Background and aimsHepatitis B virus (HBV) replication generates a double-stranded linear DNA (dslDNA) byproduct. This dslDNA can undergo intermolecular and intramolecular nonhomologous end-joining (NHEJ) recombination, resulting in viral integration and dslDNA-derived covalently closed circular DNAs (dsl-cccDNAs), respectively. The insertions and deletions (INDELs) at the end-joining site have been used to differentiate dsl-cccDNA from the authentic cccDNA. The prevalence and characteristics of dsl-cccDNA in chronic hepatitis B (CHB) patients remain unclear.Approach and resultsHBV-targeted next-generation sequencing (NGS) was used to identify 32 dsl-cccDNA-positive candidates, 22 HBeAg(+) and 10 HBeAg(-), from 56 liver biopsies of antiviral treatment-naïve CHB patients for dsl-cccDNA confirmation and characterization by PSAD-cccDNA PCR NGS. INDELs within the DR2-1 region (nt 1600-1840) of the cccDNA were analyzed. Various clonally expanded, heterogenous ~22-nt deletions in the X gene region around nt 1760 were discovered in all 32 samples. The dsl-cccDNA species were then defined and characterized by the INDELs clustered at the DR1 surrounding region (nt 1800-1840). The proportion of dsl-cccDNA in total cccDNA was higher among HBeAg(+) compared to HBeAg(-) samples. The diversity of dsl-cccDNA species positively correlated with cccDNA levels and serum viral load, and was higher in HBeAg(+) CHB.Conclusionsdsl-cccDNA is more abundant and diverse among the HBeAg(+) CHB subjects. The existence of replication-defective dsl-cccDNA may facilitate immune evasion and HBV integration, and complicate HBV pathogenesis.

BACKGROUND Cell-free DNA (cfDNA) has advanced cancer genetic profiling through liquid biopsy. Recent evidence suggests that urinary cfDNA may serve as a noninvasive alternative to plasma-derived cfDNA. AIM To comprehensively characterize transrenal DNA as a source for genetic profiling of hepatocellular carcinoma (HCC), using hepatitis B virus (HBV) DNA as a representative marker. METHODS HBV- and HCC-targeted next-generation sequencing (NGS) was performed to compare HBV fragment size distributions, 5’ 4-mer end motifs, and HCC mutation detection rates between urine and plasma cfDNA. RESULTS HBV transrenal DNA fragments from HCC patients were significantly shorter than those from patients with hepatitis or cirrhosis and displayed distinct 5’ 4-mer end motifs. Compared with plasma, urine samples contained higher levels of HCC-distinctive 5’ 4-mer end motifs. Mutation detection rates were comparable between urine and plasma, with urine higher mutation for TP53 in NGS. Among 101 HCC patients, detection of TP53 , CTNNB1 , and TERT mutations showed a 78.3% overall concordance between urine and plasma cfDNA. In a subset of 15 patients analyzed by HCC-targeted NGS, positional-level concordance between urine and plasma cfDNA reached 97%. CONCLUSION Urine cfDNA demonstrates both overlapping and unique features compared with plasma cfDNA, supporting its potential as a noninvasive and accessible source for genetic profiling of liver cancer.

