1H-NMR Based Serum Metabolomics Highlights Different Specific Biomarkers between Early and Advanced Hepatocellular Carcinoma Stages

Abstract

The application of non-targeted serum metabolomics profiling represents a noninvasive tool to identify new clinical biomarkers and to provide early diagnostic differentiation, and insight into the pathological mechanisms underlying hepatocellular carcinoma (HCC) progression. In this study, we used proton Nuclear Magnetic Resonance (¹H-NMR) Spectroscopy and multivariate data analysis to profile the serum metabolome of 64 HCC patients, in early (n = 28) and advanced (n = 36) disease stages. We found that ¹H-NMR metabolomics profiling could discriminate early from advanced HCC patients with a cross-validated accuracy close to 100%. Orthogonal partial least squares discriminant analysis (OPLS-DA) showed significant changes in serum glucose, lactate, lipids and some amino acids, such as alanine, glutamine, 1-methylhistidine, lysine and valine levels between advanced and early HCC patients. Moreover, in early HCC patients, Kaplan-Meier analysis highlighted the serum tyrosine level as a predictor for overall survival (OS). Overall, our analysis identified a set of metabolites with possible clinical and biological implication in HCC pathophysiology.

Keywords: NMR; OPLS-DA; hepatocellular carcinoma; metabolomics; radiofrequency; sorafenib.

Figure 1

Typical proton Carr–Purcell–Meiboom–Gill nuclear magnetic resonance (¹H CPMG NMR) spectra in the (a) aromatic, (b) sugars and (c) aliphatic regions, with some identified metabolites for the different groups of advanced (ADV) and early (EAR) hepatocellular carcinoma (HCC) patients referred to the specific sample spectra in the figure.

Figure 2

Serum metabolic profile discriminates between advanced and early HCC patients. (a) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plot (R²X = 0.75, R²Y = 0.58, Q² = 0.38 p[CV-ANOVA] = 9.00484 × 10⁻⁵ (Cohen’s coefficient (K) equal to 0.968, Table S1) and (b) the corresponding S-line plot for the model displaying the discriminant metabolites and the related predictive loadings (variables in the proton Nuclear Magnetic Resonance (¹H-NMR) spectra. Variables are colored according to the correlation scaled loading (p(corr)). The arrows indicate the metabolite content increase for the advanced (ADV) and early (EAR) group.

Figure 3

(a) Metabolic Pathway Analysis identifies significant differences between advanced and early HCC patients. Nodes in red indicate significance (p < 0.05), and the size of the nodes indicate impact. (b) Main pathways through which amino acids supply the Krebs cycle to furnish energy. Red arrows indicated the change direction: metabolite increased (upward arrow) and metabolite decreased (down arrow) in advanced with respect to early HCC patients.

Figure 4

Kaplan–Meier analysis for overall survival (survival probability) for the whole 28 HCC patients in the early stage enrolled in this study.