Circulating tumor and cancer stem cells in hepatitis C virus-associated liver disease

doi:10.3748/wjg.v20.i48.18240

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World Journal of Gastroenterology

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1007-9327

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Baishideng Publishing Group Inc, 7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA

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World J Gastroenterol. Dec 28, 2014; 20(48): 18240-18248
Published online Dec 28, 2014. doi: 10.3748/wjg.v20.i48.18240

Table 1 Primer sequences

Gene	Primers sequences	Annealing temp.(°C)	Base pairs
MAGE1	First round:	60	421
	5’-CGG CCG AAG GAA CCT GAC CCA G-3’
	5’-GCT GGA ACC CTC ACT GGG TTG CC-3’
	Second round (nested):	60	299
	5’-ACA GAG GAG CAC CAA GGA GAA G-3’
	5’-AGT TGA TGG TAG TGG GAA AGG C-3’
MAGE3	First round:	60	725
	5’-TGG AGG ACC AGA GGC CCC C-3’
	5’-GGA CGA TTA TCA GGA GGC CTG C-3’
	Second round (nested):	60	371
	5’-CGG AGG AGC ACT GAA GGA GAA G-3’
	5’-CCT CCT CTT CTT CGT TGC TGG-3’
CK-19	5’-CCC GCG ACT ACA GCC ACTA-3’	60	745
CK-19	5’-GCT CAT GCG CAG AGC CT-3’	60	745
CD133	P1: 5’-AAT TCT AAT ACG ACT CAC TAT AGG GAA GAA CAG GGA TGA TGT TGG GTC TCA-3’
	P2: 5’-TTT CAA GGA CTT GCG AAC TCT CTT GA-3’
	MB: 5’-CGA TCC AAG GAC AAG GCG TTC ACA GGA TCG-3’
Telomerase	5’-TGA CAC CTC ACC TCA CCC AC-3’	60	96
Telomerase	5’-CAC TGT CTT CCG CAA GTT CAC-3’	60	96
β-actin	5’-ACA CTG TGC CCA ACG AGG-3’	56	540
β-actin	5’-AGG GGC CGG TCA TAC T-3’'	56	540

Table 2 Laboratory findings

		HCC (n = 70)	CH (n = 30)	Control (n = 33)
Age, mean (range)		57.32 (20-74)^a	47.70 (43-60)^a	51.09 (31-75)
Gender (male/female)		67/3	21/9	33/0
AFP	Mean (range)	9547.54 (15-117032)^a^c	11.02 (1-30)^a	7.45 (4-10)
AFP	Median	1481.5^a^c	8.9^a	7.9
Total bilirubin	Mean (range)	2.66 (0.8-8.6)^a	4.73 (0.6-114.0)	0.60 (0.3-1.0)
Total bilirubin	Median	2.0^a	1	0.6
ALT	Mean (range)	71.39 (17-189)^a	54.23 (10-200)^a	24.91 (16-37)
ALT	Median	57.5^a	34.5^a	25
AST	Mean (range)	122.94 (35-530)^a^c	54.47 (12-140)^a	28.45 (17-39)
AST	Median	88.5^a^c	39.5^a	28
Creatinine	Mean (range)	1.09 (0.4-2.0)^a^c	0.84 (0.5-1.1)	0.81 (0.4-1.4)
Creatinine	Median	1.0^a^c	0.9	0.8
Alkaline phosphatase	Mean (range)	240.50 (76-580)^a^c	138.90 (80-220)^a	44.39 (15-71)
Alkaline phosphatase	Median	218.5^a^c	132.5^a	44

^aP < 0.05 vs controls;

^cP < 0.05 vs CH. AFP: Alpha-fetoprotein; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; CH: Chronic hepatitis; HCC: Hepatocellular carcinoma.