BACKGROUND:Hepatitis B surface antigen (HBsAg) can be derived from intrahepatic covalently closed circular DNA (cccDNA) and integrated hepatitis B virus (HBV) DNA (iDNA). OBJECTIVE:We evaluated the cccDNA and iDNA from liver tissues of 24 hepatitis B e antigen (HBeAg)(+) and 32 HBeAg(-) treatment-naïve chronic hepatitis B (CHB) participants in the North American Hepatitis B Research Network. DESIGN:For cccDNA analysis, DNA was heat-denatured and digested by plasmid-safe ATP-dependent DNase to remove relaxed circular DNA and iDNA before real-time polymerase chain reaction. For iDNA detection, total DNA was subjected to HBV hybridisation-targeted next generation sequencing assay for identification of the HBV-host junction sequences. Comparisons of HBV cccDNA and iDNA with other virological biomarkers were assessed. RESULTS:Intrahepatic cccDNA, serum HBV DNA, HBV RNA, hepatitis B core related antigen and quantitative hepatitis B surface antigen were higher in HBeAg(+) CHB. Intrahepatic hepatitis B core antigen staining was present in 87% HBeAg(+) but only 13% HBeAg(-) samples (p<0.0001). HBsAg staining was frequent in over 85% in both groups. 23 (95.8%) HBeAg(+) participants had ≤50% iDNA whereas 25 (78.1%) HBeAg(-) participants had >50% iDNA of total HBV DNA in their livers. For HBeAg(+) CHB, the iDNA integration sites were random with only 15.9% localised to the direct repeat 2 (DR2)-DR1 region. For HBeAg(-) CHB, 52.4% of the iDNA integrations were clustered at DR2-DR1. Microhomology-mediated end joining patterns of double-stranded linear DNA HBV integration was more frequent in HBeAg(+) livers. CONCLUSION:HBeAg(-) CHB was associated with high HBsAg staining concentration despite low cccDNA levels suggesting that iDNA was the primary source of HBsAg. The high frequency of DR2-DR1 iDNA distribution in HBeAg(-) CHB suggests the selection advantage and clonal expansion of this integrant in the natural history of CHB.

The persistence of covalently closed circular DNA (cccDNA) underlies chronic hepatitis B virus (HBV) infection and remains as the major barrier to achieving a functional cure. An accurate and sensitive quantification of cccDNA is critical for evaluating therapeutic strategies. The conventional quantitative PCR (qPCR) lacks specificity to distinguish cccDNA from viral relaxed circular DNA (rcDNA) and other replicative intermediates, while nuclease pretreatment (e.g., plasmid-safe ATP-dependent nuclease, exonucleases I/III, or T5 exonuclease) may either incompletely digest rcDNA and/or risk over-digesting cccDNA. Here, we evaluated a novel patented bisulfite conversion-based cccDNA (BSC-cccDNA) qPCR platform that employs bisulfite treatment to disrupt rcDNA complementarity, followed by qPCR with primer sets targeting the rcDNA gap region, to enable selective amplification of cccDNA. Using synthetic HBV DNA controls and DNA samples extracted from HBV stable cell line and HBV-infected cells, we demonstrate that the BSC-cccDNA qPCR assay achieves high specificity, with tolerance of up to 109 rcDNA copies and sensitivity to 10 cccDNA copies per reaction. Comparative analyses with sensitive digital PCR (dPCR), cross-gap qPCR, and Southern blot further validated the sensitivity and accuracy of the BSC-cccDNA assay. These findings establish the BSC-cccDNA qPCR assay as a reliable tool for standardized cccDNA measurement and highlight its potential for both basic research and clinical applications.IMPORTANCEAccurate quantification of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is critical for understanding viral persistence and evaluating curative therapeutic strategies. However, current PCR-based assays lack sufficient specificity to distinguish cccDNA from relaxed circular DNA (rcDNA). Enzymatic pretreatment approaches to remove rcDNA introduce variability, risk incomplete digestion, and potential cccDNA loss, thereby compromising quantitative accuracy. We evaluated a novel bisulfite conversion-based cccDNA (BSC-cccDNA) qPCR assay that chemically disrupts rcDNA complementarity, enabling highly specific cross-gap amplification without reliance on nuclease digestion. Using synthetic substrates, cell-derived HBV DNA, and infected hepatocyte models, we demonstrate that this assay tolerates up to 10⁹ rcDNA copies while detecting as few as 10 cccDNA copies, with performance validated against Southern blot and dPCR. By improving both specificity and quantitative reliability, the BSC-cccDNA platform provides a potential standardized tool for accurate cccDNA measurement in basic research and therapeutic development.

