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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

Cell-free DNA (cfDNA) from blood has become a promising analyte for cancer genetic liquid biopsy. Urinary cfDNA has been shown to contain mutations associated with non-genitourologic cancers including hepatocellular carcinoma (HCC). In this study, we evaluate urine as a noninvasive alternative to blood-based liquid biopsy in both germline and circulating tumor DNA (ctDNA) genotyping in HCC. Using quantitative PCR (qPCR), whole-genome sequencing (WGS), and targeted NGS, DNA isolated from blood or urine of patients with HCC was analyzed for overall genome coverage, HCC hotspot coverage, and germline or somatic mutation concordance. Targeted NGS of plasma and urine cfDNA was also performed for detection of somatic variants. We found urine cfDNA, similar to plasma cfDNA, showed a major mononucleosomal species of 150-180 bp in both healthy individuals and patients with HCC. By WGS, overall genome coverage breadth was similar between urine and plasma cfDNA, with higher fraction of covered cancer-associated mutation hotspots in urine cfDNA. qPCR analyses of HCC-associated mutations (TP53, CTNNB1, and TERT) in 101 patients with HCC revealed 78% overall concordance between plasma and urine. Targeted NGS of HCC-associated gene regions in additional 15 HCC patients showed a 97% overall position-level concordance between plasma and urine cfDNA. Collectively, urine DNA can potentially be used as a completely noninvasive liquid biopsy for HCC.Significance StatementHepatocellular carcinoma (HCC) is the most common liver cancer worldwide and the fastest growing gastrointestinal cancer in the U.S. Cell-free DNA (cfDNA) which originates from various cells undergoing apoptosis or necrosis including tumor cells, is present in all body fluids levels including urine. Urinary cfDNA isolated from patients with HCC showed a similar fragment size distribution, overall genome coverage, and comparable sensitivity for detecting HCC-associated variants compared to plasma cfDNA. Urine was also determined to be a reliable source of germline genotype information, similar to peripheral blood mononuclear cells in blood-based liquid biopsies. Urine cfDNA can be used as a completely non-invasive liquid biopsy in HCC.

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.

This study assessed three commercially available cell-free DNA (cfDNA) extraction kits and the impact of a PEG-based DNA cleanup procedure (DNApure) on cfDNA quality and yield. Six normal donor urine and plasma samples and specimens from four pregnant (PG) women carrying male fetuses underwent extractions with the JBS cfDNA extraction kit (kit J), MagMAX Cell-Free DNA Extraction kit (kit M), and QIAamp Circulating Nucleic Acid Kit (kit Q). Recovery of a PCR product spike-in, endogenous TP53, and Y-chromosome DNA was used to assess kit performance. Nucleosomal-sized DNA profiles varied among the kits, with prominent multi-nucleosomal-sized peaks present in urine and plasma DNA isolated by kits J and M only. Kit J recovered significantly more spike-in DNA than did kits M or Q (p &lt, 0.001) from urine, and similar amounts from plasma (p = 0.12). Applying DNApure to kit M- and Q-isolated DNA significantly improved the amplification efficiency of spike-in DNA from urine (p &lt, 0.001) and plasma (p ≤ 0.013). Furthermore, kit J isolated significantly more Y-chromosome DNA from PG urine compared to kit Q (p = 0.05). We demonstrate that DNApure can provide an efficient means of improving the yield and purity of cfDNA and minimize the effects of pre-analytical biospecimen variability on liquid biopsy assay performance.

Background & AimsContinued 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.MethodsCandidate 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.ResultsMutated 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 ConclusionsThe 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.

Background 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. Methods 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. Results 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 (

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.

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.

BACKGROUND: Cell-free DNA (cfDNA) from blood has become a promising analyte for cancer diagnostics. However, because blood contains high concentrations of proteins and enzyme inhibitors, plasma cfDNA quality can be variable. Urinary cfDNA has been shown to contain mutations associated with non-genitourologic cancers including hepatocellular carcinoma (HCC). In this study, we evaluate urine as a noninvasive alternative to blood-based liquid biopsy for both germline and ctDNA genotyping in HCC. METHOD: Using qPCR, whole-genome sequencing (WGS), and targeted NGS, DNA isolated from blood or urine of patients with HCC was evaluated and analyzed for overall genome coverage, HCC hotspot coverage, and germline or somatic mutation concordance. Targeted NGS of plasma and urine cfDNA was also performed for detection of somatic variants. RESULTS: Similar to plasma cfDNA, urine cfDNA showed a major mononucleosomal species of 165-170 bp, with dinucleosomal species also discernable in most healthy individuals and patients with HCC. Overall genome coverage breadth was similar between urine and plasma cfDNA, with higher fraction of covered cancer-associated mutation hotspots in urine cfDNA. Quantitative PCR analyses of HCC-associated mutations (TP53, CTNNB1, and hTERT) in 102 patients with HCC, revealed 78% overall concordance between plasma and urine. Targeted NGS of HCC-associated gene regions in additional 15 patients showed an 97% overall position-level concordance between plasma and urine cfDNA. Furthermore, urine outperformed plasma in both the sensitivity and specificity of detection of CTNNB1 and hTERT mutations. CONCLUSION urine DNA can potentially be used as a completely noninvasive diagnostic biomarker for HCC. Citation Format: Amy Kim, Selena Lin, Yixiao Cui, Terence Gade, Fwu-Shan Shieh, Max Chao, John Shieh, Surbhi Jain, Jonathan Cheng, James Hamilton, Hie-Won L. Hann, Dmitry Goryunov, Ying-Hsiu Su. Urine as a non-invasive alternative to blood for germline and somatic mutation detection in hepatocellular carcinoma [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 544.

