Hepatocellular carcinoma (HCC) is the one of the leading causes of cancer mortality in the world, mainly due to the difficulty of early detection and limited therapeutic options. The implementation...

Detection of human hepatitis B virus (HBV) DNA in the urine of patients with chronic hepatitis B infection (CHB) has been reported previously, suggesting urine could provide a potential route of horizontal HBV transmission. However, it is not clear whether the HBV DNA detected in urine is indeed full-length, infectious viral DNA. The aim of this study is to assess the potential infectivity of urine from patients with CHB and to correlate HBV DNA detection in urine with clinical parameters, such as serum viral load and HBeAg status. Urine from 60 CHB patients with serum viral loads ranging from undetectable to 10⁸ IU/mL were analyzed for HBV DNA and serum immune markers. HBV DNA was detected from total urine DNA and size-fractionated urine DNA (separated into ≤1 kb and > 1 kb fractions) by PCR analysis of six regions of the HBV genome. Twenty-seven of 59 (45.7%) patients with HBV serum viral load (≥20 IU/mL) contained at least 20 copies per mL of fragmented HBV DNA in urine detected in at least 1 of the 6 PCR assay regions. Only one patient contained HBV DNA detected by all six regions, and was found to have evidence of blood in the urine. Sixteen of 25 urine samples with high viral load (> 10⁵ IU/mL) and 11 of 34 urine samples with low viral load (< 10⁵ IU/mL) contained detectable HBV DNA. Twelve of 27 (44.44%) patients with detectable HBV DNA in urine were HBeAg positive, and only 5 of these HBeAg positive patients were in the group of 33 (15.15%) patients with no detectable HBV DNA in urine. By Fishers’ exact test, HBV DNA in urine is significantly associated with high serum viral load (P = 0.0197) and HBeAg (P = 0.0203). We conclude that urine from CHB patients with healthy kidney function should not contain full-length HBV DNA, and therefore should not be infectious.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. The beta-catenin gene, CTNNB1, is among the most frequently mutated in HCC tissues. However, mutational analysis of HCC tumors is hampered by the difficulty of obtaining tissue samples using traditional biopsy. Here, we explored the feasibility of detecting tumor-derived CTNNB1 mutations in cell-free DNA (cfDNA) extracted from the urine of HCC patients. Using a short amplicon qPCR assay targeting HCC mutational hotspot CTNNB1 codons 32–37 (exon 3), we detected CTNNB1 mutations in 25% (18/73) of HCC tissues and 24% (15/62) of pre-operative HCC urine samples in two independent cohorts. Among the CTNNB1-mutation-positive patients with available matched pre- and post-operative urine (n = 13), nine showed apparent elimination (n = 7) or severalfold reduction (n = 2) of the mutation in urine following tumor resection. Four of the seven patients with no detectable mutations in postoperative urine remained recurrence-free within five years after surgery. In contrast, all six patients with mutation-positive in post-operative urine recurred, including the two with reduced mutation levels. This is the first report of association between the presence of CTNNB1 mutations in pre- and post-operative urine cfDNA and HCC recurrence with implications for minimum residual disease detection.

Background & Aims: Continued limitations in hepatocellular carcinoma (HCC) screening have led to late diagnosis with poor survival, despite well-defined high-risk patient populations. Our aim is to develop a non-invasive urine circulating tumor DNA (ctDNA) biomarker panel for HCC screening to aid in early detection. Methods: Candidate ctDNA biomarkers was prescreened in urine samples obtained from HCC, cirrhosis, and hepatitis patients. Then, 609 patient urine samples with HCC, cirrhosis, or chronic hepatitis B were collected from five academic medical centers and evaluated by serum alpha feto-protein (AFP) and urine ctDNA panel using logistic regression, a Two-Step machine learning algorithm, and iterated 10-fold cross-validation. Results: Mutated TP53 , and methylated RASSF1a and GSTP1 , were selected for the urine ctDNA panel. The sensitivity of AFP-alone (9.8 ng/mL cut-off) to detect HCC was 71% by Two-Step. The combination of ctDNA and AFP increased the sensitivity to 81% at a specificity of 90%. The AUROC for the combination of ctDNA and AFP vs . AFP-alone were 0.925 (95% CI, 0.924-0.925) and 0.877 (95% CI, 0.876-0.877), respectively. Notably, among the patients with AFP <20 ng/mL, the combination panel correctly identified 64% of HCC cases. The panel performed superiorly to AFP-alone in early-stage HCC (BCLC A) with 80% sensitivity and 90% specificity. In an iterated 10-fold cross-validation analysis, the AUROC for the combination panel was 0.898 (95% CI, 0.895-0.901). Conclusions: The combination of urine ctDNA and serum AFP can increase HCC detection rates including in those patients with low-AFP. Given the ease of collection, a urine ctDNA panel could be a potential non-invasive HCC screening test.