Liquid biopsy of gastric cancer patients: Circulating tumor cells and cell-free nucleic acids

Table 1 Detection of circulating tumor cells in gastric cancer

Characteristic and number of patients		Control (n)	Detection method		Detection rate/statistic value		Ref.
Pre or post treatment	9 (PB)	4	RT-PCR	CEA mRNA	22.2%	(Pt.)	Funaki et al[133]
					0%	(Ctrl.)
I-IV	20 (PB)	22	RT-PCR	CEA mRNA	35%	(Pt.)	Mori et al[134]
					0%	(Ctrl.)
I-IV	49 (PB)	50	RT-PCR	CK19 mRNA	0%	(PB)	Aihara et al[135]
	21 (PV)				0%	(PV)
					0%	(Ctrl.)
I-IV	30 (PB)	58	RT-PCR	CK20 mRNA	16.7%	(Pt.)	Soeth et al[136]
					3.4%	(Ctrl.)
Inoperable/metastatic	34 (PB)	33	RT-PCR	CK19 mRNA	20.6%	(Pt.)	Yeh et al[137]
					0%	(Ctrl.)
I, III, IV	35 (PB)	9	RT-PCR	CEA mRNA	45.7%	(Pt.)	Noh et al[138]
					0%	(Ctrl.)
I-IV	52 (PB)	14	RT-PCR	CK19 mRNA	9.6%	(Pt.)	Majima et al[139]
					0%	(Ctrl.)
				CK20 mRNA	9.6%	(Pt.)
					1%	(Ctrl.)
I-IV	41 (PB)		RT-PCR	CEA mRNA	22.2%	(before curative surgery)	Nishida et al[60]
(36 with curative surgery)					33.3%	(during curative surgery)
(5 with inoperable)					80%	(inoperable Pt.)
I-IV	57 (PA)	30	RT-PCR	CEA mRNA	PA: 17.5%		Miyazono et al[59]
	49 (PV)				PV: 18.4%
	51 (SVC)				SVC: 21.6%
					8.8%	(before surgery)	At least either one positive in PA/PV/SVC
					33.3%	(during surgery)
					0%	(Ctrl.)
EGC, III	29 (PB)	15	RT-PCR	CEA mRNA	EGC: 22.2%	(after surgery)	Noh et al[140]
(paired, after surgery during surgery and follow-up)					IIIa: 20%
					IIIb: 26.7%
					Total: 24.1%
					EGC: 22.2%	(during follow-up)
					IIIa: 20%
					IIIb: 34.4%
					Total: 34.4%
					0%	(Ctrl.)
I-IV	106 (PB)		RT-PCR	CEA mRNA	40.6%		Sumikura et al[141]
(during surgery)
I-IV	41 (PB)	10	RT-PCR	CEA mRNA	24.4%	(Pt.)	Koike et al[142]
					0%	(Ctrl.)
I-IV	46 (PB) (18 EGJ caner)(28 with GC)	10 (with benign GI disease)100 (tumor-free Ctrl.)	qRT-PCROncoquick density gradient centrifugation	CK20 mRNA	27.8%	(Pt. with EGJ cancer)	Friederichs et al[143]
					21.4%	(Pt. with GC)
					0%	(with benign GI disease)
					1%	(tumor-free Ctrl.)
I-IV	59 (PB)	15	qRT-PCR	CEA mRNA	0%	(before surgery)	Ikeguchi et al[58]
					45.8%	(after surgery)
					0%	(Ctrl.)
I-IV	70 (PB)		RT-PCR	CK20 mRNA	36.6%	(Pt. with curative resection)	Illert et al[144]
(41 with curative resection)					44.8%	(Pt. with residual tumor)
(29 with residual tumor)					Total: 40%
I-III	46 (PB)	13	RT-PCR	CEA mRNA	52.2%	(before surgery)	Seo et al[145]
(with curative resection)					19.6%	(after surgery)
(paired, before and after surgery)					0%	(Ctrl.)
I-IV	52 (PB)	36	RT-PCR	c-Met mRNA	61.5%	(Pt.)	Uen et al[42]
					5.6%	(Ctrl.)
				MUC1 mRNA	71.2%	(Pt.)
					8.3%	(Ctrl.)
I-IV	42 (PB)	30	RT-PCR	hTERT mRNA	61.9%	(Pt.)	Wu et al[43]
					0%	(Ctrl.)
				CK19 mRNA	69.0%	(Pt.)
					3.3%	(Ctrl.)
				CK20 mRNA	61.9%	(Pt.)
					3.3%	(Ctrl.)
				CEA mRNA	78.6%	(Pt.)
					0%	(Ctrl.)
I-IV	64 (PB)	80	MAH	hTERT mRNA	81.3%		Wu et al[44]
				CK19 mRNA	78.1%
				CK20 mRNA	82.8%
				MUC1 mRNA	84.4%
					No detection in controls
