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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 & 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.

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.

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.

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.

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.

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 of HCC surveillance programs in well-defined, high-risk populations were only able to detect about 40–50% of HCC at curative stages (Barcelona Clinic Liver Cancer stages 0 & 1) due to the low sensitivities of the current screening methods. The advance of sequencing technologies has identified numerous modifications as potential candidate DNA markers for diagnosis/surveillance. Here we aim to provide an overview of the DNA alterations that result in activation of cancer pathways known to potentially drive HCC carcinogenesis and to summarize performance characteristics of each DNA marker in the periphery (blood or urine) for HCC screening.

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.

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) is the 7th most common cancer worldwide and remains the third leading cause of cancer deaths (Yang and Roberts 2010). It has a 5-year survival rate of less than 11% even in developed nations (Garcia, Jemal et al. 2007). The 5-year survival rate drops from 26% to 2% in patients with localized versus metastasized cancer (ACS, 2010). The poor prognosis is due mainly to late detection with the methods currently available. Thus, a better screening method to detect HCC at its early, curative stage is needed to improve its outcome. One of the major etiological factors associated with HCC development is chronic infection with hepatitis B virus (HBV). In developing countries, 59% of liver cancers are attributable to HBV and, in developed countries, 23% of liver cancers are attributable to HBV (Garcia, Jemal et al. 2007).

Hypermethylation of the glutathione S-transferase π 1 (GSTP1) gene promoter region has been reported to be a potential biomarker to distinguish hepatocellular carcinoma (HCC) from other liver diseases. However, reports regarding how specific a marker it is have ranged from 100% to 0%. We hypothesized that, to a large extent, the variation of specificity depends on the location of the CpG sites analyzed. To test this hypothesis, we compared the methylation status of the GSTP1 promoter region of the DNA isolated from HCC, cirrhosis, hepatitis, and normal liver tissues by bisulfite-PCR sequencing. We found that the 5' region of the position -48 nt from the transcription start site of the GSTP1 gene is selectively methylated in HCC, whereas the 3' region is methylated in all liver tissues examined, including normal liver and the HCC tissue. Interestingly, when DNA derived from fetal liver and 11 nonhepatic normal tissue was also examined by bisulfite-PCR sequencing, we found that methylation of the 3' region of the promoter appeared to be liver-specific. A methylation-specific PCR assay targeting the 5' region of the promoter was developed and used to quantify the methylated GSTP1 gene in various diseased liver tissues including HCC. When we used an assay targeting the 3' region, we found that the methylation of the 5'-end of the GSTP1 promoter was significantly more specific than that of the 3'-end (97.1% vs. 60%, p

Hypermethylation of the promoter of the tumor suppressor gene, adenomatous polyposis coli (APC), occurs in various malignancies, including hepatocellular carcinoma (HCC). However, reports on the specificity of the methylation of the APC gene for HCC have varied. To gain insight into how these variations occur, bisulfite PCR sequencing was performed to analyze the methylation status of both sense and antisense strands of the APC gene in samples of HCC tissue, matched adjacent non-HCC liver tissue, hepatitis, cirrhosis, and normal liver tissues. DNA derived from fetal liver and 12 nonhepatic normal tissue was also examined. These experiments revealed liver-specific, antisense strand-biased CpG methylation of the APC gene and suggested that, although methylation of the antisense strand of the APC gene exists in normal liver and other non-HCC disease liver tissue, methylation of the sense strand of the APC gene occurs predominantly in HCC. To determine the effect of the DNA strand on the specificity of the methylated APC gene as a biomarker for HCC detection, quantitative methylation-specific PCR assays for sense and antisense strand DNA were developed and performed on DNA isolated from HCC (n = 58), matched adjacent non-HCC (n = 58), cirrhosis (n = 41), and hepatitis (n = 39). Receiver operating characteristic curves were constructed. With the cutoff value set at the limit of detection, the specificity of sense and antisense strand methylation was 84% and 43%, respectively, and sensitivity was 67.2% and 72.4%, respectively. This result demonstrated that the identity of the methylated DNA strand impacted the specificity of APC for HCC detection. Interestingly, methylation of the sense strand of APC occurred in 40% of HCCs from patients with serum AFP levels less than 20 ng/mL, suggesting a potential role for APC as a biomarker to complement AFP in HCC screening.

Robust assays for the isolation and characterization of urinary FOS (free oligosaccharides) have been developed to screen patients for altered protein and/or lipid glycosylation. A FOS analysis can therefore identify potential biomarkers for hepatocellular carcinoma, since variations in glycosylation as a result of tumorigenecity should be detectable in the FOS of patients. HCC (hepatocellular carcinoma) accounts for 80–90% of all liver cancers. It occurs more often in men than women and occurs mostly in people 50–60 years old. The disease is more common in parts of Africa and Asia than in North or South America and Europe. Using a combination of solid-phase extraction techniques and affinity chromatography, followed by separation of urinary FOS by NP (normal phase)-HPLC and HIAX (hydrophilic interaction and anion-exchange)-HPLC, more than 200 different species have been identified in patient samples. The high incidence of small sialylated oligosaccharides in HCC patients suggests that pro-inflammatory markers may be detected as early indicators of disease progression. In addition, the methods developed here to isolate and analyse excreted glycoprotein- and glycosphingolipid-bound oligosaccharides have been used to characterize changes in metabolic processes that underlie a number of human genetic disorders. The ability to predict disease status in microlitre amounts of readily available non-invasive urine samples indicates that rapid methods for screening can be developed.

