Published OnlineFirst March 14, 2016; DOI: 10.1158/0008-5472.CAN-15-3199 Cancer Integrated Systems and Technologies Research Acetylcarnitine Is a Candidate Diagnostic and Prognostic Biomarker of Hepatocellular Carcinoma Yonghai Lu1, Ning Li2, Liang Gao3, Yong-Jiang Xu4, Chong Huang2, Kangkang Yu2, Qingxia Ling2, Qi Cheng2, Shengsen Chen2, Mengqi Zhu2, Jinling Fang1, Mingquan Chen2, and Choon Nam Ong1,3 Abstract The identification of serum biomarkers to improve the diag- carcinoma tumors and matched normal tissues. Post hoc analysis nosis and prognosis of hepatocellular carcinoma has been elusive to evaluate serum diagnosis and progression potential further to date. In this study, we took a mass spectroscopic approach to confirmed the diagnostic capability of serum acetylcarnitine. characterize metabolic features of the liver in hepatocellular Finally, an external validation in an independent batch of 58 carcinoma patients to discover more sensitive and specific serum samples (18 hepatocellular carcinoma patients, 20 liver biomarkers for diagnosis and progression. Global metabolic cirrhosis patients, and 20 healthy individuals) verified that profiling of 50 pairs of matched liver tissue samples from hepa- serum acetylcarnitine was a meaningful biomarker reflecting tocellular carcinoma patients was performed. A series of 62 hepatocellular carcinoma diagnosis and progression. These find- metabolites were found to be altered significantly in liver tumors; ings present a strong new candidate biomarker for hepatocellular however, levels of acetylcarnitine correlated most strongly with carcinoma with potentially significant diagnostic and prognostic tumor grade and could discriminate between hepatocellular capabilities. Cancer Res; 76(10); 2912–20. Ó2016 AACR. Introduction early and accurate diagnosis of hepatocellular carcinoma, there have been a number of previous investigations on gene expres- Hepatocellular carcinoma is the most common type of pri- sion, miRNA profiles, and protein expression of hepatocellular mary liver cancer and has become a major global health issue. carcinoma (7–12). Recently, metabolomics investigations have As the third leading cause of cancer-related deaths, hepatocel- provided a new angle for biomarker discovery. lular carcinoma causes more than half a million deaths world- Metabolomics is a powerful high-throughput platform that wide per year (1). Furthermore, it is worth noting that 5-year measures low molecular weight metabolites in biologic sam- survival rate for patients with hepatocellular carcinoma is less ples, and it has recently been used to elucidate biochemical than 5% due to its fast development and high malignancy (2, 3). pathways of various human diseases and simultaneously offers a-Fetoprotein (AFP), as the most widely used tumor marker in an opportunity for discovering novel biomarkers (13, 14). clinical practice, is not an effective marker for hepatocellular Current metabolomics studies on hepatocellular carcinoma are carcinoma diagnosis due to its poor sensitivity and low spec- mainly focused on identifying significant metabolites in serum/ ificity (4–6). To explore more sensitive and specific markers for urine samples with the aim of revealing systemic metabolic changes caused by hepatocellular carcinoma and finding poten- 1School of Public Health, National University of Singapore, Singapore. tial biomarkers with clinical application (15–22). There has 2Department of Infectious Diseases and Hepatology of Huashan been limited analysis of the effects of hepatocellular carcinoma 3 Hospital, Fudan University, Shanghai, China. NUS Environmental on liver metabolism (23, 24). Although serum/urine metabolic Research Institute, National University of Singapore, Singapore. 4Key Laboratory of Insect Developmental and Evolutionary Biology, Insti- profiling can reflect circulating metabolic characterization in tute of Plant Physiology and Ecology, Shanghai Institutes for Biolog- hepatocellular carcinoma, targeting ability of these analyses ical Sciences, Chinese Academy of Sciences, Shanghai, China. is still limited. Liver is the most important metabolic organ in Note: Supplementary data for this article are available at Cancer Research human body, and it is the primary target lesion of hepatocel- Online (http://cancerres.aacrjournals.org/). lular carcinoma. Compared with serum and urine, liver tissue Y. Lu and N. Li contributed equally to this article. metabolic profiling could provide more direct information on Corresponding Authors: Choon Nam Ong, National University of Singapore, metabolic changes during hepatocellular carcinoma develop- Tahir Foundation Building, 12 Science Drive 2, 117549, Singapore. Phone: 656- ment, with higher targeting