Prognostic biomarkers for prediction of recurrence of hepatocellular carcinoma: Current status and future prospects

Table 1 Gene expression studies of hepatocellular carcinoma for prediction of recurrence

Ref.	Sample type	Screening platform	Virus type	Characteristics for distinguishing groups	Number of marker genes	Predictive accuracy for recurrence
Iizuka et al[19] (2003)	Tumour tissues	Oligonucleotide microarray	HBV < HCV	Early IHR within 1 yr after surgery	12	25 in 27 independent patients (93%)
Kurokawa et al[20] (2004)	Tumour tissues	PCR based array	HBV < HCV	Early IHR within 2 yr after surgery	20	29 in 40 independent patients (72.5%)
Budhu et al[35] (2006)	Non-tumour tissues	cDNA microarray	Almost all HBV	Venous invasion or extrahepatic metastasis	17	87 of 95 independent patients (92%)
Ho et al[34] (2006)	Tumour tissues	cDNA microarray	HBV > HCV	Venous invasion	14	26 of 35 independent samples (74.3%)
Lee et al[37] (2006)	Tumour tissues	Oligonucleotide microarray	HBV > HCV	Hepatoblast gene signature	907	P < 0.001 in 66 patients (Probability of recurrence)
Okamoto et al[21] (2006)	Non-tumour tissues	cDNA microarray	All HCV	Single nodular HCC vs multicentric HCC	36	30 of 40 training samples (75%)
Wang et al[22] (2007)	Tumour tissues and non-tumour tissues	Oligonucleotide microarray	HBV > HCV	HCC recurrence	57	84% in 25 independent samples, sensitivity 86%, specificity 82%
Hoshida et al[23] (2008)	FFPE non-tumour tissues	DASL assay	HBV < HCV	Late recurrence more than 2 yr after resection	132	P = 0.003 in 224 patients in validation set (Probability of late recurrence)
Somura et al[24] (2008)	Tumour tissues	Oligonucleotide microarray/qRT-PCR	HBV < HCV	Early IHR within 1 yr after surgery	3	35 of 43 independent patients (81.4%)
Tanaka et al[25] (2008)	Tumour tissues	Oligonucleotide microarray	HBV > HCV	Aggressive recurrence exceeding Milan Criteria	1 (AURKB)	54 of 67 independent patients (80.5%)
Woo et al[26] (2008)	Tumour tissues	Oligonucleotide microarray	All HBV	Early IHR within 1 yr after surgery	628	P = 0.0018 in 139 independent patients (Probability of early recurrence)
Yoshioka et al[27] (2009)	Tumour tissues	Oligonucleotide microarray	HBV < HCV	Early IHR within 2 yr after surgery	172	P < 0.0001 in 97 independent patients (Probability of RFS)
Roessler et al[28] (2010)	Tumour tissues	Oligonucleotide microarray	HBV > HCV	Early IHR within 2 yr after surgery	161	P = 0.0057 for cohort 1, P = 0.017 for cohort 2 (Probability of RFS)
Tsuchiya et al[29] (2010)	Non-tumour tissues	Oligonucleotide microarray	All HCV	Late recurrence more than 1 yr after resection	38	P < 0.0001 in 44 training samples (Probability of RFS)
Woo et al[38] (2010)	Tumour tissues	Oligonucleotide microarray	HBV > HCV	Cholangiocarcinoma-like signature	625	P = 0.037 in cohort 1 of 61 patients, P = 0.004 for cohort 2 of 78 patients (Probability of RFS)
Weng et al[30] (2012)	Tumour tissues, PBMC	Oligonucleotide microarray	All HBV	Early IHR within 1 yr after surgery	3	P < 0.001 in 80 independent patients (Probability of RFS)
Xieraili et al[31] (2012)	Tumour tissues	Oligonucleotide microarray	HBV < HCV	Early IHR	1 (VIL1)	P = 0.025 in 90 independent patients (Probability of RFS)
Tsunedomi et al[32] (2013)	Tumour tissues	Oligonucleotide microarray	All HCV	Early IHR within 1 yr after surgery	1 (ABCB6)	89% sensitivity, 55% specificity, 86% PPV, 62% NPV in 20 independent patients

Under the heading “virus type”, HBV > HCV means that more patients in the study are HBV positive compared to HCV positive patients. HBV < HCV means there are more HCV positive patients in the study. DASL: Complementary cDNA-mediated annealing, selection, extension and ligation; IHR: Intrahepatic recurrence; RFS: Recurrence free survival; PBMC: Peripheral blood mononuclear cells; HBV: Hepatitis B virus; HCV: Hepatitis C virus; HCC: Hepatocellular carcinoma.

