Potential conflict of interest: Nothing to report.
Supported by grants from the Key Program of the Natural Science Foundation of the State (Grant No. 90709019), the National Specific Program on the Subject of Public Welfare (Grant No. 200807014), the National Program for Key Basic Research Projects in China (Grant No. 2005CB523406), and the National Key Program on the Subject of Drug Innovation (Grant No. 2009ZX09502-005).
Hepatocellular carcinoma (HCC) is the commonest primary hepatic malignancy and the third most common cause of cancer-related death worldwide. Incidence remains highest in the developing world and is steadily increasing across the developed world. Current diagnostic modalities, of ultrasound and α-fetoprotein, are expensive and lack sensitivity in tumor detection. Because of its asymptomatic nature, HCC is usually diagnosed at late and advanced stages, for which there are no effective therapies. Thus, biomarkers for early detection and molecular targets for treating HCC are urgently needed. Emerging high-throughput metabolomics technologies have been widely applied, aiming at the discovery of candidate biomarkers for cancer staging, prediction of recurrence and prognosis, and treatment selection. Metabolic profiles, which are affected by many physiological and pathological processes, may provide further insight into the metabolic consequences of this severe liver disease. Small-molecule metabolites have an important role in biological systems and represent attractive candidates to understand HCC phenotypes. The power of metabolomics allows an unparalleled opportunity to query the molecular mechanisms of HCC. This technique-driven review aims to demystify the metabolomics pathway, while also illustrating the potential of this technique, with recent examples of its application in HCC. (HEPATOLOGY 2013)
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide.1 The high morbidity rate associated with this cancer is mainly linked to the late diagnosis, when therapy is no longer effective, and this is particularly true for high-risk patients, such as hepatitis B and C-infected individuals, and is not easily discovered in its initial stage. Early diagnosis of this leading cause of mortality is therefore highly important.2 The burden of HCC is growing worldwide and with it a more desperate need for better tools to detect, diagnose, and monitor the disease is required. Current screening methodologies for liver cancer in at-risk patients rely on measuring the serum level of α-fetoprotein (AFP), a biomarker, as well as ultrasound imaging.3 AFP's sensitivity is very limited since many other liver diseases can result in a very high blood level of AFP similar to that observed in HCC.4, 5 Therefore, more sensitive markers of disease are needed, particularly for the early detection of HCC disease, and highly sensitive and specific biomarkers such as primary indicators are relatively more useful.
Emerging metabolomics provides a powerful platform for discovering novel biomarkers and biochemical pathways to improve diagnostic, prognostication, and therapy. Metabolomics represent a global understanding of the metabolite complement of integrated living systems and dynamic responses to the changes of both endogenous and exogenous factors and has many potential applications and advantages for research into complex systems.6-8 The general procedures in which metabolomics is used for diagnosis and biomarker discovery are shown in Fig. 1. One area of considerable interest in the field of metabolomics is to detect potential biomarkers associated with diseases, and metabolic profiling could provide global changes of endogenous metabolites of patients.9 It involves the comprehensive profiling of the full complement of low molecular weight compounds in a biological system. By applying advanced analytical and statistical tools, the “metabolome” is mined for biomarkers that are associated with the state of HCC.10 It may help to understand the mechanism of HCC occurrence and progression on the metabolic level and provide information for the identification of early and differential marker metabolites for HCC. Metabolomics offers potential advantages that classical diagnostic approaches do not, based on the following discovery of clinically relevant biomarkers that are simultaneously affected by HCC.11
Analyzing and verifying the specifically early biomarkers of a disease, metabolomics enables us to better understand pathological processes, substance metabolic pathways.12 Compared with traditional diagnostic methods, even small changes of metabolites can help to detect early pathologic changes more sensitively. These large-scale analyses of metabolites are intimately bound to novel mass spectrometry (MS) technology analyzers in combination with hyphenated techniques.13 Approaches of either high-performance liquid chromatography (HPLC) or ultra-performance liquid chromatography (UPLC) online with MS have recently been employed and became increasingly popular.14, 15 Over the last few years there has been a rapidly growing number of metabolomic applications aimed at finding biomarkers that could assist diagnosis, provide therapy guidance, and evaluate response to therapy for a particular HCC.16-18 Today, the improved efficiency and accuracy of metabolomic biomarker discovery technologies are turning diagnostics into a clinical reality.19, 20 The future goals for metabolomics are the validation of existing biomarkers, in terms of mechanism and translation to man, together with a focus on characterizing the individual healthcare. Thus, in this review particular attention will be paid to the past successes in applications of state-of-the-art technology on metabolomics to contribute to low-molecular-weight metabolites discovery in HCC diagnosis research.