ability and specificity. Thus, early 874-4982; Fax: 656-779-1489; E-mail: [email protected]; and Mingquan Chen, and accurate detection of metabolic changes in hepatocellular Department of Infectious Diseases and Hepatology of Huashan Hospital, Fudan carcinoma liver tumor tissues would be very helpful to reveal University, 12 Wulumuqi Zhong Road, Shanghai 200040, China. Phone: 8618- pathologic shifts and development of the disease, providing 0186-95670; Fax: 8621-6246-0195; E-mail: [email protected] patients with early diagnosis and in-time treatment. doi: 10.1158/0008-5472.CAN-15-3199 The primary objective of this study is to elucidate the global Ó2016 American Association for Cancer Research. metabolic features of hepatocellular carcinoma tumors by using 2912 Cancer Res; 76(10) May 15, 2016 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst March 14, 2016; DOI: 10.1158/0008-5472.CAN-15-3199 Acetylcarnitine as a Biomarker of Hepatocellular Carcinoma an integrated mass spectrometry–based nontargeted metabolo- patients were HBsAg positive, and 3 hepatocellular carci- mics approach. Briefly, 50 pairs of matched liver tissues of noma and 6 liver cirrhosis patients were anti-HCV positive. hepatocellular carcinoma patients, including hepatocellular car- The experimental procedures of targeted metabolomics were cinoma tissues (HCT), adjacent noncancerous tissues (ANT), and described in Supplementary Materials and Methods. The addi- distal noncancerous tissues (DNT), were analyzed by liquid tional 58 participants were recruited from the Jurong People's chromatography quadrupole time-of-flight mass spectrometry Hospital (Jiangsu, China) between April 2013 and June 2014. and gas chromatography quadrupole time-of-flight mass spec- All participants voluntarily joined this study, gave written in- trometry to investigate altered metabolites and pathways in formed consent, and completed a questionnaire that provided hepatocellular carcinoma tumors. When examining metabolic demographical information, including age, gender, lifestyle changes, we hope to be able to identify potential biomarkers that factors, and medical family history. The study protocol was could be used for the diagnosis of hepatocellular carcinoma. The approved by the Institutional Review Boards at the Huashan diagnostic potential of differential metabolites discovered in Hospital, Jurong People's Hospital, and National University of hepatocellular carcinoma tumors was further evaluated in two Singapore (Singapore) and was conducted in accordance with batches of serum samples: one from a randomly selected sub- the Helsinki Declaration of 1964, as revised in 1975. group of these 50 patients and the other one from a separate batch of 58 subjects. Sample preparation Tissue samples in discovery set: A piece of tissue (5 Æ 0.05 mg) Materials and Methods was mixed with 300 mL of ice-cold methanol/water (4:1, v/v), containing 20 mg/mL FMOC-glycine as internal standard. The Chemicals mixture was homogenized using a TissueLyser System (25 Hz, HPLC grade acetonitrile, methanol, and formic acid were 10 minutes). After that, the sample was placed on ice for purchased from Merck. Distilled water was purified "in-house" 20 minutes and then centrifuged at 4C (14,000 rpm for 10 min- using a Milli-Q System (Millipore). N-methyl-N-trimethylsilyl- utes). The supernatant fraction was collected and divided into trifluoroacetamide (MSTFA) and standard compounds, including two parts: one (100 mL) for LC/MS analysis and the other one D-galactose, D-glucose, L-valine, L-serine, fumarate, malate, stea- (10 mL) for gas chromatography–mass spectrometry (GC-MS) rate, uric acid, glycocholic acid, cortisone, myo-inositol, ribofla- analysis. For GC-MS analysis, the 10-mL supernatant was dried vin, and N-(9-fluorenylmethoxycarbonyl)-glycine (FMOC-gly- under nitrogen and derivatized with methoxyamine (50 mg/mL cine), were purchased from Sigma-Aldrich. Ammonium formate in pyridine) and subsequent trimethylsilylation with MSTFA. and oleate were purchased from Fluka. Isotopically labeled Serum samples in post hoc analysis: The serum specimen (100 mL) d3-acetylcarnitine, d3-propionylcarnitine, and d3-palmitoylcar- was diluted with 300 mL of ice-cold methanol with 20 mg/mL nitine were purchased from Cambridge Isotope Laboratories. FMOC-glycine. The mixture was shaken vigorously for 30 sec- onds. After centrifugation at 14,000 rpm for 10 minutes at 4C, Participants and sample collection the supernatant fraction was collected for LC/MS analysis. Fifty patients with hepatocellular carcinoma (38 males and As part of the system conditioning and quality control (QC) 12 females) were recruited at the Huashan Hospital (Shanghai, process, pooled QC samples were prepared by