Background & Aims: Cell-free DNA (cfDNA) has advanced cancer genetic profiling through liquid biopsy. While plasma is traditionally the primary source, emerging evidence highlights urinary cfDNA as a novel and noninvasive alternative. This study aimed to comprehensively assess transrenal DNA (trDNA) as a novel noninvasive biomarker source in HCC patients, compared to blood-based liquid biopsy. Approach & Results: HBV DNA was used as a biomarker for trDNA. HBV-targeted and HCC-focused next generation sequencing (NGS) and whole genome sequencing (WGS) were used to compare fragment insert-sizes, the genome coverage, and germline genotyping accuracy. Urinary cfDNA overall exhibited a predominantly mononucelosomal pattern similar to plasma cfDNA, but with shorter fragments, broader size distribution and a more pronounced 10-bp periodicity. In contrast, trDNA were shorter and more variable among all patients. In HCC patients, trDNA was even shorter, with distinct 4-mer end motifs, compared to non-HCC trDNA. Higher concentrations of HCC-distinctive 4-mer end motif and TP53 mutations were found in urine compared to plasma. The overall genome coverage breadth by WGS was similar between urine and plasma cfDNA, with a higher fraction of covered cancer-associated mutation hotspots in urine cfDNA. In 101 HCC patients, there was a 78% overall concordance of HCC-associated mutations (TP53, CTNNB1, and hTERT) and in select 15 patients, 97% overall position-level concordance by targeted NGS between plasma and urine cfDNA. Conclusion: Urine cfDNA has comparable features with distinct characteristics to plasma cfDNA and is a promising tool for liver cancer studies. ### Competing Interest Statement S.J., S.L., Z.W., and Y.S are shareholders of JBS Science Inc. ### Funding Statement This work was supported by the National Institute of Health grant NCI W81XWH-20-1-0605 (PI Kim), 1K08CA237624-01A1 (PI Kim), R44HG008700 (PI Wang), R01CA202769 (PI Su), U01CA275648 (PI Su), R44CA165312-07 (PI Lin). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Heartland IRB gave JBS Science ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Background:HBsAg can be derived from intrahepatic cccDNA and integrated HBV DNA (iDNA). We examined the iDNA from liver tissues of 24 HBeAg(+) and 32 HBeAg(-) treatment-naive CHB participants. Methods:Liver tissues were obtained from the North American Hepatitis B Research Network (HBRN). For cccDNA analysis, DNA was heat-denatured and digested by plasmid-safe ATP-dependent DNase to remove rcDNA and iDNA prior to qPCR. For iDNA detection, total DNA was subjected to HBV hybridization-targeted next generation sequencing (HBV-NGS) assay. The HBV-host junction sequences were identified by ChimericSeq. Comparison of HBV cccDNA and iDNA with serum and intrahepatic virological parameters were assessed. Results:Intrahepatic cccDNA, serum HBV DNA, HBV RNA, HBcrAg and qHBsAg were higher among the HBeAg(+) participants. Among the HBeAg(+) samples, 87% had positive intrahepatic HBcAg staining compared to 13% of HBeAg(-) samples (p<0.0001). HBsAg staining, in contrast, was present in over 85% of both HBeAg(+) and (-) livers. 23 (95.8%) HBeAg(+) participants had ≤50% iDNA of total HBV DNA whereas 25 (78.1%) HBeAg(-) participants had >50% iDNA in their livers. The iDNA junction-breakpoint distributions for the HBeAg(+) group were random with 15.9% localized to the DR2-DR1 region. In contrast, 52.4% of the iDNA were clustered at DR2-DR1 region among the HBeAg(-) participants. Microhomology-mediated end joining (MMEJ) patterns of dslDNA HBV integration was more frequent in HBeAg (+) livers. Conclusion:Serum RNA and HBcrAg reflect the intrahepatic cccDNA concentrations. HBeAg(-) CHB participants had high levels of intrahepatic iDNA and HBsAg despite lower cccDNA levels suggesting that iDNA is the primary source of HBsAg in HBeAg(-) CHB.

