Non invasive fibrosis biomarkers reduce but not substitute the need for liver biopsy

Table 1 METAVIR scoring system for liver fibrosis

Stage	Description
F0	No fibrosis
F1	Portal fibrosis without septa
F2	Portal fibrosis with few septa
F3	Septal fibrosis without cirrhosis
F4	Cirrhosis

Portal fibrosis is a stellate enlargement of portal tracts without any bridging fibrosis on the biopsy sample. Few septa mean at least one fibrous septa on the core biopsy. Theoretically, a fibrous septa is a bridge of connective tissue between two portal tracts, a portal tract and a centrolobular vein, or between two centrolobular veins. Septal fibrosis means that the liver biopsy is crossed by several septa; the transition between F2 and F3 begins when there is more fibrous septa than portal tracts without septa on the biopsy. Cirrhosis means that liver tissue is mutilated by nodular fibrosis that delineates hepatocytes nodules.

Table 2 Gene polymorphisms described as involved in fibrogenesis

Author	Etiologyof liver disease	Gene	Function ofthe gene product	Implicatedgenotype	Effect ongene product	Effecton fibrosis
Powell[39]	HCV	TGF-β1	Profibrogenic	Codon 25 (pro/arg)	Increased transcription	Increased
Powell[39] Forrest[40]	HCV	AT	Profibrogenic	-6 G/A	Increased transcription	Discordant results
Bonkovsky[41] Negro[42] Chitturi[43] Geier[44]	HCV / NASH	HFE	Iron metabolism	C282Y, H63D	Iron overload	Discordant results
Yee[45] Grove[46]	HCV /AFLD	TNF-β	Proinflammatory	-308 G/A	Increased transcription	Increased
Grove[47] Knapp[48]	HCV / AFLD	IL-10	Immune modulation	-1082 A/A; -627 C/A	Decreased transcription	Increased
Reynolds[49]	HCV	MPO	Battericide	-463 G/A	Increased transcription	Increased
Wozniak[50]	HCV	ApoE	Viral entry to cells	E4 allele	Abnormal function	Reduced
Muhlbauer[51]	HCV	MCP-1	Proinflammatory	-2518 G/A	Increased transcription	Increased
Wright[52]	HCV	Factor V Leiden	Thrombine generation	Codon 560 (arg/gln)	Resistance to activation	Increased
Romero-Gomez[53]	HCV	SLC11A1	Macrophage function	Homozygosity (GT)5AC(GT)10G (Allele 2) in the promoter region	Poor promoter	Reduced
Adinolfi[54]	HCV	MTHFR	Folate metabolism	C677T	HyperHC	Increased
Okamoto[55]	HCV	MMP	Matrix degradation	1G/2G (MMP-1), 5A/6A (MMP-3), C/T (MMP-9)	Reduced transcription	More frequent in cirrhosis than CHC
Yamauchi[56] Okamoto[57] Frenzer[58]	AFLD	CYP2E1	Ethanol metabolism	c1/c1, c2/c2 alleles	Increased activity	Discordant results
Yamauchi[56] Frenzer[58]	AFLD	ADH	Ethanol metabolism	ADH2 c1,c2,c3 alleles; ADH3 c1, c2, c3 alleles	Abnormal function	Discordant results
Yamauchi[56] Okamoto[57]	AFLD	ALDH	Ethanol metabolism	ALDH2 c2/c2 alleles	Abnormal function	Discordant results
Burim[59]	AFLD	CYP1A1	Ethanol metabolism	m2/m2 allele	Increased activity	Increased
Dixon[60]	NAFLD	AT and TGF-β	Profibrogenic	High AT and TGF- β1 producing polymorphisms	Increased activity	Increased

TGF-β = transforming factor beta; AT = angiotensinogen; HFE = hereditary haemochromatosis gene; NASH = non alcoholic steatohepatitis; TNF-β = tumor necrosis factor beta; AFLD = alcoholic liver disease; IL-10 = interleukin 10; NAFLD = non alcoholic fatty liver disease; MPO = myeloperoxidase; ApoE = apolipoprotein E; MCP-1 = monocyte chemotactic protein 1; SLC11A1 = solute carrier family 11 member 1; MTHFR = methylenetetrahydrofolate reductase; HC = homocisteinemia; MMP = metalloproteinase; CYP2E1 = cytochrome P 2E1; ADH = alcohol dehydrogenase; ALDH = aldehyde dehydrogenase; CYPA1 = cytochrome P A1.

