a reliable bioinformative method and a model which can be used for precise evaluation of the overall survival of RCC patients.At the same time,we also established a mature target sequencing and analysis process of mt DNA in human body fluid,and explored that the copy number of mt DNA in urine and the level of heteroplasmy mutation at site 72(T>C)may be the specific biological indicators of RCC patients.Combined with animal experiments,we preliminarily verified two ways for tumor-derived mt DNA entering into urine from plasma and entering into urine from renal cell carcinoma cells directly.It laid a theoretical foundation for the follow-up study of the application of liquid biopsy technology in RCC.