Risk factors and diagnostic biomarkers for nonalcoholic fatty liver disease-associated hepatocellular carcinoma: Current evidence and future perspectives

Table 1 Clinical risk factors for nonalcoholic fatty liver disease-associated hepatocellular carcinoma

Risk factors	Reported evidence	Ref.
Liver fibrosis	The annual incidence rate of HCC in NAFLD patients with cirrhosis was more than 10 times higher than in those without	[15,22,24,25]
	Non-invasive fibrosis markers (e.g., FIB-4 index, M2BPGi, and shear wave velocity in VTQ) also had significant associations with the risk of NAFLD-HCC
Diabetes	Associated with increased risk of HCC in NAFLD patients (hazard ratio: 2.2–4.2)	[22,26,27]
Hypertension	May be an independent risk factor for NAFLD-HCC	[29]
Dyslipidemia	May be an independent risk factor for NAFLD-HCC	[29]
Age	Increased age was an independent risk factor for HCC in patients with NASH-related cirrhosis	[15,34]
	NAFLD patients aged ≥ 65 had 1.83 times higher risk of HCC than those aged < 65
Male sex	Male patients with NASH-related cirrhosis had 4.34 times higher risk of HCC than female patients	[34]
Ethnicity	Hispanic ethnicity was associated with 1.59 times higher risk of HCC in NAFLD patients compared to white ethnicity (however, there have been conflicting results)	[15]
Mild alcohol intake	Associated with increased risk of HCC in NAFLD patients (hazard ratio: 3.6–4.8)	[38,39]
Elevated liver enzymes	Associated with increased risk of HCC in NAFLD patients (hazard ratio: 2.1–8.2)	[41-43]

HCC: Hepatocellular carcinoma; NAFLD: Nonalcoholic fatty liver disease; FIB-4: Fibrosis-4; M2BPGi: Mac-2-binding protein glycosylation isomer; VTQ: Virtual touch quantification; NASH: Nonalcoholic steatohepatitis.

Table 2 Genetic risk factors for nonalcoholic fatty liver disease-associated hepatocellular carcinoma

Risk factors	Reported evidence	Ref.
PNPLA3	Carriage of rs738409 GG polymorphism is associated with 5.1–6.4-fold increased risk of HCC in NAFLD patients	[51-53,60]
	PNPLA3 G variant (GG vs CG vs CC) was not significantly associated with the risk of cardiovascular events extrahepatic cancers or overall death, but was associated with HCC (HR: 2.66) and liver-related death (HR: 2.42)
	Used for developing polygenic risk scores
TM6SF2	TM6SF2 minor allele carriage (rs58542926 C>T) was associated with advanced fibrosis/cirrhosis and HCC (OR, 2.8) in NAFLD patients	[54,55,60]
	Combined assessment with PNPLA3 and HSD17B13 variants were useful for risk stratification of NAFLD-HCC
	Used for developing polygenic risk scores
MBOAT7	MBOAT7 rs641738 C>T variants were associated with higher risk of HCC in NAFLD patients (OR, 1.65–2.10)	[56,60]
	Used for developing polygenic risk scores
TLR5	TLR5 rs5744174 TT genotype was a risk factor of HCC in patients with steatohepatitis-related cirrhosis (OR, 1.9)	[57]
STAT6	STAT6 rs167769 CC genotype was inversely associated with the risk of HCC in NASH patients (OR, 0.015)	[58]
YAP1	Carriage of YAP1 rs11225163 C allele was inversely associated with the risk of HCC in NASH patients (OR, 0.047)	[58]
HSD17B13	Combined assessment with PNPLA3 and TM6SF2 variants were useful for risk stratification of NAFLD-HCC	[55]
DYSF	DYSF rs17007417 T allele carriage was associated with increased risk of HCC in NAFLD patients (OR, 2.74)	[59]
GCKR	GCKR rs1260326 T allele carriage was associated with increased risk of HCC in NAFLD patients (OR, 1.38)	[59,60]
	Used for developing polygenic risk scores

