Helicobacter pylori and EBV in gastric carcinomas: Methylation status and microsatellite instability

Table 1 Primer Sequences and PCR conditions for methylation-specific PCR (MSP) analysis

Gene	Primer (5’-3’) forward	Primer (5’-3’) reverse	Size (bp)	T (°C)	Ref.
COX2	M TTAGATACGGCGGCGGCGGC	TCTTTACCCGAACGCTTCCG	161	68	[24]
	U ATAGATTAGATATGGTGGTGGTGGT	CACAATCTTTACCCAAACACTTCCA	171	67
DAPK	M GGATAGTCGGATCGAGTTAACGTC	CCCTCCCAAACGCCGA	98	65	[25]
	U GGAGGATAGTTGGATTGAGTTAATGTT	CAAATCCCTCCCAAACACCAA	116	65
CDH1	M TTAGGTTAGAGGGTTATCGCGT	TAACTAAAAATTCACCTACCGAC	115	61	[26]
	U TAATTTTAGGTTAGAGGGTTATTGT	CACAACCAATCAACAACACA	97	59
hMLH1	M TATATCGTTCGTAGTATTCGTGT	TCCGACCCGAATAAACCCAA	153	65	[26]
	U TTTTGATGTAGATGTTTTATTAGGGTTGT	ACCACCTCATCATAACTACCCACA	124	63
CDKN2A	M TTATTAGAGGGTGGGGCGGATCGC	GACCCCGAACCGCGACCGTAA	150	70	[21]
	U TTATTAGAGGGTGGGGTGGATTGT	CAACCCCAAACCACAACCATAA	151	70

Table 2 Clinicopathological and epidemiological features of patients in the four tumor stages n (%)

Variables	Patients	Tumor stage
		0-I	II	III	IV
Age (yr)
≤ 50	19	3 (15.8)	2 (10.5)	9 (47.4)	5 (26.3)
> 50	70	8 (11.4)	11 (15.7)	30 (42.9)	21 (30)
Gender
Male	60	8 (13.3)	8 (13.3)	27 (45)	17 (28.3)
Female	29	3 (10.3)	5 (17.2)	12 (41.4)	9 (31)
Anatomic subsite
Proximal (Cardia)	13	1 (7.7)	1 (7.7)	5 (38.5)	6 (46.1)
Distal (Antrum/body)	46	2 (4.3)	9 (19.6)	21 (45.7)	14 (30.4)
Mixed	3	0 (0)	1 (33.3)	2 (66.7)	0 (0)
Laurén type
Diffuse	31	4 (12.9)	4 (12.9)	12 (38.7)	11 (35.5)
Intestinal	57	7 (12.3)	9 (15.8)	27 (47.4)	14 (24.6)
Mixed	1	0 (0)	0 (0)	0 (0)	1 (100)

Table 3 Associations between gene methylation status and clinicopathological features, age, H. pylori, MSI and EBV n (%)

	Methylated genes
	Total	CDH1	P-value	DAP-kinase	P-value	COX2	P-value	hMLH1	P-value	CDKN2A	P-value
Mean age (yr)		60.2		59.9		60.9		63.1		62.5
Age group (yr)
≤ 50	19	10 (52.6)	0.883	8 (44.4)	0.282	9 (50)	0.179	6 (33.3)	0.903	3 (15.8)	0.0351
> 50	69	35 (50.7)		41 (58.6)		45 (67.2)		21 (31.8)		29 (42)
Gender
Male	59	30 (50.8)	0.938	33 (55.9)	0.946	34 (59.6)	0.289	21 (35)	0.787	21 (35.6)	0.830
Female	29	15 (51.7)		16 (55.2)		20 (71.4)		11 (37.9)		11 (37.9)
Nodal metastases
Absent	17	8 (47.1)	0.708	9 (50)	0.586	11 (64.7)	0.910	6 (33.3)	0.903	6 (33.3)	0.764
Present	71	37 (52.1)		40 (57.1)		43 (63.2)		21 (31.8)		26 (37.1)
Tumor site
Cardia	13	8 (61.5)	0.855	9 (69.2)	0.427	10 (83.3)	0.228	4 (30.8)	0.304	8 (61.5)	0.266
Antrum	38	21 (55.3)		20 (52.6)		21 (56.8)		14 (37.8)		14 (35.9)
Body	10	5 (50)		7 (70)		7 (70)		1 (11.1)		4 (40)
Misted	3	3 (100)		2 (66.7)		3 (100)		0		0
Laurèn
Diffuse	30	17 (56.7)	0.503	10 (33.3)	0.628	19 (63.3)	0.973	8 (27.6)	0.481	10 (33.3)	0.628
Intestinal	57	28 (49.1)		22 (38.6)		34 (63)		19 (35.2)		22 (38.6)
Mixed	1	0 (0.0)		0		1 (100)		0		0
H. pylori
CagA+	55	31 (56.4)	0.261	30 (54.5)	0.782	35 (66)	0.536	15 (28.8)	0.41	22 (40)	0.468
CagA-	34	15 (44.1)		19 (57.6)		19 (59.4)		12 (37.5)		11 (32.4)
MS status
MSI	14	5 (35.7)	0.174	4 (26.7)	0.0121	8 (53.3)	0.349	10 (66.7)	0.0011	4 (26.7)	0.405
MSS	72	40 (55.6)		44 (62)		45 (66.2)		16 (23.5)		27 (38)
EBV
Positive	5	1 (20)	0.101	4 (80)	0.346	4 (80)	0.694	0	0.179	3 (60)	0.462
Negative	48	28 (58.3)		28 (58.3)		33 (71.7)		15 (31.9)		21 (42.9)

¹Statistically significant result.