Table 2 miRNA for prediction of recurrence in hepatocellular carcinoma

Ref.	Sample type	Screening platform	Virus type	Characteristics for distinguishing groups	Number of candidate miRNA	Predictive accuracy for recurrence
Fornari et al[53] (2009)	Tumour and non-tumour tissues	qRT-PCR	HBV < HCV	miR-122 levels	1 (miR-122)	P = 0.05 for 45 independent patients (Recurrence rate)
Fornari et al[54] (2010)	Tumour and non-tumour tissues	qRT-PCR	HBV < HCV	Late recurrence beyond two years after surgery	1 (miR-199a-3p)	P = 0.043 for 36 independent patients (Recurrence rate)
Augello et al[47] (2012)	FFPE tumour tissues	qRT-PCR	All HCV	Stages of HCC progression	18	P = 0.042 for 61 independent patients (Percent Recurrence)
Han et al[52] (2012)	FFPE tumour tissues	qRT-PCR	Mostly HBV	Recurrence after OLT	1 (miR-155)	P < 0.001 for 100 training patients (RFS)
Huang et al[51] (2012)	Non-tumour tissues	qRT-PCR	HBV > HCV	Early IHR within 6 mo after surgery	6	P < 0.001 for 216 independent patients
Shih et al[48] (2012)	Tumour and non-tumour tissues	qRT-PCR	Not mentioned	HCC and non-tumour	15	P = 0.005 for 68 training samples and 13 independent samples (RFS)
Xia et al[50] (2012)	Tumour and non-tumour tissues	qRT-PCR	Mostly HBV	early IHR within 2 years after surgery	1 (miR-214)	P = 0.009 for 50 independent patients (RFS)
Zhu et al[49] (2012)	FFPE tumour and non-tumour tissues	qRT-PCR	All HBV	Early IHR after surgery	1 (miR-29a-5p)	AUC = 0.708 for 112 independent patients

OLT: Orthotopic liver transplantation; IHR: Intrahepatic recurrence; RFS: Recurrence free survival; AUC: Area under curve; HCV: Hepatitis C virus; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; qRT-PCR: Quantitative reverse transcription polymerase chain reaction.

Table 3 Proteomic studies of hepatocellular carcinoma for prediction of recurrence

Ref.	Sample type	Screening platform	Characteristics for distinguishing groups	Candidate biomarkers	Validation method
Yokoo et al[55] (2007)	Tumour tissues	2D-DIGE, MALDI-TOF MS	Early IHR within 6 mo after resection	23 protein panel	2D-DIGE
Orimo et al[59] (2008)	Tumour and non-tumour tissues	2D-DIGE, LC-MS/MS	Histological differentiation of tumours	Adenomatous polyposis coli-end-binding protein 1 (EB1)	IHC
Yi et al[56] (2008)	Paired tumour and non-tumour tissues	2DE, MALDI-TOF/TOF MS	Early IHR within 1 yr after resection	Mortalin (HSPA9)	qPCR, Immunoblotting, IHC
Bai et al[58] (2009)	Tumour tissues	cICAT, 2DLC-MS/MS	Recurrence after liver transplantation	Calpain small subunit 1 (CAPN4)	qRT-PCR, Immunoblotting, TMA
Cheng et al[57] (2011)	Tumour tissues	2DE, MALDI-TOF/TOF MS	Recurrence after liver transplantation	N-myc downstream-regulated gene 1 (NDRG1)	Immunoblotting, IHC
Kanamori et al[60] (2011)	Tumour and non-tumour tissues	2DLC-MS/MS	Tumour and non-tumour	Talin-1 (TLN1)	IHC

IHR: Intrahepatic recurrence; IHC: Immunohistochemistry; TMA: Tissue microarray; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; 2D-DIGE: Two-dimensional difference gel electrophoresis; MALDI: Matrix-assisted laser desorption/ionization; LC-MS: Liquid chromatography–mass spectrometry.

Table 4 Advantages and limitations of the three different omics methodologies

Method	Advantages	Limitations
Transcriptomics	Large dataset of dysregulated genes identified, provides insights to biological mechanisms of diseaseAffordable priceHas provided successful example of translation to clinical use (e.g., Oncotype Dx™ in predicting breast cancer recurrence)	Differences in platform contribute to lack of overlap in signatures between different studiesHigh possibility of noise present in the gene listPoor correlation between transcript and protein levels
Proteomics	Direct measurement of biological effectorsValidation method (IHC and TMA) routinely performed in pathology labs	Small number of validated targetsPrice, availability and quality of antibodies for validation work
Metabolomics	Amenable to different types of samples which can be obtained in a non-invasive manner	Lack of large-scale validation of metabolite signaturesLimited biological information obtained

IHC: Immunohistochemistry; TMA: Tissue Microarray.