RUNX3 Methylation, Loss of RUNX3 Expression and Clinicopathologic Findings according to Helicobacter pylori CagA in Gastric Carcinoma

Yoon Ju Na; Ki-Nam Shim; Yang Hee Joo; Seong-Eun Kim; Hye-Kyung Jung; Sung-Ae Jung; Min Sun Cho

doi:10.4166/kjg.2015.66.2.75

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Na, Shim, Joo, Kim, Jung, Jung, and Cho: RUNX3 Methylation, Loss of RUNX3 Expression and Clinicopathologic Findings according to Helicobacter pylori CagA in Gastric Carcinoma

Original Article

Korean J Gastroenterol 2015;66(2):75-84.

Published online: 4 October 2015

DOI: https://doi.org/10.4166/kjg.2015.66.2.75

RUNX3 Methylation, Loss of RUNX3 Expression and Clinicopathologic Findings according to Helicobacter pylori CagA in Gastric Carcinoma

Yoon Ju Na^1,³, Ki-Nam Shim¹, Yang Hee Joo¹, Seong-Eun Kim¹, Hye-Kyung Jung¹, Sung-Ae Jung¹, Min Sun Cho²

¹Department of Internal Medicine, Ewha Womans University School of Medicine, Seoul, Korea

²Department of Pathology, Ewha Womans University School of Medicine, Seoul, Korea

³Center for Health Promotion, Samsung Medical Center, Seoul, Korea

Correspondence to: Ki-Nam Shim, Department of Internal Medicine, Ewha Womans University School of Medicine, 1071 Anyangcheon-ro, Yangcheon-gu, Seoul 07985, Korea. Tel: +82-2-2650-2632, Fax: +82-2-2655-2076, E-mail: shimkn@ewha.ac.kr

Received 2 February 2015 Revised 16 June 2015 Accepted 26 June 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background/Aims

Helicobacter pylori cytotoxin-associated gene A (CagA) has been suggested to be involved in the inactivation of Runt-related transcription factor 3 (RUNX3), a known gastric carcinoma tumor suppressor gene. It remains unclear how H. pylori CagA initiates or maintains RUNX3 promoter methylation and inactivates its protein expression in gastric carcinoma.

Methods

RUNX3 promoter methylation status, RUNX3 expression, and H. pylori CagA were investigated in 76 sample pairs of gastric carcinoma tissue. The patients' medical records were reviewed. The association between RUNX3 methylation or loss of RUNX3 expression and clinicopathologic variables according to H. pylori CagA status were investigated.

Results

In gastric carcinoma patients with H. pylori CagA-positive infection, RUNX3 methylation did not show association with lymphatic invasion, venous invasion, and TNM stages. However RUNX3 methylation was observed more frequently in poorly differentiated adenocarcinoma and signet ring cell carcinoma (77.8% vs. 20.0%, p=0.023) in early stage. In gastric carcinoma patients with H. pylori CagA-positive infection, loss of RUNX3 expression did not show association with lymphatic invasion, venous invasion, and TNM stages. However loss of RUNX3 expression was observed more frequently in early gastric carcinoma than in advanced gastric carcinoma (84.2% vs. 75.0%, p=0.51), but this difference was not significant.

Conclusions

In gastric carcinoma patients with H. pylori CagA-positive infection, RUNX3 methylation or loss of RUNX3 expression did not show correlation with lymphovascular invasion and TNM stages. In early gastric carcinoma patients with H. pylori CagA-positive infection, RUNX3 methylation was observed more in poorly differentiated adenocarcinoma and signet ring cell carcinoma.

Keywords: Helicobacter pylori, CagA, RUNX3, Methylation, Carcinoma

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Fig. 1.

Representative results of methylation-specific PCR for RUNX3 and PCR for CagA in human normal and gastric cancer tissue samples. (A) Methylation specific PCR for RUNX3. Positive control, negative control, molecular weighted marker for RUNX3 and un-methylated bands (U) were seen in cancer tissue. (B) PCR for CagA. Presence of band in normal gastric mucosa or gastric carcinoma tissue was considered positive for Helicobacter pylori CagA.

Fig. 2.

Immunohistochemical stain for RUNX3 protein. (A) Adjacent mucosa surrounded by tumor showed cytoplasmic positive reaction in goblet cells (black arrowhead). The lymphocytes in lamina propria showed a nuclear positive reaction of lymphocytes (blue arrowhead) (×200). (B) The tumor cells showed a negative reaction (×200). (C) The tumor cells showed a strong brownish nuclear positive reaction (×200). (D) The tumor cells showed no nuclear positive reaction but a cytoplasmic positive reaction (cytoplasmic mislocalization) (black arrowheads) (×400).

