Poster presented at American Association of the Study of Liver Diseases (AASLD) meeting in San Francisco California. 
Objective: 
Develop a urine test using a panel of select genetic and epigenetic markers for the early detection of primary and recurrent HCC.
Introduction:
Hepatocellular carcinoma (HCC) or liver cancer is an aggressive disease and one of the fastest growing cancers by incidence in the United States. Early detection is the key for effective treatment of HCC as the 5-year survival rate is 26% in early stage HCC as compared to only 2% when found after spreading to distant organs. The current marker, alpha-feto protein (AFP) and its fucosylated glycoform, L3, are of limited value with only 40-60% sensitivity.https://jdc.jefferson.edu/gastrohepposters/1000/thumbnail.jp

Chang, Ting-Tsung

Hann, Hie-Won

Hu, Chi-Tan

Jain, Surbhi

Lin, Selena

Song, Wei

Su, Ying-Hsiu

Poster presented at American Association of the Study of Liver Diseases (AASLD) meeting in San Francisco California. 
Objective: 
Develop a urine test using a panel of select genetic and epigenetic markers for the early detection of primary and recurrent HCC.
Introduction:
Hepatocellular carcinoma (HCC) or liver cancer is an aggressive disease and one of the fastest growing cancers by incidence in the United States. Early detection is the key for effective treatment of HCC as the 5-year survival rate is 26% in early stage HCC as compared to only 2% when found after spreading to distant organs. The current marker, alpha-feto protein (AFP) and its fucosylated glycoform, L3, are of limited value with only 40-60% sensitivity.http://jdc.jefferson.edu/gastrohepposters/1000/thumbnail.jp

Name not available

Detection of genetic and epigenetic DNA markers in urine for the early detection of primary and recurrent hepatocellular carcinoma