PNPLA3: Patatin-like phospholipase domain 3; HCC: Hepatocellular carcinoma; NAFLD: Nonalcoholic fatty liver disease; HR: Hazard ratio; TM6SF2: Transmembrane 6 superfamily member 2; HSD17B13: 7-beta-Hydroxysteroid dehydrogenase 13; OR: Odds ratio; MBOAT7: Membrane-bound O-acyl-transferase domain-containing 7; TLR5: Toll-like receptor 5; STAT6: Signal transducer activator of transcription 6; NASH: Nonalcoholic steatohepatitis; YAP1: Yes-associated protein 1; DYSF: Dystrophy-associated fer-1-like protein; GCKR: Glucokinase regulator.

Table 3 Currently available and potential diagnostic biomarkers for nonalcoholic fatty liver disease-associated hepatocellular carcinoma

Biomarkers	Reported evidence	Ref.
Currently available
AFP	Modest diagnostic ability for HCC in NAFLD patients (AUROC, 0.71–0.88)	[63,84]
DCP	The diagnostic ability for NAFLD-HCC was similar to that of AFP	[63,84]
	Combined use with AFP improved the diagnostic performance
	Used for calculation of GALAD score
AFP-L3	The diagnostic ability for NAFLD-HCC was similar to that of AFP	[63]
	Used for calculation of GALAD score
Under development
Iron status	Elevations of serum iron levels and transferrin saturation were associated with increased risk of HCC in NAFLD patients (HR, 2.91 and 2.02, respectively)	[64]
Proteins	Midkine increased the diagnostic yield in AFP-negative HCC in NAFLD patients; 59.2% of AFP-negative NAFLD-HCC patients had elevation of serum midkine levels	[65-67,72]
	IgM-free AIM had better diagnostic performance for NASH-HCC than AFP or DCP (AUROC, 0.905–0.929)
	Serum TSP-2 levels were significantly associated with advanced fibrosis in NASH patients. Among 164 patients with NAFLD, HCC occurred only in patients with high serum levels of TSP-2
Glycoprotein	Glycosylation patterns of alpha-1 acid glycoprotein may serve as a diagnostic biomarker for AFP-negative HCC in NAFLD patients	[69]
Proteoglycan	Glypican-3 had modest diagnostic ability (AUROC, 0.759), similar to AFP (AUROC, 0.763). When combined with age, sex, DCP and adiponectin, the AUROC increased to 0.948	[65]
Glycopeptide	Site-specific N-glycopeptides from vitronectin may serve as diagnostic biomarkers for NASH-HCC. When used together with AFP, the AUROC were 0.834 and 0.847, compared to 0.791 of AFP alone	[70,71]
	Site-specific N-glycopeptides from serum haptoglobin showed better diagnostic accuracy for NASH-HCC than AFP
Cytokine (adipokine)	Adiponectin had slightly better diagnostic ability (AUROC, 0.770) than AFP (AUROC, 0.763). When combined with age, sex, DCP and glypican-3, the AUROC increased to 0.948	[65]
Cell-free DNA	TERT promoter mutation (C228T) in serum cfDNA showed better diagnostic ability for early NAFLD-HCC than AFP and DCP	[76,78]
	Methylation biomarkers in cfDNA improved the diagnostic performance when combined with AFP
microRNA	The expression levels of exosomal miR-182, miR-301a and miR-373 in both serum and ascetic fluid were higher in NASH-cirrhosis patients with HCC than in those without HCC	[77]

AFP: Alpha-fetoprotein; HCC: Hepatocellular carcinoma; NAFLD: Nonalcoholic fatty liver disease; AUROC: Area under receiver operating characteristic curve; DCP: Des-carboxyprothrombin; AFP-L3: AFP isoform L3; HR: Hazard ratio; IgM: Immunoglobulin M; AIM: Apoptosis inhibitor of macrophages; TSP-2: Thrombospondin-2; NASH: Nonalcoholic steatohepatitis; TERT: Telomerase reverse transcriptase; cfDNA: Cell-free DNA; miR: microRNA.