Table 1.

Baseline Characteristics in Gastric Carcinoma Patients

Variable	Total (n=76)
Age (yr)	61.9±1.5
Gender (male)	57 (75.0)
H. pylori positive	59 (77.6)
H. pylori CagA positive	55 (72.4)
Differentiation
W/D, M/D	27 (35.5)
P/D, SRC	49 (64.5)
Location of tumor
Upper	12 (15.8)
Middle	17 (22.4)
Lower	45 (59.2)
Whole	2 (2.6)
Lymphatic invasion
Negative	26 (34.2)
Positive	50 (65.8)
Venous invasion
Negative	54 (71.1)
Positive	22 (28.9)
Depth of invasion
T1a (mucosa)	10 (13.2)
T1b (submucosa)	14 (18.4)
T2, 3, 4	52 (68.4)
Nodal status
N0	26 (34.2)
N1, 2, 3	50 (65.8)
Distant metastasis
M0	61 (80.3)
M1	15 (19.7)
Stage
I	22 (28.9)
II	15 (19.7)
III	24 (31.7)
IV	15 (19.7)

Values are presented as mean±SD or n (%).

W/D, tubular adenocarcinoma, well differentiated; M/D, tubular adenocarcinoma moderately differentiated; P/D, tubular adeno-carcinoma, poorly differentiated; SRC, signet ring cell cancer.

Table 2.

Correlation between Helicobacter pylori CagA Status and Clinicopathologic Variables in Gastric Carcinoma

Variable	H. pylori CagA positive (n=55)	H. pylori CagA negative (n=21)	p-value
Age (yr)	61.5±1.6	63.0±3.3	0.61
Gender (male)	45 (81.8)	13 (61.9)	0.14
RUNX3 methylation in normal tissue	8 (14.5)	2 (9.5)	0.72
RUNX3 methylation in cancer tissue	23 (41.8)	8 (38.1)	0.80
Loss of RUNX3 expression in cancer tissue	43 (78.2)	14 (66.7)	0.38

Values are presented as mean±SD or n (%).

Table 3.

Correlation between RUNX3 Methylation and Clinicopathologic Variables according to Helicobacter pylori CagA Status in Gastric Carcinoma (n=76)

Variable	H. pylori CagA positive (n=55)			H. pylori CagA negative (n=21)
Variable	RUNX3 methylation positive (n=23)	RUNX3 methylation negative (n=32)	p-value	RUNX3 methylation positive (n=8)	RUNX3 methylation negative (n=13)	p-value
Differentiation			1.00			1.00
W/D, M/D	9 (39.1)	13 (40.6)		2 (25.0)	3 (23.1)
P/D, SRC	14 (60.9)	19 (59.4)		6 (75.0)	10 (76.9)
Lymphatic invasion			0.78			0.047
Negative	8 (34.8)	13 (40.6)		4 (50.0)	1 (7.7)
Positive	15 (65.2)	19 (59.4)		4 (50.0)	12 (92.3)
Venous invasion			0.55			0.046
Negative	15 (65.2)	24 (75.0)		8 (100)	7 (53.8)
Positive	8 (34.8)	8 (25.0)		0 (0)	6 (46.2)
Depth of invasion			0.40			0.24
T1a	5 (21.7)	3 (9.3)		2 (25.0)	0 (0.0)
T1b	4 (17.4)	7 (21.9)		1 (12.5)	2 (15.4)
T2, 3, 4	14 (60.9)	22 (68.8)		5 (62.5)	11 (84.6)
Nodal status			1.00			0.012
N0	9 (39.1)	13 (40.6)		4 (50.0)	0 (0)
N1, 2, 3	14 (60.9)	19 (59.4)		4 (50.0)	13 (100)
Distant metastasis			1.00			1.00
M0	19 (82.6)	27 (84.4)		6 (75.0)	9 (69.2)
M1	4 (17.4)	5 (15.6)		2 (25.0)	4 (30.8)
Stage			0.55			0.29
I	6 (26.1)	11 (34.4)		3 (37.5)	2 (15.4)
II	8 (34.8)	6 (18.8)		1 (12.5)	0 (0)
III	5 (21.7)	10 (31.2)		2 (25.0)	7 (53.8)
IV	4 (17.4)	5 (15.6)		2 (25.0)	4 (30.8)

Values are presented as n (%).