Metabolomics has recently become one of the cornerstones of postgenomics for the quantitative analysis and unbiased identification of small molecular metabolites that could yield important information about a person's health or disease, some of which may be molecular targets for therapeutic intervention.21 More specifically, metabolomics is a global and noninvasive analysis of biomarkers that are indicators of normal biological or pathogenic process, or response to therapeutic intervention, thereby helping to monitor treatment response.22 Recent advances in metabolomics might provide various types of novel tumor markers for HCC.23 The majority of HCC occurs on a background of cirrhosis, principally caused by two major risk factors, chronic hepatitis B and hepatitis C infection.24 Early diagnosis can improve survival rates and there have been recent advances in technology that have enabled early identification of the process of HCC.25 Therefore, better biomarkers with higher selectivity and sensitivity are needed for the early diagnosis of HCC in high-risk patients. Although the clinical availability of these tumor markers is important, the molecular mechanisms underlying the production of tumor markers requires further clarification. The metabolomic approach may provide insight into the discovery and identification of new diagnostic biomarkers for HCC and associated diseases.
Bringing Metabolomics into the Forefront of HCC Research
Metabolomics is the assessment of global metabolic profiles and biomarkers in order to distinguish between diseased and nondiseased status information.26 Metabolite changes that are observed in diseased individuals as a primary indicator have been an important part of clinical practice.27 Metabolomics is the comprehensive assessment of endogenous metabolites and attempts to systematically identify and quantify metabolites from a biological sample. Metabolomics analysis will allow the testing of global or targeted signaling networks to determine the on-target efficacy of combination therapies that inhibit signaling pathways in series or in parallel. Potential roles for metabolomics in the clinical trials of HCC include biomarker discovery and validation, molecular target discovery, therapy decisions, and patient monitoring. Metabolomics has already shown promise in identifying metabolite-based biomarkers in HCC as biochemical profiling tools to provide important insight into the biology of HCC.28 In summary, integration of metabolomics-based diagnostic principles into the diagnosis of HCC would make it possible to interpret and explore the essence of personalized medicine, and might be the direction to enable a revolution for future healthcare; perhaps it is time to embrace the arrival of the “HCC-OMICS” era.
Biomarkers and Metabolomics Studies on HCC
A biomarker is a biological molecule found in blood or other body fluids or tissues that can be a sign of a disease. Biomarkers can be used routinely for population screening, prognosis, monitoring of therapy, and prediction of therapeutic response in HCC. A number of marker metabolites for diagnosis and prognosis of HCC have been reported.29, 30 A UPLC-MS-based metabolomics approach has been used to characterize serum profiles from HCC, liver cirrhosis (LC), and healthy subjects, and the accuracy of UPLC-MS profiles and AFP levels were compared for their use in HCC diagnosis.31 Thirteen potential biomarkers were identified that suggest there were significant disturbances of key metabolic pathways in HCC patients. Of note, glycochenodeoxycholic acid was suggested to be an important indicator for HCC diagnosis and disease prognosis. Metabolomics in combination with AFP levels could be an efficient and convenient tool for early diagnosis and screening of HCC. A nontarget metabolomics method was to find the potential biomarkers from the rat HCC disease model and test their usefulness in early human HCC diagnosis.32 Three metabolites, taurocholic acid, lysophosphoethanolamine 16:0, and lysophosphatidylcholine 22:5 were defined as “marker metabolites,” which can be used to distinguish the different stages of hepatocarcinogenesis and represent the abnormal metabolism during the progress of HCC in patients. Moreover, they were also effective for the discrimination of all HCC and chronic LD patients, which could achieve high sensitivity and specificity, better than those of AFP. Late diagnosis of HCC is one of the primary factors for poor survival of patients. Thereby, identification of sensitive and specific biomarkers for HCC early diagnosis is of great importance in biological medicine. In a study, serum metabolites of the HCC patients and healthy controls were investigated using improved LC/MS.33 Clustering analysis based on principal component analysis showed a clear separation between HCC patients and healthy individuals. The serum metabolite, 1-methyladenosine, was identified as the characteristic metabolite for HCC. These results indicate that the metabolomics method has the potential of finding biomarkers for the early diagnosis of HCC.
Identification of novel biomarkers in HCC remains impeded primarily because of the heterogeneity of the disease in clinical presentations as well as the pathophysiological variations derived from underlying conditions such as cirrhosis and steatohepatitis. A study was conducted to search for potential metabolite biomarkers of human HCC using serum and urine metabolomics approach.34 Metabolite profiling was performed by gas chromatography (GC)-MS and UPLC-MS in conjunction with univariate and multivariate statistical analyses. Forty-three serum metabolites and 31 urinary metabolites were identified in HCC patients involving several key metabolic pathways. Differentially expressed metabolites in HCC subjects, such as bile acids, histidine, and inosine are of great statistical significance and high fold changes, which warrant further validation as potential biomarkers for HCC. HCC patients with AFP values lower than 20 ng/mL was successfully differentiated from healthy controls with an accuracy of 100% using a panel of metabolite markers. This work shows that the metabolomic profiling approach is a promising screening tool for the diagnosis and stratification of HCC patients.