Circulating tumor cell (CTC) detection in hepatocellular carcinoma (HCC) is limited not only by the rarity of CTCs but also by a heavy reliance on cell surface markers such as EpCAM, which are variably expressed or lost during tumor progression. Detecting intracellular markers, such as cytokeratin offers an important complementary and comprehensive strategy but remains technically limited in flow cytometry due to the need for fixation and permeabilization, which often lead to cell loss and surface epitope damage. In this study, we systematically evaluated the feasibility of using fixed samples for flow cytometry, using HepG2 cells, PBMCs, and CTCs from patients with HCC. Our results demonstrate that fixation enabled intracellular staining without compromising cell surface marker detection, even after short-term storage at 4 °C and long-term storage at -80 °C. Fixed samples, particularly fixed unfrozen, exhibited comparable staining performance to fresh samples with only a 7–10% reduction in cell recovery. Clinical validation in HCC patients confirmed successful CTC detection, and tumor-specific CTNNB1 mutations were identified in CTC-derived DNA but not in matched plasma cfDNA. These findings support fixed CTC sample workflows as a reliable and practical approach for CTC analysis in HCC.

This study characterized a hepatitis B virus (HBV) hybridization-capture next-generation sequencing (HBV-NGS) assay and applied it to develop a model for estimating the integrated HBV DNA (iDNA) quantity and for HBV genetics liquid biopsy. Using HBV monomers and reconstituted cell line DNA (SNU398, Hep3B, and PLC/PRF/5), the HBV-NGS assay demonstrated high coverage uniformity, reproducibility across HBV genotypes A-D, and 0.1% sensitivity for detecting iDNA. The iDNA sequence and structures from SNU398 and Hep3B are reported. An iDNA estimation model was developed using tissue biopsies from patients with serum viral load < 4 log IU/mL and validated using SNU398 and Hep3B cell line DNA. The assay's utility for HBV genetic liquid biopsy was evaluated using matched plasma-urine samples with HBV DNA levels ranging from high to undetectable. In this pilot study, HBV-JS was detected in all body fluids, regardless of viral load. These findings indicate that the iDNA from patients with negligible or undetectable viral replication can be assessed for iDNA elimination efficacy in drug development. Moreover, a sensitive HBV genetics liquid biopsy can be feasible even for patients with undetectable serum viral load. This study underscores the potential of NGS-based methods to advance HBV management.

Background & Aims:Integrated HBV DNA (iDNA) plays a critical role in HBV pathogenesis, particularly in predicting treatment response and HCC. This study aimed to use an HBV hybridization-capture next-generation sequencing (HBV-NGS) assay to detect HBV-host junction sequences (HBV-JS) in a sensitive nonbiased manner to detect and estimate the iDNA fraction in tissue biopsies and HBV genetics by liquid biopsy. Methods:HBV DNA from plasmid monomers, HBV-HCC cell line (SNU398, Hep3B, and PLC/PRF/5), tissue biopsies of patients with serum HBV DNA <4 log IU/ml, and matched urine and plasma of HBV patients were assessed by HBV-NGS. Junction-specific qPCR (JS-qPCR) assays were developed to quantify abundant HBV-JS. Results:We demonstrated high coverage uniformity, reproducibility across all HBV genotypes A-D, and 0.1% sensitivity for detecting iDNA by the HBV-NGS assay. The sequence and structures of iDNA molecules from SNU398 and Hep3B are reported. An iDNA estimation model was developed using six abundant HBV-JS sequences identified from tissue biopsies by HBV-NGS assay and validated using total DNA of SNU398 and Hep3B cells. Furthermore, the utility of the HBV-NGS assay for HBV genetic analysis in liquid biopsies was explored using matched plasma-urine samples from three patients with serum HBV DNA levels ranging from high to undetectable. HBV-JS was detected in all body fluids tested, regardless of viral load. Conclusion:These findings suggest that the iDNA fraction in tissue biopsies from patients with limited or undetectable serum HBV DNA can be estimated using a robust HBV-NGS assay, and a sensitive HBV genetics liquid biopsy can be obtained. This study highlights the potential of NGS-based methods to advance HBV management.

