Association between Helicobacter pylori infection, mismatch repair, HER2 and tumor-infiltrating lymphocytes in gastric cancer

Table 1 Clinicopathological features and prognostic value

Feature	Total (n = 503)	%	Median	5 yr DFS	P value	Median	5 yr OS	P value
Age, yr (19-95 yr)
< 60	231	45.9	4.6	49.1	0.043	7.0	52.5	0.009
≥ 60	272	54.1	3.0	39.6		3.7	41.6
Sex
Female	252	50.1	3.3	43.7	0.582	3.8	45.3	0.319
Male	251	49.9	4.0	44.6		4.5	48.2
Bormann
I-II	90	18.1	NA	65.4	< 0.001	NA	66.0	0.002
III	311	62.7	2.9	40.6		4.0	44.4
IV-V	95	19.2	2.6	36.8		2.8	37.6
Lauren (n = 496)
Intestinal	222	44.8	4.3	39.9	0.716	4.8	49.3	0.598
Diffuse	181	36.5	3.0	46.2		4.1	43.9
Mixed	93	18.8	2.3	42.9		3.2	43.2
Grade
1	46	9.1	NA	62.2	0.025	NA	67.8	0.044
2	160	31.8	4.4	46.9		4.5	49.0
3	297	59.1	2.5	39.7		3.6	42.3
ILV
No	149	29.6	6.9	62.4	< 0.001	8.1	66.4	< 0.001
Yes	354	70.4	2.4	36.5		3.0	38.5
Antrum
Yes	323	64.2	2.7	47.9	0.064	3.5	51.0	0.104
No	180	35.8	4.5	38.8		5.1	40.8
Clinical stage
I	64	12.7	NA	80.7	< 0.001	NA	83.0	< 0.001
II	138	27.4	7.7	59.6		7.7	59.5
III	301	59.8	1.9	29.3		2.3	33.2
Node involvement
No	139	27.6	10.5	67.0	< 0.001	NA	68.2	< 0.001
Yes	364	72.4	2.3	35.4		3.0	38.4
Recurrence
No	322	64.0	-	-	-	NA	68.5	< 0.001
Yes	181	36.0	-	-		1.7	12.3
H. pylori HE (n = 415)
Absent	184	44.3	3.8	42.9	0.252	4.5	46.0	0.637
Present	231	55.7	7.3	48.3		5.3	51.2
H. pylori qPCR (n = 234)
Negative	85	36.3	2.5	36.8	0.229	3.1	39.1	0.219
Positive	149	63.7	3.8	38.3		4.0	39.7
dMMR (n = 479)
No	269	65.6	4.3	45.8	0.396	4.7	47.9	0.158
Yes	141	34.4	3.1	42.6	-	3.6	44.7	-
HER-2 status (n = 468)
Negative	434	95.0	3.5	43.4	0.868	4.3	46.2	0.892
Positive	23	5	3.1	39.8		3.3	39.3
IT TIL (n = 462)
< 10	186	40.3	2.7	41.8	0.763	3.7	43.6	0.395
≥ 10	276	59.7	3.7	44.0		4.5	47.1
ST TIL (n = 461)
< 30	258	55.9	2.6	41.5	0.458	3.8	45.0	0.853
≥ 30	203	44.0	3.8	44.8		4.4	46.3
IB TIL (n = 332)
< 70	178	53.6	3.3	46.0	0.991	4.4	47.8	0.660
≥ 70	154	46.4	4.4	46.4		4.9	49.4
IT CD3/HPF (n = 453)
< 58	126	27.8	2.4	34.3	0.028	2.9	36.5	0.021
≥ 58	327	72.2	4.4	47.0		5.4	50.5
IT CD8/ HPF (n = 443)
< 70	231	52.1	4.4	47.4	0.060	4.8	49.9	0.142
≥ 70	212	47.9	2.6	40.4		4.0	43.8
IT CD163/HPF (n = 205)
< 240	183	89.3	2.8	42.7	0.002	3.8	45.3	0.003
≥ 240	22	10.7	1.4	0.0		1.5	0.0
IT CD8/CD3 ratio (n = 432)
< 0.63	210	48.6	4.5	49.1	<0.001	6.0	52.9	0.001
≥ 0.63	222	51.4	2.3	36.0		2.8	38.5
IT CD3/CD163 (n = 179)
< 8	137	76.5	22	33.9	0.004	2.6	36.6	0.002
≥ 8	42	23.5	NA	59.5		NA	64.3
ST CD3/HPF (n = 363)
< 95	180	49.6	2.6	36.6	0.014	2.9	38.7	0.003
≥ 95	183	50.4	5.2	50.7		7.0	54.6
ST CD8/HPF (n = 341)
< 68	216	63.3	3.8	43.5	0.578	4.0	45.0	0.382
≥ 68	125	36.7	3.6	43.0		4.5	47.5
ST CD8/CD3 ratio (n = 328)
< 1.2	277	84.5	4.3	45.9	0.005	4.6	49.1	0.002
≥ 1.2	51	15.5	1.9	26.1		2.0	26.6

NA: Not available; HE: Hematoxylin and eosin; IT: Intratumoral; ST: Stromal; IB: Invasive border; TIL: Tumor-infiltrating lymphocytes; dMMR: Deficient mismatch-repair; H. pylori: Helicobacter pylori; HPF: High power field; DFS: Disease-free survival; OS: Overall survival; qPCR: Quantitative PCR.