Aims: The use of circulating cell-free DNA for detection of cancer genetics has been studied extensively. Liquid biopsy often refers to the use of blood as a minimally invasive source of body fluid for detecting circulating tumor DNA (ctDNA). However, urine collection, which is completely noninvasive, has been shown to also have great promise to serve as an alternate body fluid source for ctDNA. In this review article, we focus on the clinical utility of urine for genetic liquid biopsy of nonurological cancers. Conclusion: Although still in early stages as compared with blood-based liquid biopsy, recent studies have demonstrated the value of urine-based liquid biopsies for: nonurological cancer screening; early detection; monitoring for recurrence and metastasis; and therapeutic efficacy. Overall, the completely noninvasive and patient-friendly nature of the urine-based biopsy warrants further development and offers a promising alternative to blood-based biopsies.

Circulating tumor DNA (ctDNA) from blood and peripheral blood mononuclear cells (PBMCs) are used to determine comprehensive cancer genetics and germline genomics for precision medicine. To evaluate the use of urine samples as a noninvasive alternative to blood samples, we performed a comprehensive characterization of urine DNA as an alternative to PBMC DNA for germline genomics. Whole genome sequencing (WGS) was performed in PBMC and total urine DNA in six normal subjects to compare the quality and comprehensiveness of the genomic data. There was no significant difference between the sequencing data obtained from PBMC and urine DNA upon comparing the variant calling, percent reads passing Phred score Q30 (p>0.05, paired t-test), and coverage of human genome. Similar to plasma cfDNA, mononucleosomal-sized DNA was the most predominant species present in urine cfDNA. Samples from three hepatocellular carcinoma (HCC) patients were further analyzed for insert sizes by NGS, which demonstrated predominantly mononucleosomal-sized DNA fragments, with a series of peaks occurring with 10-bp periodicity, in both urine and plasma cfDNA. With shallow WGS, the genome coverage overlap was 53-61%, and by increasing the depth of coverage the overlap increased from 60% to 74%. Interestingly, 8-10% of the genome covered in plasma DNA was not detected in urine DNA, and 4-8% of the genome covered in urine DNA was not covered in plasma DNA. Next, we compared the detectability of three HCC-associated genetic mutations in the matched urine and plasma DNA obtained from 76 HCC patients including mutations at TP53 codon 249 (TP53 249T), CTNNB1 exon 3 regions 32-37 (CTNNB1 32-37), and hTERT promoter region position 124 (hTERT 124). We observed higher levels of the TP53 mutation, similar levels of CTNNB1 mutation, and lower levels of the TERT mutation in urine cfDNA compared to plasma ctDNA. This suggests a potential for urine as a potential noninvasive source for ctDNA analysis, and a combination of both urine and plasma ctDNA may increase the overall sensitivity of ctDNA detection. In conclusion, our data suggest that (1) total urine DNA can replace PBMC DNA for providing comprehensive genomic sequencing data and (2) urine can complement blood for liver cancer liquid biopsy, precision medicine, and potential applications in other cancers. Citation Format: James V. Vowles, Amy K. Kim, James P. Hamilton, Selena Y. Lin, Fwu-Shan Shieh, Harry Luu, Gabrielle Villafana, Chi-Tan Hu, Ying-Hsiu Su. Urine for noninvasive liquid biopsy for germline and somatic mutations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 722.

Despite current antiviral therapy success in reducing viral load in patients with chronic hepatitis B virus (HBV), persistent viral surface antigen (HBsAg) remains a risk factor of HCC. Recent evidence has shown that integrated HBV can serve as a source for continued viral antigen expression, despite the lack of detectable replication. Given the oncogenicity of pre-S mutant proteins and its expression in type II ground glass hepatocytes (GGH) even in the absence of viral replication, we assessed the likelihood of GGH as a pre-malignant state as compared to other adjacent-to-tumor non-GGH “normal” tissue by investigating the integrity of integrated HBV DNA. We developed a sensitive HBV-targeted NGS and downstream bioinformatics pipeline, ChimericSeq, to identify HBV-host junctions. A pilot study of 10 HBV-HCC patients, each with six micro-dissected FFPE tissue sections (2 tumor, 2 GGH, 2 HBsAg-negative), was subjected to HBV-targeted NGS and ChimericSeq analysis. We identified integrated HBV DNA in 90% of HCC as well as its matched GGH, and identified PreS2 deletions, for instance, deletion at nt. 43-52. Surprisingly, the recurrent insertional driver mutations of TERT, CCNE1, and ABCC13 integration were detected in 9 of 10 (90%) of this GGH/HCC patient cohort. Next we performed PacBio amplicon sequencing of integrated HBV DNA including Pre-S sequences to comprehensively profile the integrity of the integrated HBV DNA, such as insertional mutations or deletion, and mutations across the different frozen tissue sections (tumor, GGH, HBsAg-negative). Our data suggest that HBV integration of cancer driver genes occurs early during course of infection and integrated DNA evolved over time during carcinogenesis. Overall, a sensitive workflow to comprehensively profile integrated HBV DNA in HCC patients enables further investigation of the role of HBV integration during tumorigenesis. Citation Format: Yu-Lan Kao, Yih-Ping Su, Hung-Wen Tsai, Ih-Jen Su, Selena Lin, Wei Song, Ying-Hsiu Su. Comprehensive integrated HBV DNA profiling in HCC patients with GGH by HBV-targeted NGS and PacBio sequencing analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3630.

