Disease monitoring of hepatocellular carcinoma through metabolomics

Table 1 Significantly altered metabolites in hepatocellular carcinoma patients vs cirrhosis controls

Ref.	Platform	Tissue (organism)	HCC etiology	Significantly altered metabolites in HCC patients vs cirrhosis controls	Main pathways distinguishing HCC from cirrhosis
Patterson et al[29]	UPLC/QTOF-MS; UPLC/ESI-TQMS; GC/MS	Plasma (human)	HCC n = 20:	LPC (14:0) ↓	LPC metabolism
			EtOH n = 6	LPC (18:1) ↓
			HBV n = 3	LPC (20:4) ↓
			HCV n = 5	LPC (20:3) ↓
			NASH n = 3	LPC (22:6) ↓
			NASH/alcoholic steatohepatitis n = 1	FFA (24:0) - lignoceric acid ↓	Very long chain fatty acid metabolism
			HH n = 2	FFA (24:1) - nervonic acid ↓	Hemoglobin metabolism
				Bilirubin ↑
				Biliverdin ↑
Xiao et al[37]	UPLC/QTOF-MS	Serum (human)	HCV	3β, 6β-dihydroxy-5β-cholan-24-oic acid ↓	Bile acid biosynthesis
				3α, 7β-dihydroxy-5β-cholest-24-en-26-oic acid ↓
				GCA ↓
				GDCA ↓
				GCDCA ↓
				TCDCA ↓
				Linoelaidyl carnitine ↓	CPT shuttle system
				Oleoylcarnitine ↓
				Palmitoyl carnitine ↓
				O-octanoyl-R-carnitine ↓
				LPC (20:1) ↓	LPC metabolism
				LPC (20:4) ↓
				PE (20:4/18:1) ↓
				4E;15Z-Bilirubin IXa ↓	Hemoglobin metabolism
				15,16-dihydrobiliverdin ↓
				3-ganidinopropionicacid ↓
				Tetracosahexaenoic acid ↓	Eicosanoid metabolism
				3-hydroxy-eicosanoic acid ↓
				Oleamide ↓
				Phe-Phe ↑
Wang et al[38]	UPLC/MS-MS; LC/QTOF-MS	Serum (human)	HCC n = 82:	LPC-16:0 ↑	LPC metabolism
			HBV-cirrhosis n = 41	LPC-18:0 ↑
			HBV-only n = 41	16:0/18:1-PC ↑
				16:0/18:2-PC ↑
				16:0/20:4-PC ↑
				16:0/22:6-PC ↑
				18:0/18:2-PC ↑
				Phenylalanine ↓	Gut flora metabolism
				GCDCA ↓	Bile acid metabolism
				Canavaninosuccinate ↑↑	Organic acid metabolism
Zhou et al[39]	UPLC/QTOF-MS	Serum (human)	HCC n = 69:	LPC (14:0) HCC-C ↓	LPC metabolism
			HBV-HCC (HCC-B) n = 38	LPC (16:1) HCC-C ↓
			HCV-HCC (HCC-C) n = 31	LPC (18:3) HCC-B ↓
				HCC-C ↓
				LPC (18:2) HCC-B ↓
				HCC-C ↓
				LPC (18:1) HCC-B ↓
				HCC-C ↓
				LPC (18:0) HCC-C ↓
				LPC (20:5) HCC-C ↓
				LPC (20:4) HCC-B ↓
				HCC-C ↓
				LPC (20:3) HCC-B ↓
				HCC-C ↓
				LPC (20:2) HCC-C ↓
				LPC (20:0) HCC-B ↓
				HCC-C ↓
				LPC (22:6) HCC-C ↓
				LPC (22:5) HCC-B ↓
				HCC-C ↓
				LPC (22:4) HCC-B ↓
				HCC-C ↓
Wu et al[31]	SELDI-TOF-MS; HPLC/MS	Serum (human)	HBV	GRO-α↑	Cytokine
				Thrombin light chain ↑	Protease cleavage
Ressom et al[36]	UPLC/QTOF-MS	Serum (human)	HCC n = 78:	GDCA ↓	Bile acid metabolism