Table 2 Relationship between level of infiltrating lymphocytes and clinicopathological features

Features	IT TILs	P value	ST TILs	P value	IB TILs	P value
Median	> 10%		> 30%		> 70%
Age		0.762		< 0.001		0.279
< 60	59.1		33.3		43
≥ 60	60.4		54.8		48.9
Sex		0.382		0.074		0.678
Female	57.7		39.8		45.1
Male	61.7		48.1		47.4
Gastric region location		0.450		0.049		0.160
No antrum	62		50		51.1
Antrum	58.4		40.5		43.3
Bormann		0.221		0.379		0.094
1	54.5		50		44.4
2	47.3		36.4		38.8
3	63.3		47.6		52.2
4	61		38.3		33.3
5	50		50		28.6
Lauren histology		0.891		< 0.001		0.002
Intestinal	58.7		58.74		53.1
Diffuse	58.9		20.4		28.6
Mixed	61.6		50		42.6
Grade		0.038		< 0.001		0.716
1	50		58.3		52.8
2	53.5		55.5		45.6
3	64.6		35.6		45.8
Lymphovascular invasion		0.028		0.520		0.968
No	51.9		41.7		46.2
Yes	62.9		44.9		46.5
Lymph node involvement		0.403		0.714		0.029
Yes	60.9		43.5		42.5
No	56.6		45.5		55.6
Pathology stage		0.016		0.001		0.001
I	42		28		41.5
II	65.4		55.8		60.4
III	60.3		41.5		38.9
Recurrence		0.230		0.003		0.369
No	61.9		49.5		48
Yes	56.3		35.2		42.7
H. pylori HE		0.009		0.445		0.411
Absent	65.9		47.6		47.5
Present	52.4		43.6		42.5
H. pylori qPCR		0.002		0.047		0.010
Absent	67.1		48.6		50.9
Present	43.7		34.1		29.9
CagA/H. pylori+		0.761		0.275		0.002
CagA-	40		46.7		64.3
CagA+	44.1		32.4		24.1
VacAs/H. pylori+		0.332		0.719		0.045
VacAs-	51.9		37		47.6
VacAs+	41.4		33.3		25
VacAs1/H. pylori+		0.530		0.887		0.894
VacAs1-	38.5		32.7		24.4
VacAs1+	44.7		34		25.7
VacAm/H. pylori+		0.824		0.535		0.265
VacAm-	41.9		38.7		38.5
VacAm+	44.2		32.6		26.8
VacAm1/H. pylori+		0.154		0.093		0.508
VacAm1-	55.2		44.8		21.7
VacAm1+	39.4		27.3		29.2
VacAs1+m1+/H. pylori+		0.937		0.953		0.527
No VacAs1+m1+	44.2		32.7		24.4
VacAs1+m1+	43.3		66.7		31.8
dMMR		0.875		0.019		0.010
No	58.7		38.9		38.6
Yes	59.6		51.5		54.2
HER2 positive		0.498		0.420		0.55
No	60.1		44.1		47.2
Yes	52.4		47.6		35.3

HE: Hematoxylin and eosin; IT: Intratumoral, ST: Stromal; TIL: Tumor-infiltrating lymphocytes; dMMR: Deficient mismatch-repair; H. pylori: Helicobacter pylori; HPF: High power field.

Table 3 Relationship between density of infiltrating immune cells and clinico-pathological features