Blood liquid biopsy is a powerful tool to provide cancer genetics via detection of biomarkers like circulating tumor DNA (ctDNA) to reduce cancer death through precision medicine and has shown potential in the early detection of cancer. However, urine analysis of ctDNA, a completely noninvasive and possibly more practical procedure for cancer screening, has not been thoroughly explored. Using hepatocellular carcinoma (HCC) as a model, we examined if urine can be an alternative to blood for liquid biopsy. Our study indicated that quantification of TP53 and CTNNB1 genes by qPCR assays in matched plasma and urine samples showed that the plasma samples generally had more DNA but both were detected in significant quantities. Employing a panel of HCC biomarkers including genetic mutations at TP53 codon 249 [G:C to T:A transversion (TP53 249T)], CTNNB1 exon 3 regions 32-37 (CTNNB1 32-37), and hTERT promoter region position 124 (hTERT 124) and aberrant methylation of RASSF1A mRASSF1A), detection of these modifications in urine and plasma from HCC patients were confirmed. We observed that ctDNA marker detection was independent of detection using serum AFP, the most used biomarker for liver cancer screening. Thus, the sensitivity of screening was improved when combining ctDNA markers with AFP as shown in the receiver operating characteristic curve (ROC) analysis for performance of ctDNA markers for distinguishing HCC from non-HCC (cirrhosis and hepatitis), with an area under the ROC (AUROC) increase from 0.799 to 0.904 and 20% more liver cancer detected as compared to AFP alone. In conclusion, our data suggests that urine can complement blood for liver cancer liquid biopsy and precision medicine with potential applications to other cancers as well.

Hepatocellular carcinoma (HCC) is the world's second leading cause of cancer death; 82.4% of patients die within 5 years. This grim prognosis is the consequence of a lack of effective early detection tools, limited treatment options, and the high frequency of HCC recurrence. Advances in the field of liquid biopsy hold great promise in improving early detection of HCC, advancing patient prognosis, and ultimately increasing the survival rate. In an effort to address the current challenges of HCC screening and management, several studies have identified and evaluated liver-cancer-associated molecular signatures such as genetic alterations, methylation, and noncoding RNA expression in the form of circulating biomarkers in body fluids and circulating tumor cells of HCC patients. In this review, we summarize the recent progress in HCC liquid biopsy, organized by the intended clinical application of the reported study.

We have investigated amplification-free in situ double-stranded mutation detection in urine in the concentration range 10(−19)M – 10(−16) M using piezoelectric plate sensors (PEPs). The detection was carried out in a close-loop flow with two temperature zones. The 95°C high-temperature zone served as the reservoir where the sample was loaded and DNA de-hybridized. The heated urine was cooled flowing through a 1 m long tubing immersed in room-temperature water bath at a flow rate of 4 ml/min to reach the detection cell at the desire detection temperature for the detection to take place. With hepatitis B virus double mutation (HBVDM) and KRAS G12V point mutation as model double mutations, it is shown that PEPS was able to detect double-stranded HBVDM and KRAS with 70% detection efficiency or better at concentration as low as 10(−19)M against single-stranded mutation detection at the same concentrations, which was validated by the following in situ fluorescent reporter microspheres (FRMs) detection as well as microscopic visualization of the FRMs bound to the captured mutant on the PEPS surface. Furthermore, the same double-stranded mutation detection efficacy was demonstrated at 10(−19) M – 10(−16) M in a background of 250-fold wildtype for HBVDM and 1000-fold wildtype for KRAS. Also demonstrated was detection of KRAS mutation at 10(−19) M – 10(−16) M of SW480 DNA fragments in urine.

Hepatocellular carcinoma is one of the fastest growing cancers in the US and has a low survival rate, partly due to difficulties in early detection. Because of HCC’s high heterogeneity, it has been suggested that multiple biomarkers would be needed to develop a sensitive HCC screening test. This study applied random forest (RF), a machine learning technique, and proposed two novel models, fixed sequential (FS) and two-step (TS), for comparison with two commonly used statistical techniques, logistic regression (LR) and classification and regression trees (CART), in combining multiple urine DNA biomarkers for HCC screening using biomarker values obtained from 137 HCC and 431 non-HCC (224 hepatitis and 207 cirrhosis) subjects. The sensitivity, specificity, area under the receiver operating curve, and variability were estimated through repeated 10-fold cross-validation to compare the models’ performances in accuracy and robustness. We show that RF and TS have higher accuracy and stability; specifically, they reach 90% specificity and 86%/87% sensitivity respectively along with 15% higher sensitivity and 10% higher specificity than LR in cross-validation. The potential of RF and TS to develop a panel of multiple biomarkers and the possibility for self-training, cloud-based models for HCC screening are discussed.