			→ HCV 67%	TCA ↓
			→ HBV 15%	TCDCA ↓
			→ Alcoholism 29%	Sphingosine 1-phosphate ↑	Sphingolipid metabolism
			→ NASH 13%	LPC (16:0) ↑	LPC metabolism
			→ Cryptogenic (8%)	LPC (17:0) ↑
			→ Autoimmune (3%)	LPC (18:0) ↑
				LPC (15:0) ↑
				LPC (22:6) ↑
				LPE (22:6) ↑
				LPE (20:4) ↑
				LPE (20:3) ↑
				PS ↑
Yang et al[28]	HRMAS 1H NMR	Biopsy (human)	HCC n = 17:	Glucose ↓	Glycolysis
			→ Cirrhosis n = 9	Creatine ↓
			→ No cirrhosis n = 8	PE ↑	LPC metabolism
				Glutamine ↑	Amino acid metabolism
				Glutamate ↑
				PC + GPC ↑	Bile acid metabolism
Nahon et al[40]	NMR	Serum (human)	EtOH cirrhosis	High density lipoproteins	HDL biosynthesis
				Acetate ↑	Ketone body metabolism
				N-acetyl-glycoproteins ↑	N-acetylglycoprotein
				Glutamate ↑	Amino acid metabolism
				Glutamine ↓
Budhu et al[43]	GC/MS, UPLC/MS-MS	Biopsy samples (human)	HCC n = 356	Study reported on markers involved in cancer aggressivity through comparison of stem-like HCC to less benign mature hepatocyte HCC	N/A
			Training cohort n = 30
			Testing cohort n = 217
			Validation cohort n = 139
Beyoğlu et al[44]	GC/MS	Biopsy samples (human)	Six HCC subtypes, liver fibrosis status unknown	Glucose ↓	Glycolysis
				Glycerol 3-phosphate ↓
				Glycerol 2-phosphate ↓
				Malate ↓
				Alanine ↓
				Myo-inositol ↓	PI3K pathway
				Linoleic acid ↓	Prostaglandin biosynthesis
Fitian et al[45]	UPLC/MS-MS and GC/MS	Serum (human)	HCV cirrhosis-associated HCC n = 30	Sphingosine ↑	Sphingolipid
			HCV-cirrhosis n = 27	Xanthine ↑	Oxidative stress metabolism
			Healthy volunteers n = 30	2-Pyrrolidinone ↑	GABA metabolism
				2-Hydroxybutyrate ↑	Oxidative stress metabolism
				Serine ↑	Amino acid
				Glycine ↑
				Aspartate ↑
				12-HETE ↑	Inflammation pathway
				15-HETE ↑
				Isovalerate ↑	Gut microflora metabolism
				Dihomo-linolenate ↑	Inflammation pathway
Gao et al[46]	GC-TOF/MS	Serum (human)	HBV cirrhosis-associated HCC n = 39	Stearic acid	Fatty acid biosynthesis
			HBV-cirrhosis (n = 52)	Heptadecanoic acid
				Palmitic acid
				5-Aminovaleric acid	Gut microflora metabolism
				Cholesterol ↑	Cholesterol metabolism
				3-hydroxybutyric acid ↑	Ketogenesis
				Malic acid ↑	TCA metabolism
				Glutamine ↑	Amino acid
				Asparagine ↓
				Alanine ↑
				Threonine ↓
				Leucine ↓
				Glutamic acid ↑
				β-glutamate ↑
				5-oxoproline ↓	Glutathione metabolism
				1,2,4-cyclopropranodicarboxylic acid ↓	Dicarboxylic acid metabolism