I-IV	32 (PB)		FACS/ICC	CK8/18/19	21.9%	(before chemotherapy)	Kolodziejczyk et al[146]
(paired, before and after chemotherapy)					15.6%	(after chemotherapy)
I-IV	57 (PB)		FACS/ICC	CK8/18/19	54.4%	(before surgery)	Pituch-Noworolska et al[61]
	(before surgery)				21.2%	(after surgery)
	52 (PB)
	(after surgery)				26.8%	(TDB sample)
	56 (TDB)
I-IV	52 (PB)	20	qRT-PCR	CEA mRNA	5.0%	(before surgery)	Tani et al[147]
	(40 pre-ope)				16.7%	(after surgery)
	(12 post-ope)
I-IV	41 (PB)	41	Cell search system	EpCAM	14.3%	(Nonmetastatic GC)	Hiraiwa et al[55]
				CK8/18/19	55.6%	(metastatic GC)
					0%	(Ctrl.)
I-IV	101 (PB)	14	qRT-PCR	CK19 mRNA↑	P = 0.0127	Curative resection (n = 69) vs Ctrl. (n = 14)	Koga et al[148]
(69 with curative ope)					P = 0.0087	Non-curative resection (n = 32) vs Ctrl. (n = 14)
(32 with non-curative ope)				CK20 mRNA↑	P = 0.0022	Non-curative resection (n = 32) vs Ctrl. (n = 14)
I-IV	810 (PB)	29	RT-PCR	MT1-MMP mRNA	22.8%		Mimori et al[57]
					No data for Ctrl.
I-IV	55 (PB)	86	RT-PCR ELISA	Survivin mRNA↑	45.5%	(Pt.)	Yie et al[149]
					AUC = 0.772	Pt. (n = 55) vs Ctrl. (n = 86)
I-IV	70 (PB)	20	qRT-PCR	CEA mRNA	45.7%		Bertazza et al[150]
				CK19 mRNA	97.1%
				VEGF mRNA	38.6%
				Survivin mRNA	98.6%
					(Control samples were used the calibrator source)
I-IV	846 (PB)	25	qRT-PCR	uPAR mRNA↑	404/846 47.8%		Kita et al[151]
					P < 0.0001	Pt. (n = 846) vs Ctrl. (n = 25)
Advanced	52 (PB)		Cell search system	EpCAM	32.7%	(baseline)	Matsusaka et al[56]
(paired, before and during chemotherapy)				CK8/18/19	13.7%	(2 wk after chemotherapy)
					18.8%	(4 wk after chemotherapy)
I-IV	123 (PB)	30	qRT-PCR	CEA mRNA	36.6%	(Pt.)	Qiu et al[152]
					30%	(Ctrl.)
I-IV	30 (PB)		qRT-PCR	CK18 mRNA	I/II: 81.8%		Saad et al[153]
(after curative surgery)					III/IV: 31.6%
					Total: 50%
N/A	90 (PB)			miR-106a↑	P = 0.006	Pre-ope vs Ctrl.	Zhou et al[48]
(90 before surgery)					AUC = 0.684
(41 preoperative)					P = 0.016	Post-ope vs Ctrl.
(49 postoperative)				miR-17↑	P = 0.001	Pre-ope vs Ctrl.
					AUC = 0.743
					P = 0.019	Post-ope vs Ctrl.
I-IV	95 (PB)	21	qRT-PCR	B7-H3 mRNA↑	50.5%		Arigami et al[154]
					P < 0.0001	Pt. (n = 95) vs Ctrl. (n = 21)
					AUC = 0.86
I-IV	98 (PB)	30	RT-PCR ELISA	Survivin mRNA	I/II: 25%		Cao et al[155]
					III/IV: 56.1%
					I-IV: 45.9%
I-IV	93 (PB)	32	qRT-PCR	piR-651↓	P < 0.001	Pt. (n = 93) vs Ctrl. (n = 32)	Cui et al[49]
	(42 preo-ope)				AUC = 0.841
	(51 post-ope)			piR-823↓	P < 0.001
					AUC = 0.822
II-IV	35 (PB)	50	qRT-PCR	CEA mRNA	22.9%	(Pt.)	Dardaei et al[156]
				CK20 mRNA	37.1%	(Pt.)
				TFF1 mRNA	31.4%	(Pt.)
				MUC2 mRNA	22.9%	(Pt.)
					No detection in controls
I-IV	53 (PB)	20	qRT-PCR	miR-21↑	P < 0.0001	Pt. (n = 53) vs Ctrl. (n = 20)	Zheng et al[50]
					AUC = 0.853
I-IV	52 (PB)	15	qRT-PCR	miR-200c↑	P = 0.018	Pt. (n = 52) vs Ctrl. (n = 15)	Valladares-Ayerbes et al[51]
					AUC = 0.715
I-IV	40 (PB)	17	qRT-PCR	miR-421↑	P < 0.01	Pt. (n = 40) vs Ctrl. (n = 17)	Zhou et al[52]
					AUC = 0.773