Table 3 Expression of CD133, CD90 and CK19

		HCC (n = 70)	CH (n = 30)	Control (n = 33)
CD133	mean ± SD	75.34 ± 83.67	1205.57 ± 952.59	205.97 ± 71.88
CD133	Median	47.5^a^c	1245.0^a	200
CK19	mean ± SD	358.09 ± 335.30	392.20 ± 886.61	0.00
CK19	Median	215.5^a	64.5^a	0.0
CD90	mean ± SD	82.75 ± 15.30	43.20 ± 18.50	0.00
CD90	Median	65.3^a^c	34.1^a	0.0

CK: Cytokeratin; CH: Chronic hepatitis; HCC: Hepatocellular carcinoma; SD: Standard deviation.

^aP < 0.05 vs controls;

^cP < 0.05 vs CH.

Table 4 Expression of telomerase and melanoma antigen encoding genes n (%)

Gene		HCC (n = 70)	CH (n = 30)	Control (n = 33)	P value	χ²
Telomerase	+++	26 (37.14)	0 (0.00)	0 (0.00)	< 0.001	49.663
	++	13 (18.57)	0 (0.00)	0 (0.00)
	-	31 (44.29)	30 (100)	33 (100)
MAGE1	+++	9 (12.86)	0 (0.00)	0 (0.00)	< 0.001	55.246
	++	33 (47.14)	0 (0.00)	0 (0.00)
	-	28 (40.00)	30 (100)	33 (100)
MAGE3	+++	24 (34.29)	0 (0.00)	0 (0.00)	< 0.001	55.246
	++	20 (28.57)	0 (0.00)	0 (0.00)
	-	26 (37.14)	30 (100)	33 (100)

CH: Chronic hepatitis; HCC: Hepatocellular carcinoma; MAGE: Melanoma antigen encoding.

Table 5 Marker expression in relation to tumor stage and grade n (%)

Gene		Stage			Grade
Gene		Early (n = 18)	Late (n = 52)	P value	Low (n = 43)	High (n = 27)	P value
Telomerase	+++	2 (11.11)	24 (46.15)	0.019	8 (18.60)	18 (66.67)	< 0.001
	++	6 (33.33)	7 (13.46)		11 (25.58)	2 (7.41)
	-	10 (55.56)	21 (40.37)		24 (55.81)	7 (25.93)
MAGE1	+++	0 (0.00)	9 (17.31)	0.099	3 (6.98)	6 (22.22)	0.123
	++	8 (44.44)	25 (48.08)		20 (46.51)	13 (48.15)
	-	10 (55.56)	18 (34.62)		20 (46.51)	8 (29.63)
MAGE3	+++	2 (11.11)	22 (42.31)	< 0.001	6 (13.95)	18 (66.67)	< 0.001
	++	13 (72.22)	7 (13.46)		19 (44.19)	1 (3.70)
	-	3 (16.67)	23 (44.23)		18 (41.86)	8 (29.63)
CD133	+	0 (0.00)	28 (53.85)	< 0.001	9 (20.93)	19 (70.37)	< 0.001
CD133	-	18 (100.00)	24 (46.15)	< 0.001	34 (79.07)	8 (29.63)	< 0.001
CK19	+	10 (55.56)	52 (100.00)	< 0.001	35 (81.40)	27 (100.00)	< 0.017
CK19	-	8 (44.44)	0 (0.00)	< 0.001	8 (18.60)	0 (0.00)	< 0.017
CD90	+	6 (33.30)	31 (59.60)	0.015	9 (20.93)	19 (70.37)	< 0.001
CD90	-	12 (66.70)	11 (21.15)	0.015	34 (79.07)	8 (29.63)	< 0.001

CK: Cytokeratin; MAGE: Melanoma antigen encoding.

Citation: Bahnassy AA, Zekri ARN, El-Bastawisy A, Fawzy A, Shetta M, Hussein N, Omran D, Ahmed AAS, El-Labbody SS. Circulating tumor and cancer stem cells in hepatitis C virus-associated liver disease. World J Gastroenterol 2014; 20(48): 18240-18248
URL: https://www.wjgnet.com/1007-9327/full/v20/i48/18240.htm
DOI: https://dx.doi.org/10.3748/wjg.v20.i48.18240