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

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

Hepatocellular carcinoma (HCC) or liver cancer is an aggressive disease and one of the fastest growing cancers in incidence in the United States. The 5 year survival rate drops dramatically from a poor 26% in early stages to a mere 2% in later stages of the disease. Currently, the only available biomarker for early detection is serum alpha-fetoprotein which can identify only 40-60% of cases. We aim to improve the poor disease prognosis by developing a screening test capable of detecting liver cancer in its earlier stages. Since cancer is a disease of the genome and epigenome, if we can detect these underlying genetic mutations and epigenetic modifications in the periphery, we should be able to effectively detect cancer early. We have previously shown that urine contains fragmented, cell-free, cancer related DNA, both mutated and methylated, derived from the circulation of cancer patients. We have also demonstrated that the concentration of tumor-derived DNA in plasma and in urine is similar in patients with tumors. Hence, we propose the use of a panel of HCC-associated genetic and epigenetic methylation DNA biomarkers to develop a sensitive, noninvasive urine screening test for HCC. In order to detect these markers in the circulation derived urine DNA, we developed short amplicon (∼50 bp) PCR based assays targeting HCC-associated mutations in TP53, CTNNB1, hTERT genes and HCC associated methylation in GSTP1, RASSF1A and CDKN2A. Urine DNA isolated from hepatitis, cirrhosis and HCC patients were tested in each of the assays and analyzed. Panel performance parameters such as sensitivity, specificity and area under the receiver operating curve were calculated for individual markers and then as a group in order to evaluate their ability to distinguish HCC patients from hepatitis and cirrhosis subjects. The potential of this urine test in the early detection of HCC is discussed. Citation Format: Sitong Chen, Surbhi Jain, Selena Lin, Ying-Hsiu Su, Wei Song. Development of a urine DNA based marker panel for early detection of liver cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 888. doi:10.1158/1538-7445.AM2014-888

Hepatocellular carcinoma (HCC), the 5th most frequent cancer worldwide, has a 5-year survival rate of 14% because it is difficult to diagnose at its curative stages. The goal of this project is to determine if the detection of clonally expanded HBV DNA integration sites can be used as a marker for early detection of HBV related HCC (HBV-HCC) in order to improve its prognosis Hepatitis B virus (HBV) infections are associated with over 80% of HCC cases worldwide. Upon infection, the HBV genome integrates into the host chromosome at a random site, thus creating a unique genetic signature for each HBV infected cell. Certain HBV infected cells undergo progressive “uncontrolled clonal expansion” leading to the development of tumors, resulting in an over expansion of the unique HBV integration sites making them the predominant or “major HBV integration sites” (MIS). Cell-free circulating DNA from cancer patients reflects the characteristics of the tumor DNA. We and others have shown that urine contains DNA from the circulation and can be used as a source for cell-free circulating DNA. Successful detection of HBV integration sites in the circulation of HBV-HCC patients has not been demonstrated. In order to detect cell free circulating HBV DNA integration sites, a targeted enriched Next Generation Sequencing (NGS) assay was developed. Targeted enrichment was performed using an in-solution hybridization platform with biotinylated HBV RNA baits surrounding the HBV DNA nucleotide positions 1571 to 1960, the most frequent HBV breakpoint region. The assay was tested using library constructed DNA controls: Hep3B, which contains integrated DNA, and HepG2, which contains no HBV DNA. To determine the amount of captured DNA containing HBV DNA, the captured DNA was quantified for HBV containing DNA and total captured DNA. Almost all captured DNA contained HBV DNA. To confirm targeted capture of integrated HBV DNA, the captured DNA was cloned followed by sequencing. 3 out of 3 clones that were sequenced contain integrated HBV DNA. This assay demonstrated targeted capture and enrichment of HBV integration sites. This assay will be tested for sensitivity in detecting HBV integration sites and used to establish the criteria for the appearance of MIS as a biomarker for HBV-HCC. Overall, if successful, this cancer related HBV DNA marker would offer a high specificity and sensitivity in screening for HBV-HCC. Citation Format: Selena Lin, Surbhi Jain, Timothy Block, Frank Song, Ying Hsiu Su. Detection of clonally expanded HBV DNA integration sites as a marker for early detection of HBV related HCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1158. doi:10.1158/1538-7445.AM2013-1158