AUC: Area under the receiver operating characteristic curve; BM: Bone marrow; EGC: Early gastric cancer; EGJ: Esophagogastric junction; GI: Gastrointestinal; MAH: Membrane-array hybridization; PA: Peripheral artery; PB: Peripheral blood; PV: Portal vein; SVC: Superior vena cava; TDB: Tumor-draining blood; N/A: Not available; qRT-PCR: Quantitative real-time polymerase chain reaction; FACS: Fluorescence activated cell sorter; ICC: Intracellular cytokine flow cytometry.

Table 2 Prognostic value of circulating tumor cells in gastric cancer

Characteristic and number of patients		Detection method		Statistic value				Ref.
	17	RT-PCR	CK19 mRNA	OS	P = 0.014	CK19 (+) vs (-)		Yeh et al[137]
(non-responsive to chemotherapy)
I-IV	57	RT-PCR	CEA mRNA	Liver metastasis recurrence	P = 0.03	CEA (+) vs (-)	(a)	Miyazono et al[59]
I-IV	106	RT-PCR	CEA mRNA	Recurrence/metastasis	P = 0.02	CEA (+) vs (-)	(a)	Sumikura et al[141]
I-IV	46	qRT-PCR	CK20 mRNA	2-year-survival	P < 0.05	CK20 (+) vs (-)	N/A	Friederichs et al[143]
I-IV	41	RT-PCR	CK20 mRNA	OS	P = 0.0363	CK20 (+) vs (-)	(b)	Illert et al[144]
(with curative resection)
I-III	46	RT-PCR	CEA mRNA	Recurrence	P ≤ 0.00022	CEA after sugery (+) vs (-)	(a)	Seo et al[145]
				Recurrence	P = 0.015		(c)
I-IV	52	RT-PCR	C-Met mRNA	OS	P = 0.0178	C-Met (+) vs (-)	(b)	Uen et al[42]
			MUC1 mRNA	OS	P = 0.0352	MUC1 (+) vs (-)	(b)
I-IV	42	RT-PCR	CEA mRNA	Recurrence/metastasis	P = 0.032	CEA (+) vs (-)	(c)	Wu et al[43]
I-IV	64	MAH	hTERT/CK19/CEA/MUC1	Recurrence/metastasis	P = 0.009	All marker (+) vs the others	(c)	Wu et al[44]
				OS	P = 0.0223		(b)
Metastatic	27	CellSearch	EpCAM	OS	P = 0.039	CTC ≥ 2 vs < 2	(b)	Hiraiwa et al[55]
		System	CK8/18/19
I-IV	69	qRT-PCR	CK19 mRNA	OS	P = 0.0347	CK19 (+) vs (-)	(b)	Koga et al[148]
(with curative resection)			CK20 mRNA	OS	P = 0.049	CK20 (+) vs (-)	(b)
I-IV	810	RT-PCR	MT1-MMP	Recurrence/metastasis	P = 0.0018	MT1-MMP (+) vs (-)	(c)	Mimori et al[57]
I-IV	55	RT-PCR ELISA	Survivin mRNA	RFS	P = 0.026	Survivin (+) vs (-)	(b)	Yie et al[149]
					P = 0.026		(d)
I-IV	70	qRT-PCR	Survivin mRNA	OS	P = 0.036	Survivin high vs low	(b)	Bertazza et al[150]
					P < 0.001		(d)
Advanced	51 (2 wk after chemotherapy)	Cell search system	EpCAM	PFS (2 wk after chemotherapy)	P < 0.001	CTC ≥ 4 vs < 4	(b)	Matsusaka et al[56]
			CK8/18/19		P < 0.001		(d)
	48 (4 wk after chemotherapy)			OS (2 wk after chemotherapy)	P < 0.001		(b)
					P < 0.001		(d)
				PFS (4 wk after chemotherapy)	P < 0.001		(b)
					P < 0.001		(d)
				OS (4 wk after chemotherapy)	P < 0.001		(b)
					P = 0.004		(d)
I-IV	123	qRT-PCR	CEA mRNA	Recurrence	P = 0.001	CEA (+) vs (-)	(a)	Qiu et al[152]
				DFS	P = 0.001		(b)
					P = 0.02		(d)
I-IV	30	qRT-PCR	CK18 mRNA	RFS	P < 0.001	CK18 (+) vs (-)	(b)	Saad et al[153]
(after curative surgery)					P = 0.04		(d)
				OS	P = 0.001		(b)
					P = 0.06		(d)
I-IV	95	qRT-PCR	B7-H3 mRNA	OS	P = 0.02	B7-H3 high vs low	(b)	Arigami et al[154]
					P = 0.046		(d)
I-IV	98	RT-PCR ELISA	Survivin mRNA	DFS	P < 0.001	Survivin (+) vs (-)	(b)	Cao et al[155]
					P < 0.001		(d)
I-IV	52	qRT-PCR	miR-200c	OS	P = 0.016	miR-200c high vs low	(b)	Valladares-Ayerbes et al[51]
					P = 0.028		(d)
				RFS	P = 0.044	miR-200c high vs low	(b)
					P = 0.028		(d)

a: χ² test/Fisher’s exact test; b: Kaplan-Meier survival curves, Log-rank test/Breslow-Wilcoxon test; c: Logistic regression model (multivariate); d: Multivariate Cox proportional hazard regression model. DFS: Disease-free survival; MAH: Membrane-array hybridization; OS: Overall survival; PFS: Progression-free survival; qRT-PCR: Quantitative real-time polymerase chain reaction; RFS: Relapse-free survival.

