Onco-proteogenomics identifies urinary S100A9 and GRN as potential combinatorial biomarkers for early diagnosis of hepatocellular carcinoma Chun-Hao Huang a,b,1 , Chao-Jen Kuo a,1 , Shih-Shin Liang c , Shu-Wen Chi a , Edward Hsi d , Chi-Chao Chen e , King-Teh Lee f, ⁎, Shyh-Horng Chiou a,b,g, ⁎⁎ a Quantitative Proteomics Center and Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan b Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan c Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan d Department of Genome Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan e Physiology Biophysics and Systems Biology Program, Weill Graduate School of Medical Sciences, Cornell University, NY 10065, USA f Division of Hepatobiliary Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan g Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung 807, Taiwan abstract article info Article history: Received 2 January 2015 Received in revised form 23 February 2015 Accepted 24 February 2015 Available online 3 March 2015 Keywords: Hepatocellular carcinoma Proteogenomics Quantitative proteomics Urinary biomarkers Protein S100-A9 Granulin Hepatocellular carcinoma (HCC), the major type of liver cancer, is among the most lethal cancers owing to its aggressive nature and frequently late detection. Therefore, the possibility to identify early diagnostic markers could be of significant benefit. Urine has especially become one of the most attractive body fluids in biomarker discovery as it can be obtained non-invasively in large quantities and is stable as compared with other body fluids. To identify potential protein biomarker for early diagnosis of HCC, we explored protein expression profiles in urine from HCC patients and normal controls (n = 44) by shotgun proteomics using nano-liquid chromatog- raphy coupled tandem mass spectrometry (nanoLC–MS/MS) and stable isotope dimethyl labeling. We have systematically mapped 91 proteins with differential expressions (p b 0.05), which included 8 down-regulated microtubule proteins and 83 up-regulated proteins involved in signal and inflammation response. Further integrated proteogenomic approach composed of proteomic, genomic and transcriptomic analysis identified that S100A9 and GRN were co-amplified (p b 0.001) and co-expressed (p b 0.01) in HCC tumors and urine samples. In addition, the amplifications of S100A9 or GRN were found to be associated with poor survival in HCC patients, and their co-amplification was also prognosed worse overall survival than individual ones. Our results suggest that urinary S100A9 and GRN as potential combinatorial biomarkers can be applied to early diagnosis of hepatocellular carcinoma and highlight the utility of onco-proteogenomics for identifying protein markers that can be applied to disease-oriented translational medicine. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Hepatocellular carcinoma (HCC), the most common type of liver cancer, is the third leading cause of cancer related mortality worldwide [1]. A major etiological factor for HCC is cirrhosis, frequently caused by chronic infection with hepatitis B or C virus (HBV, HCV), nonalcoholic fatty liver disease, and alcohol abuse [2]. Many patients detected with HCC were initially found to have chronic liver disease and cirrhosis, leading to an increase in the replacement of normal tissue with fibrous tissue leading to the development of HCC [3]. HCC encompasses different pathological manifestations and etiology coupled with multiple genomic aberrations leading to high heterogeneity and intrac- table treatment. Although the introduction of the multikinase inhibitor sorafenib represents the biggest therapeutic advancement in the past decade, extending life expectancy from 8 to 11 months [4], its limited therapeutic efficacy emphasizes an urgent need for improved targeted therapies, such as CDK9 [5] or MET [6] inhibition. Moreover, these hurdles in developing therapies against HCC highlight the importance of the early detection and biomarker development for non-invasive diagnosis and prognosis. An abdominal ultrasonography and measurement of serum alpha- fetoprotein (AFP) are two of major tools to detect HCC at an early stage [7]. However, the need of an operator's expertise generally BBA Clinical 3 (2015) 205–213 Abbreviations: HCC, hepatocellular carcinoma; S100A9, protein S100-A9; GRN, granulins; nanoLC–MS/MS, nano-liquid chromatography coupled tandem mass spectrome- try; D/H, deuterium/hydrogen labeling; HILIC, hydrophilic interaction chromatography ⁎ Corresponding author. Tel.: +886 7 3121101x7651; fax: +886 7 3216992. ⁎⁎ Correspondence to: S.-H. Chiou, Quantitative Proteomics Center and Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. Tel.: +886 7 3220377; fax: +886 7 3133434. E-mail addresses: ktlee@kmu.edu.tw (K.-T. Lee), shchiou@kmu.edu.tw (S.-H. Chiou). 1 Chun-Hao Huang and Chao-Jen Kuo contributed equally to this paper. http://dx.doi.org/10.1016/j.bbacli.2015.02.004 2214-6474/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Contents lists available at ScienceDirect BBA Clinical journal homepage: http://www.journals.elsevier.com/bba-clinical/