Hepatocellular carcinoma (HCC) is an aggressive malignancy that is responsible for between 250,000 and 800,000 deaths annually worldwide. HCC has a poor prognosis with a 5-year survival rate of less than 15% mainly due to a lack of early detection. The current biomarker for HCC is the alpha-fetoprotein (AFP), which has a low sensitivity of 40-60%. There is a necessity for non-invasive, convenient, and sensitive methods to detect the cancer in its curative stages to improve prognosis. Previous studies from our laboratory showed that the location of the CpG site methylation studied could impact the marker performance in distinguishing HCC from other liver diseases. The aberrant methylation of the tumor-suppressor RASSF1A gene has been extensively studied as a marker for a variety of cancers, including liver cancer. However, some studies found that the gene was methylated even in non-cancerous liver tissues. Thus, in order to explore the possibility of detecting aberrant methylation of the tumor-suppressor RASSF1A gene as a biomarker for HCC screening, we analyzed the methylation profiles of 57 CpG sites in three regions of the promoter - P1, P2, and E1 - in normal, hepatitic, cirrhotic, adjacent non-HCC, and HCC tissue samples through bisulfite DNA sequencing. We discovered that the P1 region was most specifically methylated to HCC. We then confirmed the findings by developing a methylation specific PCR and analyzing a larger sample size. The data were analyzed and the P1 test was found to have an area under the curve (AUROC) of 0.90, whereas the E1 and P2 tests had AUROC of 0.84 and 0.72, respectively. At a cutoff of 10 copies for our analysis, the P1 MSP gave a sensitivity of 80.8% and specificity of 85.7%. Furthermore, we found that out of 62 AFP-negative samples, mRASSF1A detected 54 (87.1%). By combining AFP and mRASSF1A, 107 of 115 (93.0%) of HCC samples were detected as cancerous. Subsequently, we developed a PCR assay to detect the methylated RASSF1A gene in the urine of patients. This assay was then used to test the urine DNA isolated from patients with hepatitic, cirrhotic, or HCC after which the data were statistically evaluated. The potential of a urine DNA test for HCC screening is discussed. Citation Format: Surbhi Jain, Lijia Xie, Batbold Boldbaatar, Ying-Hsiu Su, Wei Song. Detection of aberrant methylation of the RASSF1A gene as a biomarker for hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1154. doi:10.1158/1538-7445.AM2013-1154

Hepatocellular carcinoma (HCC), the 5th most frequent cancer worldwide, has a 5-year survival rate of 14% because it is difficult to diagnose early. The goal of this project is to construct a panel of circulation-derived DNA markers to use in a urine test for the early detection of HCC to improve its prognosis. The HCC-specific p53 codon 249T mutation was the first candidate DNA marker used to explore the criteria needed to detect HCC-derived DNA markers in urine of patients with HCC in a sensitive, noninvasive manner. We showed previously that urine contains circulation-derived DNA fragments that are mostly fewer than 300 bp, designated as low-molecular-weight (LMW) urine DNA. The LMW urine DNA contains DNA from tumor tissues when tumors are present. The tumor-derived DNA fragments offer the potential to develop absolutely noninvasive urine tests for the detection of any cancer with known DNA biomarkers. A locked nucleic acid clamp-mediated PCR assay for the p53 249T mutation, targeting only 41 nucleotides of the template, followed by melting curve analysis, was developed and tested using patient urine samples. Total urine DNA samples from 17 patients with HCC were fractionated into high-molecular-weight (HMW) (>1 kb, mostly cell-associated) DNA from the urinary tract and LMW (< 1 kb, mostly circulation derived) DNA and subjected to the p53 249T mutation assay. Encouragingly, samples from 9 of 17 patients with HCC contained detectable p53 249T mutation in the LMW urine DNA fraction and only 1 of 17 matching HMW urine DNA fraction samples was found to contain p53 249T mutated DNA. These findings suggest that the assay target template size of 41bp is suitable to detect HCC-derived DNA markers in urine and that the p53 249T mutation detected in LMW urine DNA is derived from the circulation. Next, we tested a larger number of samples of LMW urine DNA from patients with HCC. Overall, we detected the p53 249T mutation in 50.8% of the LMW urine DNA samples from patients with HCC (60/118). Of the 49 subjects whose urine was positive for the p53 mutation before surgery, the p53 mutation was no longer detectable in the urine of 29 of those patients after surgical removal of the tumor. This result suggested that the p53 249T mutations detected in the urine before surgery were derived from the surgically removed HCC. For controls, we tested LMW urine DNA from patients with hepatitis and cirrhosis and from normal subjects or patients with colorectal cancer and found that 0 of 32 normal (0%), 0 of 31 colorectal cancer (0%), 7 of 69 hepatitis (10.1%), and 6 of 47cirrhosis (12.8%) samples contained detectable amounts of p53 249T mutated DNA. A urine test that includes multiple DNA markers for HCC screening and recurrence monitoring is currently being explored. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 715. doi:1538-7445.AM2012-715