Aim This study aimed to explore the potential of detecting hepatocellular carcinoma (HCC)-associated DNA markers, TP53 249T mutations and aberrant methylation of RASSF1A and GSTP1 genes, for monitoring HCC recurrence. HCC remains a leading cause of death worldwide, with one of the fastest growing incidence rates in the US. While treatment options are available and new ones emerging, there remains a poor prognosis of this disease mostly due to its late diagnosis and high recurrence rate. Although there are no specific guidelines addressing how HCC recurrence should be monitored, recurrence is usually monitored by serum-alpha fetal protein and imaging methods such as magnetic resonance imaging (MRI). However, early detection of recurrent HCC remains limited, particularly at the site of treated lesion. Methods Here, the authors followed 10 patients that were treated for a primary HCC, and monitored for months or years later. At these follow-up visits, urine was collected and tested retrospectively for 3 DNA biomarkers that associate with HCC development. Results This 10-patient study compared detection of urine DNA markers with MRI for monitoring HCC recurrence. Five patients were confirmed by MRI for recurrence, and all 5 had detectable DNA biomarkers up to 9 months before recurrence confirmation by MRI. Conclusion Overall, this suggests that detection of HCC-associated DNA markers in urine could provide a promising tool to complement detection of recurrent HCC by imaging.

Aim Aberrant methylation of the promoter, P2, and the first exon, E1, regions of the tumor suppressor gene RASSF1A, have been associated with hepatocellular carcinoma (HCC), albeit with poor specificity. This study analyzed the methylation profiles of P1, P2 and E1 regions of the gene to identify the region of which methylation most specifically corresponds to HCC and to evaluate the potential of this methylated region as a biomarker in urine for HCC screening. Methods Bisulfite DNA sequencing and quantitative methylation-specific polymerase chain reaction assays were performed to compare methylation of the 56 CpG sites in regions P1, P2 and E1 in DNA isolated from normal, hepatitic, cirrhotic, adjacent non-HCC, and HCC liver tissue and urine samples for the characterization of hypermethylation of the RASSF1A gene as a biomarker for HCC screening. Results In tissue, comparing HCC (n = 120) with cirrhosis and hepatitis together (n = 70), methylation of P1 had an area under the receiver operating characteristics curve (AUROC) of 0.90, whereas methylation of E1 and P2 had AUROC of 0.84 and 0.72, respectively. At 90% sensitivity, specificity for P1 methylation was 72.9% versus 38.6% for E1 and 27.1% for P2. Methylated P1 DNA was detected in urine in association with cirrhosis and HCC. It had a sensitivity of 81.8% for α-fetoprotein negative HCC. Conclusion Among the three regions analyzed, methylation of P1 is the most specific for HCC and holds great promise as a DNA marker in urine for screening of cirrhosis and HCC.

Currently, blood is the body fluid of choice to provide comprehensive germline genomics via Peripheral Blood Mononuclear Cells (PBMC) and cancer genetics via the detection of circulating tumor DNA (ctDNA, liquid biopsy) for precision medicine. In this study, we examined if urine can be an alternative to blood for comprehensive germline genomics and for liquid biopsy. We have previously shown that human urine contains DNA derived both from sloughed-off cell debris of the urinary tract (greater than 1 KB/High Molecular weight or HMW) and trans-renal DNA from the circulation (less than 1 KB/Low molecular weight or LMW). We performed whole genome sequencing (WGS) to compare the quality and comprehensiveness of the genomic data between 3 sets of matched PBMC and HMW urine DNA. There was no statistically significant difference between the NGS data obtained from PBMC and urine DNA upon comparing the number of reads, average coverage, percent aligned reads, percent reads passing Phred score Q30 (p value less than 0.05, paired t-test) and coverage of human genome. Next, we compared the detectability of ctDNA in the matching LMW urine and plasma DNA obtained from 44 HCC patients by employing a panel of HCC biomarkers including genetic mutations at TP53 codon 249 [G:C to T:A transversion (TP53 249T)], CTNNB1 exon 3 regions 32-37 (CTNNB1 32-37), and hTERT promoter region position 124 (hTERT 124) and aberrant methylation of RASSF1A (mRASSF1A) promoter. In this study, a systematic analysis of this HCC biomarker panel demonstrated that urine can complement blood for liver cancer screening. A combination of the plasma and urine biomarkers with serum AFP the current most widely used biomarker for HCC can provide higher sensitivity (up to 30% more) for liver cancer detection. In order to characterize and determine if LMW urine DNA is derived from plasma DNA, we performed WGS to compare the size profiles and coverage of these two sources of cell-free DNA in 3 sets of matching HCC urine and plasma samples. Overall shorter read lengths were obtained from urine DNA as compared to the corresponding plasma DNA. A series of peaks occurring at 10 bp periodicity was displayed in both sources of cell-free DNA Further analysis regarding the genome coverage, overlapping fraction and variant detection is ongoing. In conclusion, our data suggests that (i) HMW urine DNA can replace PBMC DNA for providing comprehensive genomic sequencing data and (ii) urine can complement blood for liver cancer liquid biopsy, precision medicine, and potential applications to other cancers. Citation Format: Surbhi Jain, Tai-Jung Lee, Adam Zhang, Jonathan Cheng, Jamin D. Steffen, Chi-Tan Hu, James P. Hamilton, Harry Luu, Hie-Won Hann, Fwu-Shan Shieh, Selena Y. Lin, Wei Song, Ying-Hsiu Su. Urine as an alternative to blood for germline genomics and cancer genetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 417.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer worldwide and a leading cause of global cancer mortality. Lack of efficient early detection and limited therapeutic options are two key contributing factors for the dismal prognosis of this disease. We have previously shown that urine contains fragmented, circulation-derived cell-free DNA that can be used for detection of circulating tumor DNA (ctDNA) if a tumor is present, suggesting urine as an alternative body fluid to plasma for cancer liquid biopsy. This study compared the detectability of five known HCC-associated DNA modifications, including genetic mutations (TERT -124 G>A, TP53 249 G>T and CTNNB1 hotspot at codon 32-45) and epigenetic methylation (mGSTP1, mRASSF1A), in matched urine and plasma from patients with liver cancer. In order to increase the sensitivity of detecting DNA modifications from the short, fragmented DNA templates, specialized short amplicon PCR assays were developed. To compare the detectability of HCC ctDNA markers in urine and plasma, 26 pairs of urine and plasma samples from HCC patients were identified and tested for five HCC ctDNA markers. In this cohort, 17 of 26 (65%) HCC samples had serum AFP above 20 ng/mL and were considered AFP-positive HCC. By using our HCC DNA biomarker panel composed of 5 assays, 22/26 (84.6%) urine and 23/26 (88.5%) plasma samples were found to have at least one DNA marker detected. In the combination of our HCC DNA biomarker panel with serum-AFP, the sensitivity of HCC detection in a liquid biopsy of urine and plasma as compared to serum-AFP alone (the current most used screening marker) increased from 65% to 92.3% for both urine plus AFP and plasma plus AFP. Further studies to include non-HCC samples (such as cirrhosis) to determine the specificity of the HCC DNA biomarker panel for liquid biopsy are in progress. In conclusion, our research provides the promising potential for HCC liquid biopsy screening in at-risk populations and precision medicine by using plasma, urine, or both as body fluids. Citation Format: Tai-Jung Lee, Surbhi Jain, Jamin D. Steffen, Adam M. Zhang, Chi-Tan Hu, James P. Hamilton, Ying-Hsiu Su. Comparison of ctDNA markers in matched urine and plasma from patients with liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3661.