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Thomas Jefferson UniversityJefferson Digital CommonsDepartment of Gastroenterology and HepatologyPosters Division of Gastroenterology and Hepatology11-2015Detection of genetic and epigenetic DNA markersin urine for the early detection of primary andrecurrent hepatocellular carcinomaHie-Won HannThomas Jefferson University Hospital, Hie-Won.Hann@jefferson.eduSurbhi JainJBS Science Inc.Ting-Tsung ChangNational Cheng Kung University Medical CenterChi-Tan HuBuddist Tzu Chi General Hospital and Tzu Chi UniversitySelena LinDrexel University, College of MedicineSee next page for additional authorsFollow this and additional works at: http://jdc.jefferson.edu/gastroheppostersPart of the Gastroenterology Commons, and the Hepatology CommonsThis Article is brought to you for free and open access by the Jefferson Digital Commons. The Jefferson Digital Commons is a service of ThomasJefferson University's Center for Teaching and Learning (CTL). The Commons is a showcase for Jefferson books and journals, peer-reviewed scholarlypublications, unique historical collections from the University archives, and teaching tools. The Jefferson Digital Commons allows researchers andinterested readers anywhere in the world to learn about and keep up to date with Jefferson scholarship. This article has been accepted for inclusion inDepartment of Gastroenterology and Hepatology Posters by an authorized administrator of the Jefferson Digital Commons. For more information,please contact: JeffersonDigitalCommons@jefferson.edu.Recommended CitationHann, Hie-Won; Jain, Surbhi; Chang, Ting-Tsung; Hu, Chi-Tan; Lin, Selena; Song, Wei; and Su, Ying-Hsiu, "Detection of genetic andepigenetic DNA markers in urine for the early detection of primary and recurrent hepatocellular carcinoma" (2015). Department ofGastroenterology and Hepatology Posters.http://jdc.jefferson.edu/gastrohepposters/1AuthorsHie-Won Hann, Surbhi Jain, Ting-Tsung Chang, Chi-Tan Hu, Selena Lin, Wei Song, and Ying-Hsiu SuThis book is available at Jefferson Digital Commons: http://jdc.jefferson.edu/gastrohepposters/1TEMPLATE DESIGN © 2008 www.PosterPresentations.com Hie-Won Hann1, Surbhi Jain2, Sooji Yi1, Ting-Tsung Chang3, Chi-Tan Hu4, Selena Lin5, Wei Song2, and Ying-Hsiu Su6 1Thomas Jefferson University Hospital, 2JBS Science, Inc, 3National Cheng Kung University Medical College, 4Buddhist Tzu Chi General Hospital and Tzu Chi University, 5Drexel University, College of Medicine, and 6TheBaruch S. Blumberg Institute,   Detection of genetic and epigenetic DNA markers in urine for the early detection of primary and recurrent hepatocellular carcinoma Introduction Case 3: Detection of recurrent HCC Detection of AFP-negative HCC Conclusion Case 2: Detection of recurrent HCC Case 4: Detection of recurrent HCC Acknowledgements and Disclosures Figure  6. Detection of AFP-negative HCC by 5 HCC DNA markers in (A) tissue and (B) 3 cfDNA markers in urine. Marker distribution in individual HCC with available AFP value in our study population. Filled boxes represent a marker detected "positive", higher than cut-off value at 90% specificity distinguishing HCC from cirrhosis or positive for AFP. Empty boxes represent a marker value below the cut-off at 90% specificity or negative for AFP.  Hepatocellular carcinoma (HCC) or liver cancer is an aggressive disease and one of the fastest growing cancers by incidence in the United States. Early detection is the key for effective treatment of HCC as the 5-year survival rate is 26% in early stage HCC as compared to only 2% when found after spreading to distant organs. The current marker, alpha-feto protein (AFP) and its fucosylated glycoform, L3, are of limited value with only 40-60% sensitivity.   Urine contains fragmented, cell-free, cancer associated DNA, both mutated and methylated, derived from the circulation of cancer patients (Lin, Dhillon et al. 2011, Song, Jain et al. 2012, Jain, Xie et al. 2015) and the concentration of tumor-derived DNA in plasma and in urine is similar in patients with tumors (Su, Wang et al. 2008).  Results Urine samples were collected with written informed consent and institutional review board approvals from National Cheng Kung University (Tainan, Taiwan), Tzu-Chi Hospital, (Hualien, Taiwan), and Thomas Jefferson University Hospital (Philadelphia, PA), at visits with a hepatologist,  Note the samples from TJU were barcoded for disease status as a blinded study. Total urine DNA was isolated (Su, Wang et al. 2004) and the low molecular weight urine DNA, DNA less than 1 kb, was obtained from total urine DNA using carboxylated magnetic beads (Agentcourt Bioscience Corporation) as previously developed by us (Su, Wang et al. 2008). Bisulfite (BS) treatment was performed using EZ DNA Methylation-Lightning™ Kit (Zymo Research) following manufacturer’s guidelines. In order to detect circulation-derived, cell-free DNA markers in urine, we have developed short amplicon (~50 bp) PCR-based assays for mutations in TP53 (249T) (Lin, Dhillon et al. 2011), CTNNB1 (exon 3 - codons 32-37), TERT (-124 promoter) and for aberrant DNA methylation in GSTP1 (mGSTP1) and RASSF1A (mRASSF1A) (Jain, Xie et al. 2015).  