Table 3 Features of the ideal marker of liver fibrosis

Specific for fibrosis of the liver

Providing measurement of: A) stage of fibrosis, B) fibrogenesis activity

Not influenced by comorbidities (e.g. renal, reticulo-endothelial)

Known half-life

Known excretion route

Sensitive

Reproducible

Table 4 Direct non invasive markers of liver fibrosis

Author	Liver disease	Marker	Characteristics	Pathophisiology
McHutchison[61] Murawaki[62] Halfon[63] Pares[64] Suzuki[65] Naveau[66] Santos[67] Montazeri[68]	HCV, AFLD, NAFLD, HBV	Hyaluronic acid	Component of the ECM in every tissue	Synthesized by HSCs and degraded by sinusoidal endothelial cells
Santos[67] Walsh[69]	HCV, NAFLD	Laminin	Co-expressed with type IV collagen in basement membranes	Increased deposition in viral disease
Tran[70] Saitou[71]	AFLD, HCV	YKL-40	Human cartilage glicoprotein	Involved in remodelling and degradation of ECM
Sakugawa[72] Murawaki[73] Walsh[69] Saitou[71] Santos[67]	HCV, NAFLD	Type IV collagen/ 7s domain	Main collagen component of the basement membrane	Increase with severity of liver fibrosis
Guechot[74] Pares[64]	HCV, AFLD	Procollagen III	Propeptide	Released during matrix deposition and remodelling
Boeker[75] Murawaki[62]	HCV	MMP-2	Collagenase	Correlates with fibrosis
Boeker[75]	HCV	TIMP-1	Metalloproteinase	Inhibitor of collagenase
Patel[76]	HCV	Three-marker panel	Combination of hyaluronic acid, TIMP-1, α2M	Better accuracy by combined markers

AFLD = alcoholic fatty liver disease; NAFLD = non alcoholic fatty liver disease; ECM = extracellular matrix; HSCs = hepatic stellate cells; YKL-40 = human cartilage glycoprotein-39; MMP-2 = metalloproteinase 2; TIMP-1 = tissue inhibitor metalloproteinase 1; α₂M = alfa-2-macroglobulin.

Table 6 Diagnostic performance of non invasive markers of liver fibrosis in discriminating between no-mild fibrosis (F0-F1 by METAVIR) and moderate-advanced fibrosis (F ≥ 2 by METAVIR)

Marker	Disease	Sensitivity	Specificity	AUC	References
Direct markers of liver fibrosis
Hyaluronic acid	HCV	75-79	80-100	0.82-0.92	[61-62] [69,74]
	HBV	91	98.1	0.98	[68]
	AFLD	87	93	0.79-0.91	[64,66]
	NAFLD	66-85	68-91	0.78-0.87	[65,67] [72]
Laminin	HCV	80	83	0.82	[69,81]
	NAFLD	82	89	n.a.	[67]
YKL-40	AFLD	88.5	50.8	n.a.	[70]
	HCV	78	81	0.81	[71]
Type IV collagen	HCV	73-80	81-85	0.83	[69,73]
	NAFLD	64	89	n.a.	[67]
Type IV collagen-7s	HCV	74-83	75-88	n.a.	[73]
	NAFLD	70	81	0.83	[72]
Procollagen III	HCV	60-78	74-75	0.69	[71,74]
	AFLD	80	87	0.87	[64]
MMP-2	HCV	7-75	70-100	0.59	[62,75]
TIMP-1	HCV	67	68	0.71	[75]
Three marker panel	HCV	77	73	0.83	[76]
Indirect markers of liver fibrosis
APRI	HCV	41-91	47-95	0.69-0.88	[86,95-97]
	HIV/HCV	51	91	0.8	[87]
Forns’ index	HCV	79.8-94	95-98.3	0.78-0.86	[88,98] [99,97]
	HIV/HCV	43	96	0.77	[87]
Fibrotest	HCV	65-87	59-80.6	0.74-0.87	[90,97]
					[100,101]
	HIV/HCV	90	60	0.85	[91]
	HBV	34	93	0.78	[92]
	AFLD	88	60	0.84	[66]
FPI	HCV	85-96	94-98	0.77	[93]

AUC=area under the curve; AFLD=alcoholic fatty liver disease; NAFLD=non-alcoholic fatty liver disease; n.a.=not available; YKL-40=human cartilage glycoprotein-39; MMP-2=metalloproteinase 2; TIMP-1=tissue inhibitor metalloproteinase 1; APRI=AST to platelet ratio index; FPI= fibrosis probability index.