Infection with Hepatitis B virus (HBV) is a major etiology of hepatocellular carcinoma (HCC), the third leading cause of cancer-related deaths worldwide. Studies have shown that some cells infected with HBV contain integrated viral DNA, and integrated viral DNA has been observed more frequently in HCC than in non-HCC infected liver. Although integration occurs randomly throughout host chromosomes, recurrent integration sites have been reported. Interestingly, these integration sites are located in genes known to be involved in carcinogenesis. During HCC carcinogenesis, tumor cells undergo uncontrolled clonal expansion in which a particular integration junction will become dominant (or a major junction). We have adapted a method known as primer extension capture (PEC) to enrich integrated HBV DNA for next-generation sequencing (NGS) to detect these integration junctions that occur at low frequency. We next developed a novel program, “HccDriverFinder,” to identify major junctions, incorporate PubMed data mining for driver identification, detect HBV mutations (revealing a striking 98.8% HCC-linked mutations identified in cancer), and visualize integration events. By analyzing NGS data generated from 13 matched HCC and adjacent non-HCC tissue samples, we found specific integrations in known targets such as TERT and CCNE1, and uncovered new recurrent HBV integration sites in genes including CSAD and ABCC13 (both of which are reportedly linked with tumorigenesis). We have further optimized the PEC approach to enrich for the whole HBV genome and have applied it to urine DNA from 31 hepatitis, 27 cirrhosis, and 29 HCC patients for the potential of identifying HBV junctions in circulation. Our approach holds the promise of building a driver identification kit for HCC detection, drug development and precision medicine. Citation Format: SHIH-CHUN SHEN, Amy Jiaen Lu, John Caleb Shieh, Yih-Ping Su, Bilal Nasir, Ning Ling, Daryl T. Lau, Jamin D. Steffen, Fwu-Shan Shieh, Wei Song, Ying-Hsiu Su. Development of a novel approach to identify cancer drivers for hepatitis B virus-associated hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 559.

Identification of viral integration sites has been important in understanding the pathogenesis and progression of diseases associated with particular viral infections. The advent of next-generation sequencing (NGS) has enabled researchers to understand the impact that viral integration has on the host, such as tumorigenesis. Current computational methods to analyze NGS data of virus-host junction sites have been limited in terms of their accessibility to a broad user base. In this study, we developed a software application (named ChimericSeq), that is the first program of its kind to offer a graphical user interface, compatibility with both Windows and Mac operating systems, and optimized for effectively identifying and annotating virus-host chimeric reads within NGS data. In addition, ChimericSeq's pipeline implements custom filtering to remove artifacts and detect reads with quantitative analytical reporting to provide functional significance to discovered integration sites. The improved accessibility of ChimericSeq through a GUI interface in both Windows and Mac has potential to expand NGS analytical support to a broader spectrum of the scientific community.