Ground glass hepatocytes (GGHs) have been associated with hepatocellular carcinoma (HCC) recurrence and poor prognosis. We previously demonstrated that pre-S expression in some GGHs is resistant to current hepatitis B virus (HBV) antiviral therapies. This study aimed to investigate whether integrated HBV DNA (iDNA) is the primary HBV DNA species responsible for sustained pre-S expression in GGH after effective antiviral therapy. We characterized 10 sets of micro-dissected, formalin-fixed-paraffin-embedded, and frozen GGH, HCC, and adjacent hepatitis B surface antigen-negative stained tissues for iDNA, pre-S deletions, and the quantity of covalently closed circular DNA. Eight patients had detectable pre-S deletions, and nine had detectable iDNA. Interestingly, eight patients had integrations within the TERT and CCNE1 genes, which are known recurrent integration sites associated with HCC. Furthermore, we observed a recurrent integration in the ABCC13 gene. Additionally, we identified variations in the type and quantity of pre-S deletions within individual sets of tissues by junction-specific PacBio long-read sequencing. The data from long-read sequencing indicate that some pre-S deletions were acquired following the integration events. Our findings demonstrate that iDNA exists in GGH and can be responsible for sustained pre-S expression in GGH after effective antiviral therapy.

Hepatocellular carcinoma (HCC) is among the world's third most lethal cancers. In resource-limited settings (RLS), up to 70% of HCCs are diagnosed with limited curative treatments at an advanced symptomatic stage. Even when HCC is detected early and resection surgery is offered, the post-operative recurrence rate after resection exceeds 70% in five years, of which about 50% occur within two years of surgery. There are no specific biomarkers addressing the surveillance of HCC recurrence due to the limited sensitivity of the available methods. The primary goal in the early diagnosis and management of HCC is to cure disease and improve survival, respectively. Circulating biomarkers can be used as screening, diagnostic, prognostic, and predictive biomarkers to achieve the primary goal of HCC. In this review, we highlighted key circulating blood- or urine-based HCC biomarkers and considered their potential applications in resource-limited settings, where the unmet medical needs of HCC are disproportionately highly significant.

An optimized hepatocellular carcinoma (HCC)-targeted methylation next generation sequencing assay was developed to discover HCC-associated methylation markers directly from urine for HCC screening. Urine cell-free DNA (ucfDNA) isolated from a discovery cohort of 31 non-HCC and 30 HCC was used for biomarker discovery, identifying 29 genes with differentially methylated regions (DMRs). Methylation-specific qPCR (MSqPCR) assays were developed to verify the selected DMRs corresponding to 8 genes ( GRASP , CCND2 , HOXA9 , BMP4 , VIM , EMX1 , SFRP1 , and ECE) . Using archived ucfDNA, methylation of GRASP , HOXA9 , BMP4 , and ECE1 , were found to be significantly different ( p < 0.05) between HCC and non-HCC patients. The four markers together with previously reported GSTP1 and RASSF1A markers were assessed as a 6-marker panel in an independent training cohort of 87 non-HCC and 78 HCC using logistic regression modeling. AUROC of 0.908 (95% CI, 0.8656–0.9252) was identified for the 6-marker panel with AFP, which was significantly higher than AFP-alone (AUROC 0.841 (95% CI, 0.778–0.904), p = 0.0026). Applying backward selection method, a 4-marker panel was found to exhibit similar performance to the 6-marker panel with AFP having 80% sensitivity compared to 29.5% by AFP-alone at a specificity of 85%. This study supports the potential use of methylated transrenal ucfDNA for HCC screening.

This study evaluated the impact of cell debris and 7-day room temperature storage on the quality and yield of transrenal DNA. Archived urine specimens collected from five hepatocellular carcinoma (HCC) patients and two pregnant women carrying male fetuses were used to assess the impact of cell debris on urine cell-free DNA (ucfDNA) isolation as measured by quantitative PCR for Y-chromosome DNA, or HCCassociated mutation and methylation markers, and by capillary electrophoresis. Prospectively collected urine from 21 HCC patients was aliquoted after collection for paired immediate freezing versus 7-day room temperature storage followed by freezing for further analysis. Cell debris contained more Ychromosome DNA than supernatant in three of the six urine specimens tested from pregnant women, suggesting that cell debris can be associated with 20.6% to 84.9% of transrenal ucfDNA. Ninety-five percent (20 of 21) of frozen and room temperature urine pairs had overlapping DNA size distribution. ucfDNA quantity determined by quantitative PCR for TP53, CTNNB1, TERT, and HCC-associated urine circulating tumor DNA markers were statistically similar between room temperature and frozen samples. This suggests no significant difference in DNA degradation between the groups. The association of transrenal ucfDNA with cell debris and HCC circulating DNA stability at room temperature is significant to further the understanding of transrenal circulating tumor DNA pre-analytical handling for HCC screening. (J Mol Diagn 2023, 25: 913-920; https://doi.org/10.1016/j.jmoldx.2023.08.006)

