Blood Gene Signature for Early Hepatocellular Carcinoma Detection in Patients With Chronic Hepatitis B Haniza Omar, MD,* Chun Ren Lim, PhD,w Samuel Chao, MSc,z Michelle Mei Lin Lee, MSc,w Chin Wei Bong, PhD,w Edie Jian Jiek Ooi, BSc,w Choon Geok Yu, MD,w Soek Siam Tan, MD,* Muhammad Radzi Abu Hassan, MD,y Jayaram Menon, MD,8 Raman Muthukaruppan, MD,8 Mandeep Singh, MD,8 Nik Azim Nik Abdullah, MD,z Boon Phoe Ooi, MD,# Robert Phooi Huat Ding, MD,** Eng Joo Low, MD,ww Francis Tan, MD,ww David Novak, MSc,z David F. Harris, MD,zz Hengxuan Yang, MD,z Ismail Merican, MD,* and Choong-Chin Liew, PhDzyy Purpose: Up to 25% of chronic hepatitis B (CHB) patients even- tually develop hepatocellular carcinoma (HCC), a disease with poor prognosis unless detected early. This study identifies a blood- based RNA biomarker panel for early HCC detection in CHB. Materials and Methods: A genome-wide RNA expression study was performed using RNA extracted from blood samples from Malaysian patients (matched HCC, CHB, controls). Genes differ- entiating HCC from controls were selected for further testing using quantitative real-time polymerase chain reaction. Finally, a 6-gene biomarker panel was identified and characterized using a training set (cohort I = 126), and tested against 2 test sets (cohort II = 222; cohort III = 174). The total number of samples used for each group is: HCC + CHB = 143, CHB = 211, control = 168. Results: Our gene panel displays a consistent trend distinguishing HCC from controls in our test sets, with an area under receiver- operating characteristic curve of 0.9 in cohort III. Our independent test set (cohort III) showed that the gene panel had a sensitivity of 70% with a specificity of 92%. The biomarker profile for HCC was consistently detected in a small subgroup of CHB patients, thus potentially predicting early, preclinical cases of cancer that should be screened more intensively. Conclusion: The biomarkers identified in this study can be used as the basis of a blood-based test for the detection of early HCC in CHB. Key Words: hepatocellular carcinoma, gene expression, diagnostics, blood, biomarkers (J Clin Gastroenterol 2015;49:150–157) H epatocellular carcinoma (HCC) is the fifth most com- mon cancer in men worldwide and the third leading cause of cancer-related death. According to GLOBOCAN estimates, approximately 750,000 new cases were diagnosed in 2008 and 700,000 people died of liver cancer. 1 HCC disproportionately affects populations in East and Southeast Asia, which account for >70% of all newly diag- nosed cases. The high prevalence of the disease in Asia can be explained by the strong linkage between liver cancer and chronic hepatitis B (CHB) virus infection. Of the world’s 400 million current chronic carriers of hepatitis B virus, three quarters live in China and Southeast Asia, 2 and some 15% to 25% of those infected will eventually develop HCC. 3,4 Other risk factors for HCC include hepatitis C virus infection, alcohol-related cirrhosis, and nonalcoholic steatohepatitis. The incidence of the disease is increasing in developed coun- tries. For example, HCC incidence has doubled in the United States over the past 20 years mainly because of immigrations from Asia and currently represents the most rapidly increas- ing cause of cancer-related death in that country. HCC is highly lethal; it develops gradually and has a clinically indolent course in its early stages. It often presents first at a late stage with liver failure or other serious com- plications. At this stage, a cure is no longer feasible; almost all patients who develop late-stage liver cancer will be dead within 1 year. 5 Early-stage HCC is potentially curable. Therefore, the focus in hepatocellular oncology has been the early detec- tion of small lesions more amenable to resection, trans- plantation, or radiofrequency ablation. 6 To this end, sev- eral groups have recommended surveillance programs to monitor at-risk populations. 7,8 The American Association for the Study of Liver Diseases 7 and the Asian Pacific Association for the Study of the Liver 8 recommend sur- veillance of certain defined populations at high risk of developing liver cancer, such as Asian men and women with CHB infection over age 40 and age 50, respectively. In this study we describe a blood-based gene-expres- sion profile for HCC which is particularly directed to surveillance strategies for HCC in CHB patients. Our hypothesis is that a subgroup of CHB patients has already Received for publication August 12, 2013; accepted February 4, 2014. From the *Selayang Hospital, Lebuhraya Selayang-Kepong, Batu Caves, Selangor Darul Ehsan; wGeneNews (Malaysia) Sdn. Bhd., Mount Miriam Cancer Hospital, Tanjung Bungah; **Island Hos- pital, Penang; ySultanah Bahiyah Hospital, Jalan Langgar, Alor Setar, Kedah; 8Queen Elizabeth Hospital, Kota Kinabalu, Sabah; zUmum Kuching Sarawak Hospital, Jalan Tun Ahmad Zaidi Adruce, Kuching, Sarawak; #Penang Hospital, Jalan Residensi; wwLam Wah Ee Hospital, Pulau Pinang; zzGeneNews Diagnostics, Office/Lab 9, Biotechnology Incubation Centre, Technology Park Malaysia, Kuala Lumpur, Malaysia; zGeneNews Ltd., Richmond Hill, ON, Canada; and yyBrigham and Women’s Hospital, Harvard Medical School, Boston, MA. Supported by GeneNews Ltd. and the Malaysian Ministry of Health (NMRR-09-317-3991 Ministry of Health, Government of Malaysia). C.R.L., H.Y., S.C., C.W.B., C.G.Y., E.J.J.O., M.M.L.L., D.F.H., D.N., and C.-C.L. are employees of GeneNews Ltd., that also sponsored this research. C.-C.L. is the Chief Scientist of GeneNews Ltd, and holds shares in the company. The remaining authors declare that they have nothing to disclose. Reprints: Choong-Chin Liew, PhD, GeneNews Ltd., 2 East Beaver Creek Road, Building 2, Richmond Hill, ON, Canada L4B 2N3 (e-mail: cliew@genenews.com). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved. ORIGINAL ARTICLE 150 | www.jcge.com J Clin Gastroenterol  Volume 49, Number 2, February 2015