Chronic Hepatitis B Virus (HBV) infection is a major etiology of hepatocellular carcinoma (HCC), a leading cause of cancer mortality worldwide. Integration of HBV DNA into the host genome occurs during the course of chronic infection. Integrated HBV DNA is observed in most of HBV-associated HCC. The integration junctions derived from the original tumor cell become abundant (referred to as major integration junctions) in the infected liver because of clonal expansion during tumor development. Detection of circulating DNA containing these tumor-derived integration junctions may therefore be useful for cancer detection. We have adapted a method known as Primer Extension Capture (PEC) to enrich integrated HBV DNA for next-generation sequencing (NGS). Our initial studies using this approach to enrich the HBV DR1-2 region (a common site for HBV integrated breakpoints) identified major integration junctions from HBV-HCC tissue samples and matched urine. Further analysis revealed that most recurrently targeted integrations from these HCC tumors have previously reported involvement in cancer. This suggested that identification of recurrently targeted genes is applicable for driver identification. Interestingly, we show how HBV targets the TERT promoter in a localized region even though no two TERT junctions examined are identical. We have further developed this PEC to enrich for the entire HBV genome and applied it to (i) liver tissue DNA from 20 matched HCC and adjacent non-HCC samples, and (ii) DNA from urine of 20 hepatitis, 20 cirrhosis and 20 HCC patients. The HBV enriched libraries were sequenced by NGS and the integration events were analyzed using the in-house developed software. The complexity of HBV junction sites in HCC and non-HCC tissue and urine derived circulating DNA is reported. Our approach has potential to be used for liquid biopsies to study the complexities of HBV integrations in chronic HBV infection and carcinogenesis, and to identify HCC-related DNA modifications for early detection and disease management. Citation Format: Selena Y. Lin, Jamin D. Steffen, Yih-Ping Su, Surbhi Jain, Ting-Tsung Chang, Wei Song, Ying-Hsiu Su. Detection of HCC-derived major HBV integration junctions in urine and their implications for driver identification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 733. doi:10.1158/1538-7445.AM2017-733

The purpose of this study was to explore the potential of urine DNA biomarkers for the early detection of primary hepatocellular carcinoma (HCC). HCC is an aggressive disease with a 5-year survival rate of 26% in early-stage cancers, and a mere 2% in later stages. The most commonly used screening biomarker for HCC is serum alpha-fetoprotein (AFP), which detects only 40-60% of cases. We have previously shown that urine contains fragmented, circulation-derived, cell-free DNA that can be used for detection of cancer-related DNA markers, if a tumor is present. Given the molecular heterogeneity of HCC, we assembled a panel of frequently reported early detection DNA biomarkers for HCC that included genetic mutations (TERT -124 G to A, TP53 249 G to T and CTNNB1 hotspot at codon 32-45) and methylation markers (GSTP1, RASSF1A, CDKN2A, SFRP1, TFPI and MGMT). In this present study, we developed specialized short amplicon PCR assays optimized to detect these biomarkers from the urine of patients with cancer and tested the panel of biomarkers in urine of hepatitis, cirrhosis and HCC patients to demonstrate the feasibility of a breakthrough urine DNA test for HCC screening and early detection. Our evaluation indicated that the TP53 mutation, methylation of RASSF1A and GSTP1 urine DNA biomarkers were significantly higher in HCC than in non-HCC population. A correlation analysis indicated that these three markers do not correlate with each other and are suitable to be combined in a panel of biomarkers for the early detection of HCC. The 3-marker HCC urine DNA panel had an AUROC of 0.880 for distinguishing HCC from cirrhosis and hepatitis. By logistic regression, the sensitivity of the 3-DNA marker urine panel alone or in combination with AFP is 84.5% or 89.5% respectively, with a specificity of 90% for detecting HCC with AUROC of 0.951. Furthermore, these 3-DNA markers scored 43 of the 49 (87.8%) AFP-negative (less than 20 ng/mL) HCC urine samples "positive" in this study population. A novel statistic model was built by using Random Forest machine learning method as compared to full logistic regression in both open labeled urine samples and 242 blinded urine validation samples. A sensitivity of 92.3 % at a specificity of 87% was obtained by applying the random forest algorithm generated from the open labeled data (at 90% specificity cutoff) to the validation study, where as a sensitivity of 76.9% at a specificity of 84.7% was obtained from the full logistic regression derived from the open labeled data set. In conclusion, HCC DNA markers can be detected in urine of patients with HCC by short-amplicon, PCR-based assays and this urine test has the potential to become the first line of screening for HCC in high risk populations. Citation Format: Surbhi Jain, Jamin D. Steffen, Yih-Ping Su, Jeremy Wang, Ting-Tsung Chang, Chi-Tan Hu, Wei Song, Ying-Hsiu Su. Detection of genetic and epigenetic DNA markers in urine for the early detection of liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5699. doi:10.1158/1538-7445.AM2017-5699

