Abstract 5699: Detection of genetic and epigenetic DNA markers in urine for the early detection of liver cancer
Authors: Jain Surbhi , Steffen Jamin D. , Su Yih-Ping , Wang Jeremy , Chang Ting-Tsung , Hu Chi-Tan , Song Wei , Su Ying-Hsiu 
Published: July 2017

Abstract
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