Features	IT CD3	P value	ST CD3	P value	IT CD8	P value	ST CD8	P value	IT CD163	P value
Median	> 58		> 95		> 70		> 68		> 240
Age		0.714		0.275		0.026		0.224		0.162
< 60	73		47		42.1		32.9		7.4
≥ 60	71.5		52.8		52.7		39.3		13.5
Sex		0.039		0.557		0.814		0.699		0.354
Female	76.5		52		48.4		37.7		8.7
Male	67.8		49		47.3		35.7		12.7
Gastric region location		0.503		0.585		0.795		0.901		0.031
No antrum	74.1		52.4		48.7		36.2		16.7
Antrum	71.1		49.4		47.4		36.9		7.1
Bormann		0.648		0.441		0.338		0.006		0.462
I-II	70.4		47.4		41.8		22.6		5.3
III	73.6		55.6		50.7		43		11.7
IV-V	68.9		52.9		46.1		30		13
Lauren histology		0.03		0.003		0.308		0.003		0.038
Intestinal	66.2		57.4		43.7		40.3		8.2
Diffuse	78		36.6		50		22.2		17.8
Mixed	75.9		50.8		52.7		46.3		2.9
Grade		<0.001		0.311		<0.001		0.083		0.02
1	59		55.3		26.8		20.5		3.8
2	62.9		54.2		40.7		38.2		4.3
3	79.5		46.4		55.3		39.2		16.4
Lymphovascular invasion		0.602		0.241		0.092		0.117		0.865
No	73.9		55.2		41.8		30.4		11.3
Yes	71.5		48.4		50.5		39.3		10.5
Lymph node involvement		0.723		0.983		0.487		0.141		0.252
Yes	72.6		50.4		48.9		39.1		12.1
No	71		50.5		45.2		30.6		6.3
Pathology stage		0.942		0.101		0.674		0.058		0.197
I	70.9		50		42.9		20		9.5
II	73.2		59		47.2		41		4.1
III	72		46.3		49.2		37.8		13.3
Recurrence		0.02		0.079		0.448		0.475		0.389
No	76		53.9		46.6		35.2		9.3
Yes	65.9		44.4		50		39.1		13.2
H. pylori HE		0.547		0.817		0.356		0.97		0.637
Absent	71.1		51.4		49.1		36		13.4
Present	73.9		52.8		44.2		36.2		11.1
H. pylori qPCR		0.143		0.335		0.001		0.001		0.105
Absent	83.1		55.9		62.7		49.1		26.1
Present	74		48.1		37		22.8		14.1
CagA/H. pylori+		0.254		0.603		0.023		0.54		0.729
CagA-	82.9		42.9		57.5		21.2		14.3
CagA+	74.2		48		37.2		26.6		17.2
VacAs/H. pylori+		0.992		0.843		0.071		0.855		0.823
VacAs-	76.8		45.8		51.7		24.4		15.2
VacAs+	76.9		47.6		37.3		25.9		16.9
VacAs1/H. pylori+		0.397		0.432		0.942		0.234		0.543
VacAs1-	79.2		53.5		36.4		18.4		14.7
VacAs1+	72.2		44.7		37		30		20.8
VacAm/H. pylori+		0.1		0.248		0.255		0.884		0.865
VacAm-	69.4		40.4		47.6		24.5		17.1
VacAm+	80.6		50.6		38.7		25.6		15.8
VacAm1/H. pylori+		0.145		0.098		0.242		0.822		0.877
VacAm1-	89.7		65.2		48.1		23.8		16.7
VacAm1+	77		44.8		35.4		26.3		15.2
VacAs1+m1+/H. pylori+		0.113		0.289		0.913		0.253		0.345
No VacAs1+m1+	86		57.5		37.7		21.6		12.9
VacAs1+m1+	72.2		44		38.9		34.6		23.5
dMMR		0.393		0.049		0.095		0.05		0.169
No	75.4		47.6		45.5		32.3		11.5
Yes	71.3		59.5		54.7		43.9		5.1
HER2 status		0.06		0.204		0.009		0.404		0.835
Negative	74.4		52.4		49.9		37.7		11.1
Positive	56.5		38.1		21.7		28.6		9.1

HE: Hematoxylin and eosin; IT: Intratumoral; ST: Stromal; HPF: High power field; H. pylori: Helicobacter pylori; dMMR: Deficient mismatch-repair; qPCR: Quantitative PCR.

Table 4 Multivariate analysis of factors associated with disease-free survival and overall survival

Features	DFS		OS
	HR	P value	HR	P value
Age
< 60	Reference	-	Reference	-
> 60	1.8 (1.2, 2.5)	0.002	1.9 (1.3, 2.8)	< 0.001
Histological grade
Differentiated	Reference		Reference
Undifferentiated	1.7 (1.01, 2.7)	0.043	2.0 (1.2, 3.3)	0.007
WHO classification
1-2	Reference		Reference
3-4	2.5 (1.6, 3.8)	< 0.001	2.4 (1.5, 3.7)	< 0.001
Clinical stage
I-II	Reference		Reference
III	2.1 (1.4, 3-2)	< 0.001	1.9 (1.2, 2.9)	0.004
IT CD3/HPF
High (> 58)	Reference		Reference
Low (< 58)	1.5 (1.02, 2.2)	0.037	1.6 (1.1, 2.4)	0.018
IT CD8/CD3 ratio
< 0.63	Reference		Reference
> 0.63	1.7 (1.2, 2.5)	0.002	1.6 (1.1, 2.3)	0.008
Non-significant variables
Sex	-	0.939	-	0.51
Cardia-fundus	-	0.398	-	0.212
Bormann	-	0.328	-	0.315
Lauren	-	0.311	-	0.291
Lymph nodes	-	0.836	-	0.663
lymphovascular invasion	-	0.972	-	0.277
H. pylori	-	0.123	-	0.140
Deficient mismatch-repair	-	0.973	-	0.365
HER2	-	0.222	-	0.114
IT TIL	-	0.940	-	0.547
Stromal TIL	-	0.818	-	0.806
IT CD8/HPF	-	0.077	-	0.340

DFS: Disease-free survival; OS: Overall survival; HR: Hazard ratio; WHO: World Health Organization; IT: Intratumoral; TIL: tumor-infiltrating lymphocytes; HPF: High power field; H. pylori: Helicobacter pylori.