Human urine has been shown to contain DNA from circulation. In this study, we aim to provide unambiguous evidence that urine contains hepatocellular carcinoma (HCC)-derived DNA, and thus can be used for detecting HCC-associated DNA markers. We used novel hepatitis B virus (HBV)-host junction sequences (HBV-JSs), created by viral integration in the human genome, as unique markers to track the HBV-integrated DNA from tumor tissues to their corresponding urine samples. An HBV DR1-2 enriched Next-Generation sequencing (NGS) assay was developed to identify the major HBV-JSs in 15 HBV-HCC tissues. Upon validation of the major HBV-JSs by Sanger sequencing, a short-amplicon PCR assay tailed for each HBV-JS was developed to test the junctions’ existence in the corresponding urine DNA. 15 major HBV-JSs were identified by the HBV DR1-2 enriched NGS assay from 15 HBV related HCC (HBV-HCC) tissues, 13 of which were validated by Sanger sequencing. By using HBV-JS specific short-amplicon junction PCR assays, we detected and confirmed six of nine HBV-JSs for which there were matching urine samples. Urine contains detectable major HBV-JSs derived from HCC, and thus can be used for liquid biopsies to study not only the complexities of HBV-JS species during chronic HBV infection and carcinogenesis, but also other HCC-related DNA modifications for the early detection of HCC and disease management. Citation Format: Selena Lin, Evan R. Trauger, Benjamin P. Song, Ling Lan, Patrick M. Jongeneel, Emilie G. C. Thompson, Malcolm C. Hoffman, Surbhi Jain, Ting-Tsung Chang, Timothy M. Block, Wei Song, Ying-Hsiu Su. Detection of HBV-host junction DNA sequences in urine of patients with hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4934.

The purpose of this study was to explore the potential of urine DNA biomarkers for the early detection of primary and recurrent hepatocellular carcinoma (HCC). HCC is an aggressive disease with a 5-year survival rate of 26% in early-stage cancers, and a mere 2% in later stages, with an approximate 50% recurrence rate in the first 2 years of treatment. The most commonly used screening biomarker for HCC is serum alpha-fetoprotein (AFP), which detects only 40-60% of cases. We have previously shown that urine contains fragmented, circulation-derived, cell-free DNA that can be used for detection of cancer-related DNA markers, if a tumor is present. In order to detect circulation-derived, cell-free DNA markers in urine, we have developed short amplicon (∼50 bp) PCR-based assays for the three most frequent hotspot mutations in TP53 (codon 249), TERT (-124, promoter), and CTNNB1 (hotspot in exon 3, codons 32-37), and for aberrant DNA methylation in GSTP1 (mGSTP1) and RASSF1A (mRASSF1A). This five marker panel has an area under the receiver operating curve of 0.94 (92.2% sensitivity at 80% specificity) in distinguishing primary HCC (n = 77) from non-HCC (n = 91) patients by a logistic regression-based combination algorithm. Furthermore, these 5 DNA markers scored 42 of the 45 (93.3%) AFP-negative (< 20 ng/mL) HCC urine samples “positive” in this study population. To validate the urine DNA test for the early detection of recurrent HCC, an on-going blinded study was conducted. Ten patients with treated HCC were monitored for recurrence of HCC with serum AFP and urine DNA markers. Urine samples were collected from these patients at visits with a hepatologist, barcoded, and tested for the five DNA markers. Of the 10 patients, 4 developed recurrence during the study. The HCC-specific urine DNA markers were detected in 3 out of these 4 patients six months prior to MRI diagnosis, and detected concurrent with MRI diagnosis in the other patient. In conclusion, HCC DNA markers can be detected in urine of patients with HCC by short-amplicon, PCR-based assays. Furthermore, we have demonstrated that the urine DNA test detected at least 40% more HCC in an open-labeled study as compared to serum AFP alone, and has the potential to become the first line of screening for HCC in high risk populations. Citation Format: Surbhi Jain, Hie-Won Hann, Sitong Chen, Selena Y. Lin, Ting-Tsung Chang, Chi-Tan Hu, Wei Song, Ying-Hsiu Su. Detection of genetic and epigenetic DNA markers in urine for the early detection of primary and recurrent HCC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3128.

The purpose of this study was to develop a computational method of extracting viral integration events from NGS data in an intuitive way that could accommodate access from users of all disciplines. Viral integration into the host genome is a characteristic of many pathogenic viruses, including the hepatitis B virus (HBV) and human papillomavirus (HPV). Gradual insertion of viral components near proto-oncogenes of the host genome over time can induce uncontrolled cellular proliferation, eventually leading to carcinogenesis. While increased availability of high-throughput next generation sequencing (NGS) has provided tools for researchers to discover these underlying host changes due to viral integrations, there is an emerging need for analytical support of this data. Here we present ChimericSeq, a user-friendly program that can quickly identify viral integration events from NGS data. To fully evaluate this program, we compared the functionality of ChimericSeq to other current viral integration programs. A number of synthetic data sets of HBV sequence fragments integrated into 100bp fragments of random human genomic DNA were created to mimic the nature of chimeric reads of NGS data. ChimericSeq was able to correctly identify reads containing at least 25bp of viral sequence at 100% accuracy. This was a major improvement over the current programs, VirusClip and ViralFusionSeq, which could not detect reads with only 25bp of viral sequence, and even had difficulty correctly identifying viral integration sites containing additional viral sequence. Furthermore, ChimericSeq could detect viral events nearly 10X and 100X faster than these current programs, respectively. Upon testing of NGS data from actual HBV-positive human tissue, we found that ChimericSeq was not only much faster, but was also able to detect more unique viral integration events than current viral integration tools. In conclusion, ChimericSeq expands NGS analytical support to a broader spectrum of the scientific community, being the first program of its kind to offer support in an intuitive graphical user interface (GUI) for commonly used operating platforms, such as Windows and Mac. Citation Format: Patrick Jongeneel, Selena Lin, Jamin Steffen, Surbhi Jain, Ying-Hsiu Su, Wei Song. ChimericSeq: an easy-to-use program for discovery and analysis of integration events from NGS data. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5290.

e15640Background: HCC is an aggressive cancer with a 5-year survival rate of 26% if detected early (stages 1 & 2), and a mere 2% in late stages, with an approximate 50% recurrence rate in 2 years a...