Case 1: Detection of a primary HCC Figure 3: Retrospective analysis of urinary DNA markers plus serum AFP collected during doctor visits of a 55 year old male with HBV-cirrhosis who developed HCC that was undetectable by serum AFP but elevated  urine CTNNB1 mutation levels and was treated with microwave ablation. The patient, however, had a recurrence of HCC 4 months post treatment. While mGSTP1 and CTNNB1 mutation levels in urine declined post-treatment, urinary mRASSF1A, TERT 124 mutation and TP53 249T mutation levels continue to rise concurrent with the recurrence. Figure 5: Retrospective analysis of urinary DNA markers plus serum AFP collected during doctor visits of a 53 year old male with HBV-hepatitis/early cirrhosis who developed HCC that was detectable by serum AFP and was treated with microwave ablation.  The patient, however, had a recurrence of HCC 2 months post MW ablation.  While mGSTP1 level in urine declined post treatment, urinary mRASSF1A and TERT 124 mutation levels continued to rise concurrent with the recurrence. Systemic capillaries Lung Capillaries Vein Cell-free DNA in urine Mutated tumor DNA Normal DNA Normal or cancer cell death by apoptosis or necrosis-DNA released into circulation Kidney Liver cancer Special Assay Development  for LMW DNA templates Figure. 1. LMW urine DNA co-migrates with 1-2 nucleosomal sized DNA from serum and does not exist as naked DNA in urine. This indicates that the source of this circulation-derived DNA in urine is predominantly from apoptotic cells Pathway Gene DNA Alteration Incidence (%) Reported in circulation (Yes/No) Wnt signalling APC  M/G 80-93/ 1-2 Yes  SFRP1  M 42-63 Yes CTNNB1 G 19-33  No AXIN1 G 13-15  No Cell cycle/ Apoptosis RASSF1A  M 55-85 Yes  CDKN2A M/G 50-70/ 5-10 Yes  TP53  G 20-50 Yes  IRF2  G 5  No Detoxification  GSTP1 M 38-80 Yes Telomerase maintainance TERT G 59  No Table 1. Frequent genetic mutations (G) and epigenetic aberrant DNA methylations (M) reported in HCC tissues. Urinary Biomarkers copies/ml Serum AFP  and serum HBV viral load Microwave ablation Figure 2: Retrospective analysis of DNA biomarkers from urine samples, plus serum AFP and serum HBV viral load  levels  collected during doctor visits of a 74 year-old male with HBV-cirrhosis who developed HCC that was undetectable by serum AFP  and was treated with microwave ablation. Urinary mRASSF1A DNA and TERT 124 mutated DNA levels started to elevate 6 months prior to HCC detection by MRI and declined post treatment. TP53 249T  mutation level rose with the appearance of HCC and declined post treatment. Serum AFP  and serum HBV viral load Urinary Biomarkers copies/ml Figure 4: Retrospective analysis of urinary DNA biomarkers and serum AFP levels collected during doctor visits of a 73 year old male with HBV-cirrhosis and HCC that was treated with TACE. Samples were collected before TACE at the first time point. The DNA markers were undetectable but serum AFP was slightly elevated at 89 ng/ml and declined to baseline after TACE therapy. HCC recurrence was detected by MRI 11-13 months after TACE treatment. TERT 124 mutation levels are elevated 6 months prior to recurrence detection by MRI. mRASSF1A , TP53 249 T mutation, and serum AFP levels rise with the appearance of recurrent nodule and declined post treatment. This recurrence was also treated with TACE. Note, MRI at 11 months was not definitive.  Urine mGSTP1 , TERT 124 levels rise 4 months post repeat TACE concurrent with the appearance of a LIRADS category 3 lesion on MRI.  Methods Urinary Biomarkers copies/ml 1300 1350 Microwave ablation Serum AFP  and serum HBV viral load 5500 Microwave ablation 5000 Serum AFP  and serum HBV viral load Urinary Biomarkers copies/ml Results (A) HCC tissue biomarker distribution (B)  HCC urine biomarker distribution This work is supported by R44 CA165312-02. Wei Song is the President and founder of JBS Science, Inc.  Surbhi Jain is an employee of JBS Science, Inc. Develop a urine test using a panel of select genetic and epigenetic markers for the early detection of  primary and recurrent HCC. Objective Recurrence A total of 10 cases with treated HCC were monitored by serum AFP and urine DNA biomarkers in a blinded study. Of the 10, 4 developed recurrence during the study. HCC-specific urine DNA markers were detected in 3 of 4 patients six months prior to MRI diagnosis and in one patient concurrent with MRI diagnosis.   Fifteen cirrhosis and nine hepatitis patients were monitored every six months for HCC. In this group, one patient developed primary HCC and the urine DNA biomarkers were detected six months prior to MRI diagnosis in a blinded study. The urine test could lead to a paradigm shift for screening and effective management of primary and recurrent HCC and for personalized disease management. 

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Detection of genetic and epigenetic DNA markers in urine for the early detection of primary and recurrent hepatocellular carcinoma

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