Selena Lin

Abstract 876: Comprehensive analysis of the complexity of HBV DNA integration sites in the circulation of patients with HBV-related liver disease

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA HBV related liver disease ranges from hepatitis, cirrhosis, and hepatocellular carcinoma (HCC), which has a 5-year survival rate of 14% because it is difficult to diagnose at its curative stages. Chronic hepatitis B virus (HBV) infections is the major etiology of HCC, associated with over 50% of HCC cases worldwide and up to 70-80% in endemic areas. 85-90% of HBV related HCCs (HBV-HCC) contain integrated HBV DNA. During chronic HBV infection, HBV DNA has been shown to integrate at random sites in the host chromosome creating a unique genetic signature in each HBV infected hepatocytes. This makes the pool of HBV DNA integration sites highly complex in the liver. This genetic signature in HBV-infected liver along with the fundamental mechanism of carcinogenesis, which is clonal expansion, would result in HCC tissue containing only one to few clonally expanded integration sites, resulting in reduced complexity. Thus, we hypothesize the detection of reduced complexity of HBV DNA integration sites in the circulation can be a potential biomarker for HBV-HCC. In order to study the complexity of cell free circulating HBV DNA integration sites in the circulation, a targeted next generation sequencing (NGS) assay for urine DNA was developed. We chose to use urine in order to avoid detecting of HBV DNA from infectious viruses. Targeted enrichment was performed using an in-solution hybridization platform with biotinylated RNA baits covering the entire HBV genome. The assay was tested using library constructed DNA controls: Hep3B, which contains integrated DNA, and HepG2, which contains no HBV DNA to confirm targeted capture of HBV DNA. Next, matching tissue and urine DNA and DNA from urine collected after surgical removal of HCC tumor from seven HBV-HCC patients were collected and underwent library construction. The library DNA samples were subsequently enriched with HBV and HCC-specific RNA baits. Enriched library DNA was sequenced on Illuminas MiSeq platform. Although the analysis is still ongoing, we have successfully analyzed the complexity of cell-free circulating HBV DNA integration sites in urine. We detected 46 integration sites in the tissue and urine where the location of integrated HBV DNA mapped primarily (89%) to a known HBV integration breakpoint region, HBV DR1-2. Furthermore in 3 HBV-HCC patients, a pattern of reduced complexity of cell-free circulating HBV DNA integration sites was found in urine before the removal of HCC, as compared to urine collected after surgery suggesting reduced complexity of HBV DNA integration sites in HBV-HCC patients. A similar NGS analysis of the urine of hepatitis and cirrhosis patients is in progress. Overall, if successful this study will demonstrate the potential of detecting reduced complexity of HBV DNA integration sites as a biomarker for HBV-HCC screening and provide insight to the involvement of HBV DNA integration sites in HBV-HCC carcinogenesis. Citation Format: Selena Lin, Surbhi Jain, Batbold Boldbaatar, Timothy Block, Wei Song, Ying-Hsiu Su. Comprehensive analysis of the complexity of HBV DNA integration sites in the circulation of patients with HBV-related liver disease. [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 876. doi:10.1158/1538-7445.AM2014-876

Abstract 888: Development of a urine DNA based marker panel for early detection of liver cancer

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

ChimericSeq: An open-source, user-friendly interface for analyzing NGS data to identify and characterize viral-host chimeric sequences

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.

Abstract 5290: ChimericSeq: an easy-to-use program for discovery and analysis of integration events from NGS data

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.

Abstract 4934: Detection of HBV-host junction DNA sequences in urine of patients with hepatocellular carcinoma

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.

Abstract 1568: Detection of HBV-host junction DNA sequences in urine of patients with HBV-infected HCC

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-JS9s 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-JS9s 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-JS9s 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-JS9s 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-JS9s 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

Abstract 1561: Detection of colorectal cancer-associated genetic and epigenetic alterations in urine of patients with CRC

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

Abstract 1158: Detection of clonally expanded HBV DNA integration sites as a marker for early detection of HBV related HCC.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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

Abstract 715: Detection of a p53 codon 249 hotspot mutation in the urine of the patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the 5 th 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 ( 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