Hepatitis B virus (HBV)-host junction sequences (HBV-JSs) has been detected in the urine of patients with HBV infection. This study evaluated HBV-JSs as a marker of minimum residual disease (MRD) and tumor recurrence after treatment in HBV-hepatocellular carcinoma (HCC) patients. Archived serial urine DNA from two HBV–HCC with recurrence as confirmed by MRI and four HBV-related cirrhosis (LC) patients were used. Urinary HBV-JSs were identified by an HBV-targeted NGS assay. Quantitative junction-specific PCR assays were developed to investigate dynamic changes of the most abundant urinary HBV-JS. Abundant urinary HBV-JSs were identified in two cases of tumor recurrence. In case 1, a 78-year-old female with HBV- HCC underwent a follow-up MRI following microwave ablation. While MRI results were variable, the unique HBV-JS DNA, HBV-Chr17, steadily increased from initial diagnosis to HCC recurrence. In case 2, a 74-year-old male with HBV–HCC contained two HBV-JS DNA, HBV-Chr11 and HBV-TERT, that steadily increased after initial HCC diagnosis till recurrence. One LC examined had HBV-TERT DNA detected, but transiently in 3.5 years during HCC surveillance. HBV-JS DNA was persistently elevated prior to the diagnosis of recurrent HCC, suggesting the potential of urinary HBV-JS DNA to detect MRD and HCC recurrence after treatment.

Background Hepatocellular carcinoma (HCC) occurs in a well-defined high-risk patient population, but better screening tests are needed to improve sensitivity and efficacy. Therefore, we investigated the use of urine circulating tumour DNA (ctDNA) as a screening test. Methods Candidate markers in urine were selected from HCC and controls. We then enrolled 609 patients from five medical centres to test the selected urine panel. A two-stage model was developed to combine AFP and urine panel as a screening test. Results Mutated TP53 , and methylated RASSF1a , and GSTP1 were selected as the urine panel markers. Serum AFP outperformed the urine panel among all cases of HCC, but the urine panel identified 49% of HCC cases with low AFP < 20 ng/ml. Using the two-stage model, the combined AFP and urine panel identified 148 of the 186 HCC cases (79.6% sensitivity at 90% specificity), which was 30% more than the cases detected with serum AFP alone. It also increased early-stage HCC detection from 62% to 92% (BCLC stage 0), and 40% to 77% (BCLC stage A). Conclusion Urine ctDNA has promising diagnostic utility in patients in HCC, especially in those with low AFP and can be used as a potential non-invasive HCC screening test.