Pathways of importance in the comparison of (1A) HCC vs cirrhosis and (2) cirrhosis vs healthy controls are shown. Arrows indicate the metabolite’s expression in cases vs appropriate controls. P < 0.05 was used as the significance level and metabolites reported in table are those which were most significantly upregulated or downregulated in each study. EtOH: Alcohol; TOCSY: Total correlation spectroscopy; HH: Hereditary hemochromatosis; TCA: Tricarboxylic acid; UPLC: Ultrahigh-performance liquid chromatography; QTOF: Quadrupole time of flight; SELDI: Surface-enhanced laser desorption/ionization; HRMAS: High-resolution magic angle spinning; LPE: Lysophosphatidyethanolamine; LPC: Lysophosphatidylcholine; HCC: Hepatocellular carcinoma; MS: Mass spectrometry; TOF: Time-of-flight; GC: Gas chromatography; LC: Liquid chromatography; NMR: Nuclear magnetic resonance; HBV: Hepatitis B virus; HCV: Hepatitis C virus; NASH: Non-alcoholic steatohepatitis; FFA: Free fatty acids; PE: Phosphorylethanolamine; GABA: γ-aminobutyric acid.

Table 2 Significantly altered metabolites in cirrhosis patients vs healthy volunteers

Ref.	Platform	Tissue (organism)	Significantly altered metabolites in cirrhosis patients vs healthy volunteers	Main pathways distinguishing cirrhosis from healthy volunteers
Gao et al[33]	1H NMR	Serum (human)	Isoleucine ↓	Amino acid metabolism
			Leucine ↓
			Valine ↓
			Glutamine ↑
			Tyrosine ↑
			Phenylalanine ↑
			1-methylhistidine ↑
			N-acetylglycoproteins ↑
			Acetate ↑	N-acetylglycoprotein
			Acetoacetate ↓	Ketonogenesis
			Pyruvate ↑
			α-ketoglutarate ↑	Glycolysis
			Choline ↓	TCA cycle
			Taurine ↑
			Glycerol ↑	Bile acid metabolism
Li et al[42]	UPLC/QTOF-MS	Serum (mouse)	Leucine ↓	Amino acid metabolism
			Phenylpyruvic acid ↓
			Phenylalanine ↓
			Tryptophan ↓
			LPE (16:0) ↓	LPE metabolism
			LPE (18:0) ↓
			LPC (16:0) ↓	LPC metabolism
			LPC (20:1) ↓
			LPC (22:6) ↑
			PC (16:0/18:3) ↑	Phosphatidylcholine metabolism
			PC (12:1/24:3) ↑
			PC (16:0/20:4) ↑
			PC (16:0/22:6) ↑
			PC (18:0/20:4) ↑	Sphingomyelin metabolism
			SM (d18:0/16:1) ↓
Soga et al[35]	Capillary elecrtophore-sis/TOF-MS	Serum (human)	γ-glutamylalanine ↑	Glutathione metabolism
			γ-glutamylvaline ↑
			γ-glutamylglutamine ↑
			γ-glutamylphenyl-
			γ-glutamylcitrulline ↑
			Alanine ↑	Amino acid metabolism
			Methionine sulfoxide ↑