Hepatocellular carcinoma (HCC), or liver cancer, is an aggressive disease and is regarded as one of the fastest growing cancers in the United States in incidence. The 5-year survival rate of HCC patients drops dramatically from a poor 26%, in early-stage cancer, to a mere 2% in late stages of this disease. The only available early detection biomarker is serum alpha-fetoprotein, which can detect only 40-60% of cases. Our goal is to develop a screening test for early detection of liver cancer, in order to meet this urgent need for new and better biomarkers for HCC. Cancer is a disease of the genome and epigenome, and if we can detect these underlying genetic mutations and epigenetic modifications in the periphery, we can effectively detect cancer early. Hence, we chose a panel of both HCC-associated genetic and DNA methylation biomarkers. We have previously shown that urine contains fragmented cell-free DNA derived from the circulation and that we can detect cancer-related DNA, both mutated and methylated DNA, in the urine of such patients. We have also demonstrated that the concentration of tumor-derived DNA in plasma and in urine is similar in patients with tumors. In order to demonstrate the feasibility of detecting HCC DNA biomarkers in the circulation derived urine DNA, we have developed short amplicon (∼50 bp) PCR-based assays targeting HCC-specific mutations in TP53 and CTNNB1 genes and HCC-specific methylation in GSTP1, RASSF1A and CDKN2A genes. Urine DNA isolated from samples of hepatitis, cirrhosis, and HCC patients were tested in each of the assays and analyzed for individual marker and panel performance. The performance of the markers and our approach towards the development of a targeted next generation urine DNA based HCC screening test is discussed. Citation Format: Surbhi Jain, Sitong Chen, Selena Y. Lin, Adam Clemens, Hei-won L. Hann, Ting-Tsung Chang, Chi-Tan Hu, Shun-Hua Chen, Wei Song, Ying-Hsiu Su. Development of a noninvasive and sensitive urine screening test for liver cancer targeting circulation-derived cancer DNA biomarkers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1569. doi:10.1158/1538-7445.AM2015-1569

Colorectal cancer (CRC) has been the second leading cause of cancer-related deaths due in part to a low compliance rate ( Citation Format: Adam Winfield Clemens, Selena Lin, Surbhi Jain, Ying-Hsiu Su, Wei Song. Detection of colorectal cancer-associated genetic and epigenetic alterations in urine of patients with CRC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1561. doi:10.1158/1538-7445.AM2015-1561

Hepatitis B virus (HBV) related liver diseases often progresses from hepatitis to cirrhosis and eventually to hepatocellular carcinoma (HCC), which has a poor prognosis, mostly due to late detection. Chronic HBV infections are associated with >50% of HCC cases worldwide, and up to 90% of HBV-related HCCs (HBV-HCC) were found to contain integrated HBV DNA. During infection, HBV DNA integrates in the host chromosome at variables sites, generating a unique HBV-host junction sequence (HBV-JS) that can serve as a DNA finger print of an infected hepatocyte. During hepatocarcinogenesis, tumorigenic hepatocyte(s) undergo uncontrolled clonal expansion resulting in one to few particular HBV-JS's becoming dominant. Thus, we hypothesize that the appearance of expanded HBV-JS species is a marker of clonal expansion and can be a potential marker for early detection of HBV-HCC. A PCR-based enrichment assay and next generation sequencing (NGS) approach were developed for the HBV DR1-2 integration hotspot region to explore the feasibility of detecting HBV-JS's in the circulation (i.e. urine) of patients with HBV-HCC. HBV-HCC tissue DNA samples underwent library construction and were enriched for DNA containing the HBV DR1-2 sequences. Enriched DNA fragments were then cloned and sequenced, from which 5 HBV-JS's were discovered at Chr5 (hTERT promoter), Chr7 (AOAH intronic transcript 1), Chr12 (KCN2), Chr19 (miRNA 512-1,-2), and Chr22 (LARGE) in four patient samples. These sites were further confirmed in matched urine DNA samples by designing primers to amplify the HBV-JS. The NGS approach, which includes HBV-HCC (n = 18), HBV-hepatitis (n = 5), and HBV-cirrhosis (n = 8) urine samples to identify HBV-JS’s, is in progress. Overall, this would be the first study both to demonstrate unambiguous detection of HBV-JS's in the urine of HBV-HCC patients and to suggest the potential of detecting expanded HBV-JS species in urine as a marker for HBV-HCC screening. Furthermore, if successful, the high-throughput detection of HBV-JS's in urine by NGS offers a promising noninvasive HBV-HCC screening platform, as well as provides insight into the carcinogenic mechanisms of HBV DNA integration. Citation Format: Selena Lin, Benjamin Song, Evan Trauger, Malcolm Hoffman, Emilie Thompson, Rebecca Zhou, Surbhi Jain, Wei Song, Ying-Hsiu Su. Detection of HBV-host junction DNA sequences in urine of patients with HBV-infected HCC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1568. doi:10.1158/1538-7445.AM2015-1568