W/D, tubular adenocarcinoma, well differentiated; M/D, tubular adenocarcinoma, moderately differentiated; P/D, tubular adenocarcinoma, poorly differentiated; SRC, signet ring cell cancer.

Table 4.

Correlation between RUNX3 Methylation and Clinicopathologic Variables according to Helicobacter pylori CagA Status in Early Gastric Carcinoma (n=24)

Variable	H. pylori CagA positive (n=19)			H. pylori CagA negative (n=5)
Variable	RUNX3 methylation positive (n=9)	RUNX3 methylation negative (n=10)	p-value	RUNX3 methylation positive (n=3)	RUNX3 methylation negative (n=2)	p-value
Differentiation			0.023			0.40
W/D, M/D	2 (22.2)	8 (80.0)		0 (0)	1 (50.0)
P/D, SRC	7 (77.8)	2 (20.0)		3 (100)	1 (50.0)
Lymphatic invasion			0.65			0.10
Negative	5 (55.6)	7 (70.0)		3 (100)	0 (0)
Positive	4 (44.4)	3 (30.0)		0 (0)	2 (100)
Venous invasion			0.58			0.40
Negative	7 (77.8)	9 (90.0)		3 (100)	1 (50.0)
Positive	2 (22.2)	1 (10.0)		0 (0)	1 (50.0)
Depth of invasion			0.37			0.40
1a	5 (55.6)	3 (30.0)		2 (66.7)	0 (0)
1b	4 (44.4)	7 (70.0)		1 (33.3)	2 (100)
Nodal status			0.65			0.10
N0	5 (55.6)	7 (70.0)		3 (100)	0 (0)
N1, 2, 3	4 (44.4)	3 (30.0)		0 (0)	2 (100)
Stage			0.63			1.00
I	6 (66.7)	8 (80.0)		3 (100)	2 (100)
II	3 (33.3)	2 (20.0)		0 (0)	0 (0)

Values are presented as n (%).

W/D, tubular adenocarcinoma, well differentiated; M/D, tubular adenocarcinoma, moderately differentiated; P/D, tubular adenocarcinoma, poorly differentiated; SRC, signet ring cell cancer.

Table 5.

Correlation between RUNX3 Expression and Clinicopathologic Variables according to Helicobacter pylori CagA Status in Gastric Carcinoma (n=76)

Variable	H. pylori CagA positive (n=55)			H. pylori CagA negative (n=21)
Variable	RUNX3 expression positive (n=12)	RUNX3 expression negative (n=43)	p-value	RUNX3 expression positive (n=7)	RUNX3 expression negative (n=14)	p-value
Differentiation			0.51			0.22
W/D, M/D	6 (50.0)	16 (37.2)		2 (28.6)	3 (21.4)
P/D, SRC	6 (50.0)	27 (62.8)		5 (71.4)	11 (78.6)
Lymphatic invasion			0.78			0.63
Negative	4 (33.3)	17 (40.0)		1 (14.3)	4 (28.6)
Positive	8 (66.7)	26 (60.0)		6 (85.7)	10 (71.4)
Venous invasion			0.30			0.12
Negative	7 (58.3)	32 (74.4)		3 (42.9)	12 (85.7)
Positive	5 (41.7)	11 (25.6)		4 (57.1)	2 (14.3)
Depth of invasion			0.89			0.43
T1a	1 (8.3)	7 (16.3)		0 (0)	2 (14.3)
T1b	2 (16.7)	9 (20.9)		2 (28.6)	1 (7.1)
T2, 3, 4	9 (75.0)	27 (62.8)		5 (71.4)	11 (78.6)
Nodal status			0.74			0.12
N0	4 (33.3)	18 (41.9)		0 (0)	4 (28.6)
N1, 2, 3	8 (66.7)	25 (58.1)		7 (100)	10 (71.4)
Distant metastasis			0.66			1.00
M0	11 (91.7)	35 (81.4)		5 (71.4)	10 (71.4)
M1	1 (8.3)	8 (18.6)		2 (28.6)	4 (28.6)
Stage			0.20			1.00
I	2 (16.7)	15 (34.9)		2 (28.6)	3 (21.4)
II	6 (50.0)	8 (18.6)		0 (0)	1 (7.1)
III	3 (25.0)	12 (27.9)		3 (42.8)	6 (42.9)
IV	1 (8.3)	8 (18.6)		2 (28.6)	4 (28.6)

Values are presented as n (%).

W/D, tubular adenocarcinoma, well differentiated; M/D, tubular adenocarcinoma, moderately differentiated; P/D, tubular adenocarcinoma, poorly differentiated; SRC, signet ring cell cancer.

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