Characterization of Urinary Biomarkers of Hepatocellular Carcinoma Using Magnetic Resonance Spectroscopy in a Nigerian Population Mohamed I. F. Shariff,*,† Nimzing G. Ladep,‡ I. Jane Cox,§ Horace R. T. Williams,† Edith Okeke,‡ Abraham Malu, Andrew V. Thillainayagam,† Mary M. E. Crossey,† Shahid A. Khan,† Howard C. Thomas,† and Simon D. Taylor-Robinson† Liver Unit, Department of Medicine, Imperial College London, 10th Floor QEQM Building, St. Mary’s Hospital Campus, South Wharf Road, London W2 1NY, United kingdom, Department of Medicine, Jos University Teaching Hospital, Plateau State, Nigeria, and Imaging Sciences Department, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0HS, United Kingdom Received November 18, 2009
Hepatocellular carcinoma (HCC) is the commonest primary hepatic malignancy worldwide. Current serum diagnostic biomarkers, such as R-fetoprotein, are expensive and insensitive in early tumor diagnosis. Urinary biomarkers differentiating HCC from chronic liver disease would be practical and widely applicable. Using an 11.7T nuclear magnetic resonance system, urine was analyzed from three well-matched subject groups, collected at Jos University Teaching Hospital (JUTH), Nigeria. Multivariate factor analyses were performed using principal components analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). All patients were of Nigerian descent: 18 hepatitis B surface antigen (HBsAg)-positive patients with HCC, 10 HBsAg positive patients with cirrhosis, and 15 HBsAg negative healthy Nigerian controls. HCC patients were distinguished from healthy controls, and from the cirrhosis cohort, with sensitivity/specificity of 100%/93% and 89.5%/88.9%, respectively. Metabolites that most strongly contributed to the multivariate models were creatinine, carnitine, creatine and acetone. Urinary 1 H MRS with multivariate statistical analysis was able to differentiate patients with HCC from normal subjects and patients with cirrhosis. Creatinine, carnitine, creatine and acetone were identified as the most influential metabolites. These findings have identified candidate urinary HCC biomarkers which have potential to be developed as simple urinary screening tests for the clinic. Keywords: Hepatocellular carcinoma • metabonomics • urinary biomarkers • magnetic resonance spectroscopy • hepatitis B virus
Introduction Hepatocellular carcinoma (HCC) is the third commonest cause of cancer death worldwide.1-3 The majority of cases of HCC occur in sub-Saharan Africa and Asia, where several countries display a high incidence of over 20 cases per 100 000 population. The majority of HCCs develop on the background of cirrhosis. Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) are the most important risk factors for HCC with distinct geographical patterns of HCC distribution, reflecting the prevalence of these infections.4 In Nigeria, the prevalence of HBsAg has been shown to be as high as 30% in some areas, with HCC reported to be the most prevalent cause of cancer death in the general population (median age of diagnosis 45 years).5,6 This is primarily due to high prevalence of HBV carriage, which is * Corresponding Author: Dr. Mohamed Shariff; Liver Unit, Faculty of Medicine, Imperial College London, 10th Floor QEQM Building, St. Mary’s Hospital, South Wharf Road, London, W2 1NY. Tel: (44) (0)207 886 6454. Fax: (44) (0) 207 749 3436. E-mail:
[email protected]. † Department of Medicine, Imperial College London. ‡ Department of Medicine, Jos University Teaching Hospital. § Imaging Sciences Department, Imperial College London.
1096 Journal of Proteome Research 2010, 9, 1096–1103 Published on Web 12/08/2009
vertically acquired at birth, or horizontally acquired in infancy. Population-based studies have shown mortality and morbidity benefit from screening for HCC using serum R-fetoprotein (AFP),7 a fetal glycoprotein normally undetectable soon after birth. Most HCC secrete AFP, but as a tumor marker, AFP has poor sensitivity and specificity of less than 70%.8-10 In addition, serum AFP testing is prohibitively expensive and unavailable in many parts of Africa. Other serum tumor markers for HCC such as des-gamma-carboxyprothrombin, anti-p53, gammaglutamyl-transpeptidase, and isoferritin have been found to be no more effective than serum AFP for HCC diagnosis.11,12 A sensitive and specific urinary biomarker of HCC would be practical and widely applicable, not only in the developing world, but also as a surveillance tool for patients with chronic liver disease in the developed world. “Metabonomics” describes the study of metabolic responses to physiological, drug and disease stimuli.13 1H MRS is the most commonly used analytical method for metabonomic studies: comprehensive metabolic profiles may be generated from biofluids including urine,14,15 serum,16-18 bile19 and intact tissue.20 The defining characteristics of patients with a particular disease may be a combination of metabolite alterations, 10.1021/pr901058t
2010 American Chemical Society
research articles
Characterization of Urinary Biomarkers of HCC Table 1. Subject Characteristics characteristic
healthy controls
HCC
cirrhosis
p-value
n Median age (range) (years) Male n (%) Ethnicity HBsAg +
14 37 (27-80) 7/7 (50%) Nigerian 0/14
18 46.5 (25-85) 12/18 (66.7%) Nigerian 17/18 (94.4%)
10 37 (23-62) 8/10 (80%) Nigerian 10/10 (100%)
0.44 0.22 50% e30 g50 Present
30