Table 3 Circulating cell-free DNA in gastric cancer

Characteristic and number of patients		Controls (n)	Plasma/serum	Detection method		Detection rate/statistic value			Ref.
Unresectable	198	78 (peptic ulcer)	Serum	Immuno-PCR	MG7-Ag	82.8%	(GC)		Ren et al[157]
		118 (chronic gastritis)		Semi quantitative		7.7%	(peptic ulcer)
				PCR
		236 (healthy donors)				5.9%	(chronic gastritis)
						0.8%	(healthy donors)
						P < 0.01	Correlation with metastasis	(a)
N/A	51	30 (gastritis)	Serum	qPCR	EBV DNA	100%	(Pt. with EBER (+)		Lo et al[158]
							in primary tumor)
		197 (healthy controls)				92.9%	(Pt. with EBER (+)
							in filtrating lymphocytes)
						0%	(Pt. with EBER (-)
							in primary tumor)
						23.3%	(gastritis)
						3.6%	(Ctrl.)
I-IV	54	30	Serum	MSP	DAP-kinase	48.1%			Lee et al[85]
					E-cadherin	57.4%
					GSTP1	14.8%
					p15	55.6%
					p16	51.9%
						No detection in controls
I-IV	60	16	Serum	MSP	p16	26.1%	With p16 methylation		Kanyama et al[159]
							in primarily tumor
						0%	Without p16 methylation
							in primarily tumor
						0%	(Ctrl.)
I-IV	109	10	Serum	MSP	p16	18.3%			Ichikawa et al[160]
					E-cadherin	23.9%
					p16 + E-cadherin	36.7%
						No detection in controls
I-IV	41	10	Serum	MSP	p16	22.0%			Koike et al[142]
					E-cadherin	22.0%
					RARb	14.6%
				p16 + E-cadherin + RARb		24.4%
						No detection in controls
I-IV	63	10	Serum	MSP	p16	27.0%			Koike et al[161]
					E-cadherin	23.8%
					RARb	17.5%
				p16 + E-cadherin + RARb		50.8%
						No detection in controls
I-IV	60	22	Serum	qMSP	APC	16.7%			Leung et al[162]
					E-cadherin	13.3%
					hMLH1	41.7%
					TIMP3	16.7%
				Four markers combined		55%
						13.6%	(Ctrl.)
					APC + E-cadherin	OS: P = 0.006	Methylation (+) vs (-)	(b)
I-IV	109	10	Serum	MSP	RARb	23.8%			Ikoma et al[163]
				p16 + E-cadherin + RARb		47.7%
I-IV	53	21	Plasma	qPCR	β-actin (102 bp)	P = 0.03	Pt.(n = 53) vs Ctrl. (n = 21)	(c)	Sai et al[83]
					β-actin (253 bp)	P < 0.0001		(c)
						AUC = 0.75
				DNA integrity (253 bp/102 bp)		P = 0.07		(c)
N/A	4	10	Serum	MSP	RUNX3	100%			Tan et al[164]
					p16	50%
					RASSF1A	25%
					CDH1	25%
						No detection in controls
I-IV	52	20	Serum	MSP	p16	9.6%			Tani et al[147]
	(40 pre-ope)				E-cadherin	9.6%
	(12 post-ope)				RARb	3.8%
				p16 + E-cadherin + RARb		23.1%
I-IV	52	50	Serum	MSP	p16	26.9%	(all Pt.)		Abbaszadegan et al[165]
						60.9%	(Pt. with p16 methylation in primary tumor)
						0%	(Ctrl.)