Wang et al[38]	UPLC/MS-MS; LC/QTOF-MS	Serum (human)	LPC-16:0 ↓	LPC metabolism
			LPC-18:0 ↓
			16:0/18:1-PC ↓
			16:0/18:2-PC ↓
			16:0/20:4-PC ↓
			16:0/22:6-PC ↓
			18:0/18:2-PC ↓
			Oleamide ↑	Fatty acid metabolism
			Phenylalanine ↑
			GCDCA ↑	Bile acid metabolism
			Canavaninosuccinate ↓	Arginosuccinate synthetase pathway
Zhou et al[39]	UPLC/QTOF-MS	Serum (human)	Phenylalanine ↑	Amino acid metabolism
			GCA ↑	Bile acid metabolism
			GDCA ↑
			Bilirubin ↑	Hemoglobin metabolism
			LPE (18:2) ↓	Lysolipid metabolism
			LPC (22:6) ↓
			LPC (18:2) ↓
			LPC (20:4) ↓
			LPC (16:0) ↓
			LPC (18:0) ↓
			C18:1-CN ↑	CPT shuttle system
Chen et al[30]	UPLC/QTOF-MS	Serum (human);	Inositol ↓	TCA cycle
		Urine (human)	2,2-bipyridine ↓
			Methionine ↓	Amino acid metabolism
			Tyrosine ↓
			Arginine ↓	Fatty acid metabolism
			Stearic acid ↓
			Palmitic acid ↓
			Citric acid ↓
			2-piperidine carboxylic acid ↓
			5-Hydroxy-tryptophan ↓
Cao et al[32]	UPLC/MS	Fecal (human)	Chenodeoxycholic	Bile acid metabolism
			Acid dimeride ↓
			Urobilin ↓	Hemoglobin metabolism
			Urobilinogen ↓
			7-ketolithocholic acid ↓	Microbiome metabolism
			LPC C18:0 ↑	LPC metabolism
			LPC C16:0 ↑
Yin et al[41]	RPLC/MS	Serum (human)	Hypoxanthine ↓	Purine synthesis
			Inosine ↓
			Bilirubin ↑	Hemoglobin metabolism
			GCA ↑	Bile acid metabolism
			GCDCA ↑
			Taurine ↓
			LPC C18:2 ↓	LPC metabolism
			LPC C18:3 ↓
			LPC C16:1 ↓
			LPC C18:0 ↓
			LPC C16:1 ↓
			L-acetylcarnitine ↑	CPT shuttle system
			6-Methylnicotinic acid ↓	Nicotine metabolism
Fitian et al[45]	Integrated UPLC/MS-MS and GC/MS	Serum (human)	Glycocholate (GCA) ↑	Bile acid metabolism
			Tauroursodeoxycholate ↑
			Glychochemodeoxycholate ↑
			Azelate (nonanedioate) ↑	Dicarboxylic acid metabolism
			Undecanedioate ↑
			Sebacate (decanedioate) ↑
			Hexadecanedioate↑
			Tetradecanedioate↑
			DSGEGDFXAEGGGVR ↑	Fibrinogen cleavage peptide
			ADSGEGDFXAEGGGVR ↑
			Bilirubin (Z,Z) ↑	Hemoglobin catabolism metabolite
			Biliverdin ↑
			1,2-propanediol ↑	Ketogenesis
			Succinylcarnitine ↑	CPT shuttle system
			Acetylcarnitine ↑
			Glutarylcarnitine ↑
Gao et al[46]	GC-TOF/MS	Serum (human)	Palmitic acid ↑	Fatty acid metabolism
			Stearic acid ↑
			Oleic acid ↑
			Arachidic acid ↑	Arachidonic acid metabolism
			Aminomalonic acid ↑	Dicarboxylic acid metabolism
			Phenylalanine ↑	Amino acid metabolism
			Cysteine ↑
			Leucine ↑
			Citric acid ↑
			Oxoproline ↑