Introduction: Hepatocellular carcinoma (HCC) is one of the most common and deadly cancers worldwide. A common risk factor for liver cancer is chronic infection with the hepatitis B virus (CHB). HCC screening and surveillance is limited by diagnostic accuracy of current testing modalities. Prior studies suggest the potential of urine-based biomarkers for HBV-associated HCC (HBV-HCC), as DNA fragments from both virus and tumor have been detected in urine from those with CHB and HCC. As most HBV-HCC tumors contain integrated components of the HBV genome, unique HBV-host junctional sequences (HBV-JS) represent a viable molecular signature to identify HCC. This study evaluated a novel urine-based biomarker platform, utilizing HBV-JS DNA as a possible marker of tumor recurrence in patients with HBV-HCC. Case Description/Methods: Urine sample from HBV-HCC patients with HCC recurrence confirmed by MRI was obtained. HBV-JS were detected by an HBV-targeted NGS assay (JBS Science Inc., Doylestown, PA) followed by ChimericSeq for junction detection. The most abundant NGS-detected junction sequences were then validated by PCR-Sanger sequencing. Quantitative junction-specific PCR assays were developed to track dynamic changes of HBV-JS in the urine specimens. HBV-JS sequences were detected from 2 cases of HBV-HCC with tumor recurrence (Figure). Case 1: A 78-year-old female with HBV-related cirrhosis was diagnosed with HCC in 2015. After microwave ablation, follow-up MRI revealed a new LI-RADS 3 lesion 1 year later. Subsequent imaging remained radiographically indeterminate until 2018 when the lesion was classified as definite HCC (LI-RADS 5). While serial AFP levels were negligible and MRI results variable, the unique HBV-JS DNA, HBV-Chr17, steadily increased from initial diagnosis to HCC recurrence. Case 2: A 74-year-old male with HBV-related cirrhosis was diagnosed with HCC in 2014, which recurred with a LI-RADS 5 lesion after 1 year despite loco-regional therapy. While AFP levels were negligible, two HBV-JS DNA, HBV-Chr11 and HBV-Tert, rapidly increased after initial HCC diagnosis. Discussion: Unique HBV-JS sequences were detectable in the urine of patients with HBV- HCC. These sequences were detectable at increasing levels prior to diagnosis of recurrence of HCC by MRI imaging or AFP elevation. Together, our data suggest that HBV-JS DNA in urine maybe a further biomarker for the detection of HCC recurrence in patients with HBV-HCC.Figure 1.: HBV-host junctional sequences (HBV-JS) were detected from 2 cases of HBV-associated HCC with tumor recurrence

Abstract Integrated hepatitis B virus (HBV) DNA detected in more than 85% of HBV-infected hepatocellular carcinoma (HBV-HCC) can cause insertional mutagenesis, chromosomal instability, sustained viral protein expression, and/or immune-mediated inflammation leading to HCC carcinogenesis. An opportunistic outcome of HBV DNA integration events, which occur randomly into the host genome, is the creation of a unique HBV-host junction sequence (HBV-JS) in individually infected hepatocytes representing a specific molecular signature of that cell. A noninvasive approach to detect HBV-JS's will enable frequent monitoring and aid in assessing HBV treatment efficacy and HCC disease progression. Here, we assessed the feasibility of detecting HBV-JS's in urine of HBV-infected patients as a proof-of-concept for utilizing urine as a noninvasive HBV-JS liquid biopsy for HBV-related disease monitoring. Utilizing an in-house developed HBV primer extension capture (PEC) NGS assay, we assessed early-stage HBV-patient tumor tissue and matched urine (n=8). Five of 8 urine samples contained detectable HCC tissue-derived HBV-JS's, including a TERT junction sequence. Next, we assessed 32 urine specimens collected from 28 HBV-infected patients including hepatitis (n=5), cirrhosis (n=11), HCC (n=4), and post-HCC (n=8). Interestingly, all urine samples contained HBV-JS sequences with 30 of 32 urines containing integrations in gene-coding regions. Of 351 unique HBV-JS in gene-coding regions identified in urine, 11 HBV-JS's have also been previously reported in tissue of HCC patients, including TERT. All eleven HBV-JS's were identified in cirrhosis or HCC patient cohorts. Furthermore, the distribution of urinary integrated HBV DNA in the HBV genome in all disease categories was found predominantly clustered in HBV DR1-2 (>70%), an integration hotspot, consistent with findings in tissue. Altogether this support the potential of urine as a noninvasive HBV-JS liquid biopsy to monitor HBV-infected patients for disease progression and treatment efficacy. Citation Format: Selena Lin, Yu-lan Kao, Robin Su, Ting-Tsung Chang, Yih-Jyh Lin, Yixiao Cui, Hie-Won Hann, Grace Park, Wei Song, Ying-Hsiu Su. Detection of liver-derived hepatitis B virus-host junction sequences in urine of hepatitis B infected patients for noninvasive disease monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 545.