I-IV	20	22	Plasma	Fluorescence-based assay	DNA concentration↑	P < 0.005	Pt.(n = 20) vs Ctrl. (n = 22)	(d)	Kolesnikova et al[166]
				MSP	MGMT	70%	(Pt.)
						36.4%	(Ctrl.)
					p15	50%	(Pt.)
						18.2%	(Ctrl.)
					hMLH1	25%	(Pt.)
						9.1%	(Ctrl.)
I-IV	47	30 (benign gastric disease)	Serum	MSP	RASSF1A	24.0%	(Pt.)		Wang et al[167]
		30 (healthy controls)				3.3%	(benign gastric disease)
						0%	(healthy controls)
I-IV	20	21	Serum	MSP	HSulf-1	55%	(Pt.)		Chen et al[168]
						19.0%	(Ctrl.)
I-IV	57	79	Plasma	qPCR	MYC/GAPDH↑	P < 0.001	Pt. (n = 57) vs Ctrl. (n = 79)		Park et al[91]
						AUC = 0.841
I-IV	65	50	Serum	qMSP	RUNX3	29.2%	(Pt.)		Sakakura et al[169]
						10%	(Ctrl.)
						AUC = 0.8651	Pt. (n = 65) vs Ctrl. (n = 50)
I-IV	65	40 (benign gastric disease)	Serum	MSP	DLEC1	33.8%	(Pt.)		Zhang et al[170]
		20 (healthy controls)				5%	(benign gastric disease)
						0%	(healthy controls)
I-IV	73	20	Serum	qMSP	TFPI2	9.6%	(Pt.)		Hibi et al[171]
						0%	(Ctrl.)
						P = 0.004	Correlation with LN meta.	(a)
						P = 0.0115	Correlation with distant meta.	(a)
I-IV	65	80	Plasma	qMSP	SLC19A3	P < 0.0001	Pt.(n = 45) vs Ctrl. (n = 60)		Ng et al[172]
							(Validation 1)
						AUC = 0.82	Pt. (n = 20) vs Ctrl. (n = 20)
							(Validation 2)
I-IV	46	30 (healthy controls)	Serum	Methylation CpG island microarray	BX141696	56.5%			Zheng et al[86]
		46 (benign gastric disease)		MSP	WT1	50%
					CYP26B1	73.9%
					KCNA4	67.4%
I-IV	58	30 (healthy controls)	Serum	MeDIP	CHRM2	31.0%			Chen et al[87]
		46 (gastric precancerous lesions)		Methylation CpG island microarray	FAM5C	31.0%
				MSP		P < 0.001	Pre- vs post-operation
					MYLK	70.7%
						P < 0.001	Pre- vs post-operation
					FAM5C + MYLK	77.6%	(GC Pt.)
						10%	(healthy controls)
						30.4%	(gastric precancerous lesions)
I-IV	59	54	Plasma	qPCR	ALU↑	P < 0.001	Pt. (n = 59) vs Ctrl. (n = 54)	(c)	Park et al[84]
						AUC = 0.784
N/A	25	9	Plasma	MSP	ATP4B	64%	(Pt.)		Raja et al[173]
						0%	(Ctrl.)
I-IV	71	21	Serum	qMSP	Vimentin	P = 0.018	Pt. (n = 73) vs Ctrl. (n = 21)	(c)	Shirahata et al[174]
Operable	73	20	Serum	MSP	SOX17	58.9%	(Pt.)		Balgkouranidou et al[175]
						0%	(Ctrl.)
						OS: P = 0.049	Methylation (+) vs (-)	(b)
I-IV	73		Plasma	qPCR	HER2	64.4%	Pt. with 2+/3+ score in HER2 IHC assay		Lee et al[92]
Pt. with 2+/3+ score in HER2 IHC assay
I-IV	202	88	Serum	qMSP	XAF1	69.8%	(Pt.)		Ling et al[88]
						0%	(Ctrl.)
						AUC = 0.909	Pt. (n = 202) vs Ctrl.(n = 88)
						DFS: P < 0.0001	Methylation (+) vs (-)	(b)

