Low Coverage Sequencing Of Urine Sediment DNA For Detecting Copy Number Aberrations In Bladder Cancer

BackgroundBladder cancer is the ninth most common cancer in the world,and its incidence has increased in recent years.Even the non-invasive bladder cancer has a recurrence rate of up to 30%,and the five-year survival rate is even insufficient as the disease progresses.50%.Malignant tumor cells usually have genomic instability and appear as copy number amplification or deletion of the entire chromosome or fragment,and may thus change normal somatic cells,increase their ability to increase,invade,and metastasize,thereby transforming into malignant tumor cells.The phenomenon of increasing such aneuploidy changes in chromosomes is called copy number variation(CNA,copy number aberration).The genome of bladder cancer exhibits strong genomic instability characteristics,with strong mutation load and extensive copy number variation(CNA)affecting multiple chromosomes,making it a clear target for exploring new diagnostic methods.PurposesTo explore a method for detecting DNA copy number variation in bladder cancer urine sediment using low-coverage whole genome sequencing.To explore the significance of this method in the non-invasive diagnosis and postoperative tumor progression monitoring of bladder cancer.MethodWe performed DNA extraction on urine sediment and plasma samples from patients with bladder cancer,and used low-coverage whole-genome sequencing to detect DNA copy number variation,and finally combined with clinical information for analysis.The study was divided into three parts:The first part we recruited 54 patients with bladder cancer,11 patients with benign bladder tumors,and 43 normal people,and collected their urine sediment samples,using low-coverage whole genome sequencing and analysis the DNA copy number aberrations of these three groups.The second part we compared the differences of copy number aberrations between urine sediment DNA and plasma cfDNA in 12 patients with bladder cancer.The third part we compared the copy number changes of bladder cancer in urinary sediment DNA before and after treatment,and explores whether this method can be used to detect the progression of bladder cancer.ResultWe sequenced the urine-sediment DNA of bladder cancer,bladder benign tumor,and normal control groups by low-coverage whole-genome sequencing.It was found that the proportion of CNA samples in the bladder cancer group was significantly higher than the other two groups(P<0.005).In the bladder cancer group,CNA detective proportion is much more higher in the high-grade group than in the lowgrade or benign group(P<0.005).The sequencing method is highly sensitive and can detect minimal micro-repetitive microdeletion changes in urine-precipitated DNA.DNA copy number variation in urine of bladder cancer,the repeat region mainly occurs in chromosomes 1q,5p,6p,7p,8q,13q;the deletion region mainly occurs in 2q,8p,9q,9p,and part of chromosome 11p region.The analysis revealed that highfrequency copying repeat regions involved,such as STK3,COX6C,SPAG1,CDKAL1,etc.;and genes involved in bladder cancer tumor gene-related pathways such as C9orf53,CDKN2A,CDKN2B,MIR31,and IFNA1 in the high-frequency deletion region.By comparing the results of urine-precipitated DNA and plasma cfDNA in bladder cancer,and the results of DNA copy number changes in urine sediment before and after treatment,it is speculated that low-coverage sequencing technology can be used to detect bladder cancer and can be used for diagnosis and monitoring of bladder.The progress of cancer is progressing.ConclusionThe use of 0.1～0.3×low-coverage whole genome sequencing can be used to detect the DNA copy number aberrations in urine sediment of bladder cancer.This method provides a new idea for the future development of non-invasive diagnosis of bladder cancer.The method also requires further verification and method improvement in a larger sample size.