With the technique of metabolomics, GC/MS, urine or serum metabolites can be assayed to explore disease biomarkers. In a recent work, the metabolomic method was used to investigate the urinary metabolic difference between HCC male patients and normal male subjects.35 The urinary endogenous metabolome was assayed using chemical derivatization followed by GC/MS. After GC/MS analysis, 103 metabolites were detected, of which 18 metabolites were shown to be significantly different between the HCC and control groups. A diagnostic model was constructed with a combination of 18 marker metabolites or together with AFP, using principal component analysis and receiver-operator characteristic curves. This noninvasive technique of identifying HCC biomarkers from urine may have clinical utility. Nuclear magnetic resonance (NMR)-based metabolomics was used to characterize metabolic profiles of LC and HCC.36 Compared to healthy humans, LC and HCC sera had higher levels of acetate, n-acetylglycoproteins, pyruvate, glutamine, alpha-ketoglutarate, glycerol, tyrosine, 1-methylhistidine, and phenylalanine, together with lower levels of low-density lipoprotein, isoleucine, valine, acetoacetate, creatine, choline, and unsaturated lipids. A score plot of pattern recognition analysis was capable of distinguishing LC and HCC patients from healthy humans. These results indicate that serum NMR spectra combined with pattern recognition analysis techniques offer an efficient, convenient way of depicting tumor biochemistry, which may be of great benefit to early diagnosis of human malignant diseases. Overall, these results suggested that metabolomic study is a potent and promising strategy for identifying novel biomarkers of HCC.
Metabolomics was used to identify serum biomarkers for HCC in patients with LC, and provided useful insight into HCC biomarker discovery with metabolomics as an efficient and cost-effective platform.37 The results confirmed that metabolites involved in sphingolipid metabolism and phospholipid catabolism such as sphingosine-1-phosphate and lysophosphatidylcholine are up-regulated in sera of HCC versus those with LC. Down-regulated metabolites include those involved in bile acid biosynthesis such as glycochenodeoxycholic acid 3-sulfate, glycocholic acid glycodeoxycholic acid, taurocholic acid, and taurochenodeoxycholate. This work showed that metabolomics is a promising tool to identify candidate metabolic biomarkers for early detection of HCC cases in a high-risk population of cirrhosis patients. Serum metabolome was detected through chemical derivatization followed by GC/MS. The acquired GC/MS data were analyzed by stepwise discriminant analysis and a support vector machine.38 The metabolites including butanoic acid, ethanimidic acid, glycerol, L-isoleucine, L-valine, aminomalonic acid, D-erythrose, hexadecanoic acid, octadecanoic acid, and 9,12-octadecadienoic acid in combination with each other gave the strongest segregation between the two groups. By applying these variables, it provided a diagnostic model that could well discriminate between HCC patients and normal subjects, and appears to be a useful tool in the area of HCC diagnosis.
Discovery of biomarkers is a core research to develop more efficient therapeutic strategies and diagnostic criteria for HCC patients. Therefore, development of biomarkers with higher sensitivity and specificity is expected to emerge. Recent advances in metabolomic technology made it possible to identify the metabolites in clinical samples and thus extensive efforts are now being made to search for the biomarkers.39 Metabolomics represents an emerging and powerful discipline concerned with comprehensive analysis of small molecules and provides a powerful approach to discover biomarkers in biological systems.40 Therefore, these observations support that metabolomics is an ideal approach to reveal the scientific and intrinsic connotation of liver syndromes. In conclusion, the metabolomics study discriminated patients with high sensitivity and specificity, thereby demonstrating this model as a potential tool for use in medical practice in the near future.
Conclusions and Future Perspectives
Metabolomics has significantly increased in recent years and enabled mapping of early biochemical changes in disease and hence can provide an opportunity to develop predictive biomarkers that can trigger earlier interventions. High-throughput metabolomics approaches have revolutionized HCC research and moved it into a stage where many metabolites can be studied simultaneously. Valuable information regarding HCC development, therapy, and diagnosis can now be obtained with microliter sample volumes. This approach also opens the door to biomarker discovery, disease diagnosis, and treatment. So far, biomarker discovery using metabolomic approaches is in its technology-optimization stage. Any findings associated with relevance to HCC, once passed to the clinical level, will be eventually combined with other diagnosis approaches to hopefully reach the 100% detection level for high-risk patients. Metabolomic research has the potential to generate novel noninvasive diagnostic tests, based on biomarkers of disease, which are simple and cost-effective yet retain high sensitivity and specificity characteristics. Metabolomics analysis has also given us resources with which to discover possible early markers for the presence of HCC and for assessing progression throughout the course of treatment and has aided the discovery of possible prognostic indicators of outcome and disease response to therapy.