a: χ² test/Fisher’s exact test; b: Kaplan-Meier survival curves, Log-rank test/Breslow-Wilcoxon test; c: Unpaired t-test/Mann-Whitney U-test; d: Unknown statistic method. AUC: Area under the receiver operating characteristic curve; LN: Lymph node; MeDIP: Methylated DNA immunoprecipitation; MSP: Methylation specific-PCR; qMSP: Quantitative methylation specific-PCR. qRT-PCR: Quantitative real-time polymerase chain reaction; N/A: Not available; GC: Gastric cancer; EBV: Epstein-barr virus; DAP: Death-associated protein; APC: Adenomatous polyposis coli; OS: Overall survival; HER: Human epidermal growth factor.

Table 4 Circulating cell-free mRNA in gastric cancer

Characteristic and number of patients		Controls (n)	Plasma/serum	Detection method		Detection rate/statistic value			Ref.
I-IV	52	20	Plasma	qRT-PCR	hTERT	7.5%	(preoperative)		Tani et al[147]
	(40 preoperative)				MUC1	1%
	(12 postoperative)				hTERT + MUC1	15%
					hTERT	16.7%	(postoperative)
					MUC1	8.3%
					hTERT + MUC1	16.7%
						No detection in controls
I-IV	89	42	Plasma	qRT-PCR	CXCR4↑	41.6%	[before surgery (n = 89)]		Xu et al[176]
[paired, before and						23.2%	[after surgery (n = 69)]
after surgery (n = 69)]						21.4%	[Ctrl. (n = 42)]
						P < 0.05	Before surgery (n = 89) vs Ctrl. (n = 42)	(a)
					Bmi-1↑	57.3%	[before surgery (n = 89)]
						43.5%	[after surgery (n = 69)]
						28.6%	[Ctrl. (n = 42)]
						P < 0.05	Before surgery (n = 89) vs Ctrl. (n = 42)	(a)
						P < 0.05	Before (n = 89) vs after (n = 69) surgery	(a)
I-IV	118	40 (gastritis)	Plasma	qRT-PCR	hTERT mRNA↑	P < 0.05	GC (n = 118) vs gastritis (n = 40)	(a)	Kang et al[123]
		58 (healthy controls)				P < 0.05	GC (n = 118) vs Ctrl. (n = 58)	(a)
						AUC = 0.891	GC (n = 118) vs Ctrl. (n = 58)
						DFS: P < 0.001		(b)
						DFS: P = 0.001		(c)
						OS: P < 0.001		(b)
						OS: P < 0.001		(c)

a: Mann-Whitney U-test; b: Kaplan-Meier survival curves, Log-rank test/Breslow-Wilcoxon test; c: Multivariate Cox proportional hazard regression model. AUC: Area under the receiver operating characteristic curve; DFS: Disease-free survival; OS: Overall survival; qRT-PCR: Quantitative real-time polymerase chain reaction; hTERT: Human telomerase reverse transcriptase; MUC1: Mucin-1.