Table 7 Diagnostic performance of non invasive markers of liver fibrosis in detecting cirrhosis

Marker	Disease	Sensitivity	Specificity	AUC	References
Direct markers of liver fibrosis
Hyaluronic acid	HCV	80-100	79-89.4	0.85-0.92	[61,63] [71,74]
	AFLD	99	80	0.93	[66]
	NAFLD	n.a.	n.a.	0.92	[65]
YKL-40	HCV	80	71	0.79	[71]
Type IV collagen	HCV	60	61	n.a.	[71,73]
Procollagen III	HCV	60-77	66-74	0.73	[71,74]
MMP-2	HCV	74-83	96-100	0.97	[75]
TIMP-1	HCV	100	56-75	0.9	[75]
Indirect markers of liver fibrosis
AAR	HCV	47-81.3	55.3-97		[85,102] [96]
	HIV/HCV	38	77	0.6	[87]
APRI	HCV	38.4-57	86.7-93	0.61-0.94	[86,95-97]
	HIV/HCV	53	89	0.79	[87]
GUCI	HCV	80	78	0.85	[89]
Fibrotest	HCV	13-50	91-98	0.71-0.87	[90,97]
					[100,101]
	HIV/HCV	100	65	0.87	[91]
	HBV	18	99	0.78	[92]
	AFLD	99	83	0.95	[66]
Glycocirrho test	Most HCV	79	86	0.87	[94]

AUC = area under the curve; AFLD = alcoholic fatty liver disease; NAFLD = non-alcoholic fatty liver disease; YKL-40 = human cartilage glycoprotein-39; MMP-2 = metalloproteinase 2; TIMP-1 = tissue inhibitor metalloproteinase 1; AAR = aspartate to alanine aminotrasferase ratio; APRI = AST to platelet ratio index; GUCI = Goteborg University Cirrhosis Index.

Table 5 Indirect non invasive markers of liver fibrosis

Authors	Liver disease	Biomarker	Description	Rationale
Giannini[85]	HCV, NAFLD	AAR	AST to ALT ratio	AST and ALT levels increase with progressive fibrosis
Wai[86] Macias[87]	HCV, HIV/HCV	APRI	AST to platelet ratio	Statistical association with liver fibrosis
Forns[88] Macias[87]	HCV, HIV/HCV	Forns’ index	Combination of age, platelet, γGT, cholesterol	Statistical association with liver fibrosis
Islam[89]	HCV	GUCI	Combination of AST, INR, platelet	Statistical association with liver fibrosis
Imbert-Bismut[90] Myers[91] Myers[92] Naveau[66]	HCV, HIV/HCV, HBV, AFLD	Fibrotest	Combination of α2M, ApoA1, bilirubin, γGT, haptoglobin	Statistical association with liver fibrosis
Sud[93]	HCV	FPI	Combination of HOMA-IR, age, cholesterol, AST, alcohol intake	Statistical association with liver fibrosis
Callewaert[94]	CLDs (mostly HCV)	Glycocirrho test	Profiles of serum protein N-glycans	Glycoproteins are produced mainly by hepatocytes

NAFLD = non alcoholic fatty liver disease; AAR = aspartate to alanine aminotrasferase ratio; α₂M = alfa-2-macroglobulin; ApoA1 = apolipoprotein A1; APRI = AST to platelet ratio index; GUCI = Goteborg University Cirrhosis Index; INR = international normalised ratio; FPI = fibrosis probability index; HOMA-IR = homeostasis model assessment of insulin resistance; CLDs = chronic liver diseases. γGT = gamma glutamil transpeptidase.

Table 8 Diagnostic performance of APRI, Forns’ index, Fibrotest and of sequential algorithms combining the three markers in patients with chronic hepatitis C

	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)	Accuracy (%)	AUC	Classified patients(%)
Detection of significant fibrosis in chronic hepatitis C with elevated ALT
APRI	29.7	93.8	95.7	52.7	60.2	0.69	54.1
Fibrotest	65	80.6	80	66.7	72.6	0.81	100
Forns	24.3	98.3	94.7	50.9	57.1	0.79	55.5
Sequential algorithm	100	83.8	92.7	100	94.2	n.a.	100
Detection of significant fibrosis in chronic hepatitis C with PNALT
APRI	26.9	100	100	56.8	62.7	0.77	74
Fibrotest	58.3	91.3	77.7	80.7	80	0.71	100
Forns	11.5	100	100	52.1	54.9	0.58	56
Sequential algorithm	100	87.5	94.3	100	96.3	n.a.	100
Detection of cirrhosis in chronic hepatitis C
APRI	38.4	86.7	38.5	86.7	78.1	0.61	54.1
Fibrotest	50	92.9	57.9	90.5	85.9	0.71	100
Sequential algorithm	94.6	95.1	78.3	99.1	95.5	n.a.	100

PPV = positive predictive value; NPV = negative predictive value; AUC = area under the curve; APRI = aspartate aminotrasferase to platelets ratio; Forns = Forns’ index; n.a. = not available; PNALT = persistently normal ALT.