EtOH: Alcohol; TOCSY: Total correlation spectroscopy; HH: Hereditary hemochromatosis; TCA: Tricarboxylic acid; UPLC: Ultrahigh-performance liquid chromatography; QTOF: Quadrupole time of flight; SELDI: Surface-enhanced laser desorption/ionization; HRMAS: High-resolution magic angle spinning; LPE: Lysophosphatidyethanolamine; LPC: Lysophosphatidylcholine; MS: Mass spectrometry; TOF: Time-of-flight; NMR: Nuclear magnetic resonance; LC: Liquid chromatography; GC: Gas chromatography; CPT: Carnitine palmitoyltransferase; TCA: Tricarboxylic acid.

Table 3 Utility of significantly altered (P < 0.05) metabolites in accurately predicting hepatocellular carcinoma (hepatocellular carcinoma cases vs patients with cirrhosis)

Ref.	Platform	Comparison	Class prediction methodology	Classification accuracy or sensitivity/specificity	AFP sensitivity/specificity
Patterson et al[29]	UPLC/ESI-QTOF-MS	HCC (n = 20) vs cirrhosis (n = 7)	Random forest	96.3	-
Chen et al[30]	Integrated GC/QTOF-MS + UPLC/QTOF-MS	HCC (n = 82) vs healthy (n = 71)	OPLS-DA	100.0	-
Wu et al[31]	SELDI-TOF MS	HCC (n = 48) vs cirrhosis (n = 54) or healthy (n = 42)	GRO-α + thrombin light chain PS20 Protein immunoassay	89.6/89.6	69/83
Cao et al[32]	UPLC/QTOF-MS	HCC (n = 23) vs cirrhosis (n = 22)	PLS-DA	67.0	-
Gao et al[33]	NMR	HCC (n = 39) vs cirrhosis (n = 36)	PLS-DA	45.7	-
Wu et al[34]	GC/MS	HCC (n = 20) vs healthy (n = 20)	PCA with ROC curve analysis	AUC=88.3; AUCAFP = 92.5 when combined with AFP	-
Soga et al[35]	LC/MS-MS	HCC (n = 32) vs HCV-only (n = 35) or cirrhosis (n = 18)	Multiple logistic regression; ROC curve analysis	88.1	0.760
Wang et al[38]	UPLC-MS	HCC (59) vs cirrhosis (20) or NHC (20)	PLS-DA, ROC curve analysis	CSA 79.3/100 CSA + AFP20 96.4/100 UPLC-MS 100/100	AFP20 74/38 AFP200 52/90
Zhou et al[39]	UPLC-QTOF-MS	HCC (n = 69) vs cirrhosis (n = 28)	PLS-DA, ROC curve analysis	AEA 88.0 PEA 82.0 AEA + PEA 88.0	-
Nahon et al[40]	NMR	Small HCC (n = 28) vs cirrhosis (n = 93); Large HCC (n = 33) vs cirrhosis (n = 93)	OPLS	Small HCC: 61.0/100.0 Large HCC: 100.0/100.0	-
Yin et al[41]	RPLC/QTOF-MS; HILIC/QTOF-MS	HCC (n = 25) vs cirrhosis (n = 24) or healthy (n = 25)	OPLS	RPLC: 61.8 HILIC: 57.0 RPLC + HILIC = 63.6	-
Li et al[42]	UPLC/QTOF-MS	HCC (n = 8) vs cirrhosis (n = 6) or healthy (n = 6) (murine samples)	OPLS-DA	88.2	-
Budhu et al[43]	Training set1: GC/MS + UPLC/MS-MS; Testing set2: Affymetrix GeneChip	Training set: Stem-like aggressive HpSC-HCC (n = 15) vs Mature hepatocyte less aggressive MH-HCC (n = 15); Testing set: HpSC-HCC and MH-HCC (n = 217)	Multivariate analysis	172.0/83.0, AUC = 0.830 272.0/91.0, AUC = 0.860	-
Fitian et al[45]	UPLC/MS-MS + GC/MS	HCC (n = 30) vs HCV-cirrhosis (n = 27)	Random forest	72% 12-HETE 73.3/69.2	AFP20 63.3/83.6
			ROC analysis	15-HETE 83.3/59.3
				Aspartate 100/51.9
				Glycine 83.3/63.0
				Serine 73.3/85.2
				Phenylalanine 73.3/81.5
				Homoserine 70.0/85.2
				Sphingosine 58.3/86.7
				Xanthine 63.3/88.9
				2-Hydroxybutyrate 76.7/77.8
Gao et al[46]	GC-TOF/MS	HCC (n = 39) vs HBV-cirrhosis (n = 52)	Random forest (validation set)	96.8% in HCC vs HBV-cirrhosis 100% in HBV-cirrhosis vs HBV	-
				100% in HBV vs NHC
			ROC analysis (validation set)	100/95.2 HBV vs NC
				83.3/100 HBV-cirrhosis vs HBV
				76.9/83.3 HCC vs HBV-cirrhosis
			Bayes discriminant function model (validation set)	76.9% HCC 100% HBV-cirrhosis
				94.1% HBV
				100% NHC

Classification accuracy describes the capacity of the metabolomic classification technique to accurately predict the group of each study subject. UPLC: Ultrahigh-performance liquid chromatography; AEA: Anandamide; OPLS: Orthogonal projection to latent structure; PCA: Principal component analysis; PEA: Palmitylethanolamide; PLS-DA: Partial least squares-discriminant analysis; MS: Mass spectrometry; TOF: Time-of-flight; GC: Gas chromatography; HCC: Hepatocellular carcinoma; HBV: Hepatitis B virus; GRO-α: Growth related oncogene-alpha; ROC: Receiver operator characteristic; NHC: Normal healthy controls; AFP20: AFP performance at the cutoff of 20 ng/mL; AFP200: AFP performance at the cutoff of 200 ng/mL.