Table 5 Circulating cell-free microRNA/long non-coding RNA in gastric cancer

Characteristic and number of patients		Controls (n)	Plasma/serum	Detection method		Detection rate/statistic value				Ref.
microRNA
I-IV	69	30	Plasma	qRT-PCR	miR-17-5p↑	P = 0.05		Pt. (n = 69) vs Ctrl (n = 30)	(a)	Tsujiura et al[124]
					miR-21↑	P = 0.006		Pt. (n = 69) vs Ctrl (n = 30)	(a)
						P = 0.013		paired (n = 10), pre-op > post-op	(b)
					miR-106a↑	P = 0.008		Pt. (n = 69) vs Ctrl (n = 30)	(a)
					miR-106b↑	P < 0.001	AUC = 0.721	Pt. (n = 69) vs Ctrl (n = 30)	(a)
						P = 0.022		paired (n = 10), pre-op > post-op	(b)
					let-7a↓	P = 0.002		Pt. (n = 69) vs Ctrl (n = 30)	(a)
					miR-106a/let-7a↑		AUC = 0.879
I-IV	164	127	Serum	Solexa sequencing	miR-1↑	P < 0.01	Pt. (n = 164) vs Ctrl (n = 127)		(a)	Liu et al[177]
				qRT-PCR	miR-20a↑	P < 0.01			(a)
					miR-27a↑	P < 0.01			(a)
					miR-34a↑	P < 0.01			(a)
					miR-423-5p↑	P < 0.01			(a)
					Five-serum miRNA signature↑	AUC = 0.879	Pt. (n = 22) vs Ctrl (n = 22) (test study)
						AUC = 0.831	Pt. (n = 142) vs Ctrl (n = 105) (validation study)
I-IV	56	30	Plasma	Microarray	miR-451↑	P < 0.01	AUC = 0.96	Pt. (n = 56) vs Ctrl (n = 30)	(a)	Konishi et al[125]
				qRT-PCR		P < 0.01		paired (n = 29), pre-op > post-op	(b)
					miR-486↑	P < 0.01	AUC = 0.92	Pt. (n = 56) vs Ctrl (n = 30)	(a)
						P < 0.01		paired (n = 29), pre-op > post-op	(b)
I-IV	40	41	Serum	Microarray	miR-187*↑	P = 0.0016	AUC = 0.704	Pt. (n = 40) vs Ctrl (n = 41)	(a)	Liu et al[178]
				qRT-PCR	miR-371-5p↑	P = 0.0009	AUC = 0.715		(a)
					miR-378↑	P < 0.0001	AUC = 0.861		(a)
N/A	82	82	Serum	Microarray	miR-221↑	AUC = 0.74	Pt. (n = 68) vs Ctrl (n = 68) (second validation study)			Song et al[179]
				qRT-PCR	miR-376c↑	AUC = 0.71
					miR-744↑	AUC = 0.71
N/A	20		Serum	qRT-PCR	miR-196a	P = 0.012	Pre-op (n = 20) > post-op (n = 20)		(a)	Tsai et al[180]
(pre-op, post-op and recurrent)						P = 0.002	Post-op (n = 20) < at recurrent (n = 20)		(a)
