Abstract
Urothelial bladder carcinoma (UBC) is characterized by a large number of genetic alterations. DNA from urine is a promising source for liquid biopsy in urological malignancies. We aimed to assess the availability of cell-free DNA (cfDNA) and exosomal DNA (exoDNA) in urine as a source for liquid biopsy in UBC. We included 9 patients who underwent surgery for UBC and performed genomic profiling of tumor samples and matched urinary cfDNA and exoDNA. For mutation analysis, deep sequencing was performed for 9 gene targets and shallow whole genome sequencing (sWGS) was used for the detection of copy number variation (CNV). We analyzed whether genetic alteration in tumor samples was reflected in urinary cfDNA or exoDNA. To measure the similarity between copy number profiles of tumor tissue and urinary DNA, the Pearson’s correlation coefficient was calculated. We found 17 somatic mutations in 6 patients. Of the 17 somatic mutations, 14 and 12 were identified by analysis of cfDNA and exoDNA with AFs of 56.2% and 65.6%, respectively. In CNV analysis using sWGS, although the mean depth was 0.6X, we found amplification of MDM2, ERBB2, CCND1 and CCNE1, and deletion of CDKN2A, PTEN and RB1, all known to be frequently altered in UBC. CNV plots of cfDNA and exoDNA showed a similar pattern with those from the tumor samples. Pearson’s correlation coefficients of tumor vs. cfDNA (0.481) and tumor vs. exoDNA (0.412) were higher than that of tumor vs. normal (0.086). We successfully identified somatic mutations and CNV in UBC using urinary cfDNA and exoDNA. Urinary exoDNA could be another source for liquid biopsy. Also, CNV analysis using sWGS is an alternative strategy for liquid biopsy, providing data from the whole genome at a low cost.
Highlights
Urine is an ideal body fluid for liquid biopsy as it could be collected in a truly non-invasive manner with a relatively reduced limit in volume
In the shallow whole genome sequencing (sWGS) process, 92.1% and 92.6% of sequences from cell-free DNA (cfDNA) and exosomal DNA (exoDNA) were mapped in the human genome, suggesting most urinary cfDNA and exoDNA is from the host genome. (Supplement Table S3)
We found that the genetic alterations in urinary bladder cancer (UBC) were well reflected in both urinary cfDNA and exoDNA
Summary
Urine is an ideal body fluid for liquid biopsy as it could be collected in a truly non-invasive manner with a relatively reduced limit in volume. Previous studies have reported that cell-free DNA (cfDNA) in circulation passes through glomerular filtration which is known as “trans-renal DNA”10. It could be used as a source for circulating tumor DNA and urinary biomarkers[11,12]. While most studies on the utilization of nucleic acids in exosomes as biomarkers have focused on miRNAs or mRNAs, exosome contains double-stranded DNA fragments, and genomic alterations in cancer have been identified in exosomal DNA (exoDNA)[16,17,18]. To detect copy number aberrations, we used the QDNAseq algorithm, which provides high-quality DNA copy number information from data produced by sWGS This algorithms showed better performance than previous approaches for sWGS analysis, especially in low-quality samples such as DNA from formalin-fixed specimens[21]. CfDNA is fragmented into small sizes and characterized by low quality and quantity
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