I-IV	30	39	Serum	qRT-PCR	miR-21↑	P < 0.001	AUC = 0.81	Pt. (n = 30) vs Ctrl (n = 39)	(a)	Wang et al[181]
I-IV	87		Plasma	qRT-PCR	miR-17-5p↑	P < 0.001		Pre-op (n = 65) > post-op (n = 16)	(a)	Wang et al[182]
	(65 pre-op)					P = 0.003		Post-op (n = 16) < recurrent (n = 6)	(a)
	(16 post-op)					OS: P = 0.0003	miR-17-5p high vs low	Pre-op (n = 65)	(c)
	(6 recurrent)				miR-20a↑	P = 0.006		Pre-op (n = 65) > post-op (n = 16)	(a)
						OS: P = 0.0003	miR-20a high vs low	Pre-op (n = 65)	(c)
						OS: P = 0.013	miR-20a high vs low	pre-op (n = 65)	(d)
I-IV	20	20	Serum	miRNA microarray	miR-375↓	P < 0.001	AUC = 0.835	Pt. (n = 20) vs Ctrl (n = 20)	(a)	Zhang et al[183]
				qRT-PCR
	20	190	Plasma	miRNA microarray	miR-195-5p↓	P < 0.05	Fold changes 13.3	Pt. (n = 20) vs Ctrl (n = 130)	(a)	Gorur et al[184]
I-III	79				miR-21↑	P < 0.001	Correlation with pN stage		(e)	Kim et al[185]
(25 without LN meta)					miR-146a↑	P = 0.001	Correlation with pN stage		(e)
(54 with LN meta)					miR-148a↑	P < 0.001	Correlation with pN stage		(e)
I-IV	69		Plasma	qRT-PCR	miR-21↑	CSS: P = 0.0451	miR-21 high vs low		(c)	Komatsu et al[186]
						CSS: P = 0.0133			(d)
I-II	80	70	Plasma	qRT-PCR	miRNA-199a-3p↑	P < 0.001	AUC = 0.818	Pt. (n = 80) vs Ctrl. (n = 70)	(a)	Li et al[187]
		(healthy controls)				P = 0.004	Pt. (n = 80) vs pancreous disease (n = 20)		(a)
		20				P = 0.012	Pre-op > post-op (n = 30)		(b)
		(precancerous disease)
Long non-coding RNA
I-IV	43	33	Plasma	qRT-PCR	H19	P = 0.029		Pt. (n = 43) vs Ctrl. (n = 33)	(a)	Arita et al[128]
					HOTAIR	P = 0.096			(a)
					MALAT1	P = 0.14			(a)

a: Unpaired t-test/Mann-Whitney U-test; b: Wilcoxon test; c: Kaplan-Meier survival curves, Log-rank test; d: Logistic regression model (multivariate); e: Kruskal-Wallis test. AUC: Area under the receiver operating characteristic curve; CSS: Cause-specific survival; LN: Lymph node; N/A: Not available; qRT-PCR: Quantitative real-time polymerase chain reaction; OS: Overall survival.