Helicobacter pylori virulence gene expression defines a stage-associated transcriptional signature in gastric cancer

Table 1 Baseline demographics and significant clinical characteristics (n = 200), n (%)

Variable	Overall (n = 200)	Stage I (n = 68)	Stage II (n = 65)	Stage III (n = 22)	Stage IV (n = 45)	P value
Age (years)	52.9 ± 16.4	51.2 ± 15.4	54.3 ± 19.3	56.1 ± 12.4	52.0 ± 15.0	0.532
Gender						0.727
Female	88 (44.0)	27 (39.7)	32 (49.2)	10 (45.5)	19 (42.2)
Male	112 (56.0)	41 (60.3)	33 (50.8)	12 (54.5)	26 (57.8)
Histological Dx						0.034a
Adenocarcinoma	129 (64.5)	43 (63.2)	38 (58.5)	20 (90.9)	28 (62.2)
Others	71 (35.5)	25 (36.8)	27 (41.5)	2 (9.1)	17 (37.8)
Histological grade						0.001b
Well	47 (23.5)	14 (20.6)	18 (27.7)	6 (27.3)	9 (20.0)
Moderate	48 (24.0)	13 (19.1)	14 (21.5)	5 (22.7)	16 (35.6)
Poorly	47 (23.5)	9 (13.2)	14 (21.5)	10 (45.5)	14 (31.1)
Atypical	58 (29.0)	32 (47.1)	19 (29.2)	1 (4.5)	6 (13.3)
H. pylori (histology)						0.042a
Positive	130 (65.0)	36 (52.9)	48 (73.8)	17 (77.3)	29 (64.4)
Negative	70 (35.0)	32 (47.1)	17 (26.2)	5 (22.7)	16 (35.6)
Survival status						< 0.001b
Deceased	65 (32.5)	15 (22.1)	17 (26.2)	10 (45.5)	23 (51.1)
Under treatment	82 (41.0)	15 (22.1)	36 (55.4)	11 (50.0)	20 (44.4)
Cured	53 (26.5)	38 (55.9)	12 (18.5)	1 (4.5)	2 (4.4)

^aP < 0.05.

^bP < 0.01. One-way ANOVA was used to calculate P values for the continuous variable (age), whereas the χ² test or Fisher’s exact test, as appropriate, was used for categorical variables. H. pylori: Helicobacter pylori.

Table 2 Prevalence and association of Helicobacter pylori virulence markers, n (%)

Marker	Gastric cancer positive (n = 159)	Controls (n = 200)	Odds ratio	95%CI	P value
cagA	131 (82.4)	40 (20.0)	18.7	10.9-32.3	< 0.001b
vacA	148 (93.1)	74 (37.0)	22.9	11.6-45.2	< 0.001b
babA	117 (73.6)	33 (16.5)	14.1	8.6-23.2	< 0.001b
iceA	58 (36.5)	32 (16.0)	3.01	1.84-4.94	< 0.001b

^bP < 0.01.

cagA: Cytotoxin-associated gene A; vacA: Vacuolating cytotoxin A; babA: Blood group antigen-binding adhesin A; iceA: Induced by contact with epithelium gene A.

Table 3 Distribution of virulence factor accumulation by clinical stage, n (%)

Virulence factor accumulation	Stage I (n = 50)	Stage II (n = 54)	Stage III (n = 18)	Stage IV (n = 37)	P value
1 gene	5 (10.0)	2 (3.7)	0 (0.0)	5 (13.5)
2 genes	5 (10.0)	4 (7.4)	5 (27.8)	8 (21.6)
3 genes	34 (68.0)	36 (66.7)	13 (72.2)	19 (51.4)
4 genes	6 (12.0)	12 (22.2)	0 (0.0)	5 (13.5)	0.045a

^aP < 0.05.

Table 4 Fold induction of Helicobacter pylori virulence genes relative to healthy controls (n = 159)

Clinical stage	cagA	vacA	babA	iceA
Stage I	3.11	3.15	5.68	0.83
Stage II	4.81	2.90	1.80	2.35
Stage III	4.53	4.06	0.67	0.68
Stage IV	4.26	2.41	1.59	0.52

cagA: Cytotoxin-associated gene A; vacA: Vacuolating cytotoxin A; babA: Blood group antigen-binding adhesin A; iceA: Induced by contact with epithelium gene A.

Table 5 Relative mRNA expression of Helicobacter pylori virulence factors by tumor-node-metastasis stage (n = 159)

Virulence gene	Stage I median (IQR)	Stage II median (IQR)	Stage III median (IQR)	Stage IV median (IQR)	P value
cagA	1.93 (1.04-3.36)	3.20 (1.51-5.98)	3.80 (1.57-6.49)	3.11 (1.09-6.20)	< 0.001b
vacA	2.06 (1.36-3.48)	2.42 (1.41-3.92)	3.13 (1.85-3.91)	1.79 (1.12-3.20)	< 0.001b
babA	4.11 (1.62-5.41)	1.39 (1.03-2.11)	0.57 (0.30-1.11)	1.19 (0.54-1.72)	< 0.001b
iceA	0.63 (0.39-0.94)	0.86 (0.46-3.17)	0.54 (0.40-0.91)	0.65 (0.30-1.41)	0.027a

^aP < 0.05.

^bP < 0.01; the Kruskal-Wallis test was used for comparisons across the four stages. cagA: Cytotoxin-associated gene A; vacA: Vacuolating cytotoxin A; babA: Blood group antigen-binding adhesin A; iceA: Induced by contact with epithelium gene A; IQR: Interquartile range.

Table 6 Spearman correlation matrix of virulence factor expression

Gene	cagA	vacA	babA	iceA
cagA	1.00	0.31	0.08	-0.18
vacA	0.31	1.00	0.22	-0.06
babA	0.08	0.22	1.00	-0.03
iceA	-0.18	-0.06	-0.03	1.00

cagA: Cytotoxin-associated gene A; vacA: Vacuolating cytotoxin A; babA: Blood group antigen-binding adhesin A; iceA: Induced by contact with epithelium gene A.

Table 7 Multivariate ordinal logistic regression parameter estimates (n = 159)

Predictor/threshold	(β)	Std. Error	z-statistic	P value	OR	95%CI
babA	-0.624	0.107	-5.814	< 0.001b	0.536	0.428-0.654
iceA	-0.221	0.085	-2.585	0.010a	0.802	0.675-0.945
cagA	0.073	0.047	1.544	0.123	1.076	0.981-1.182
vacA	0.009	0.091	0.095	0.924	1.009	0.843-1.205
Stage I|II	-2.368	0.441	-5.376	< 0.001b	-	-
Stage II|III	-0.472	0.393	-1.201	0.230	-	-
Stage III|IV	0.176	0.389	0.452	0.651	-	-

^aP < 0.05.

^bP < 0.01.

cagA: Cytotoxin-associated gene A; vacA: Vacuolating cytotoxin A; babA: Blood group antigen-binding adhesin A; iceA: Induced by contact with epithelium gene A; OR: Odds ratio.

Table 8 Model selection and comparison statistics

Model specification	Log-likelihood	AIC (lower is better)	Model χ2	P value
Univariate (cagA)	-207.67	423.33	3.30	0.069
Toxigenic axis (cagA + vacA)	-207.17	424.34	4.29	0.117
Full multivariate panel	-181.50	377.01	55.63	< 0.001b

^bP < 0.01.

AIC: Akaike information criterion; cagA: Cytotoxin-associated gene A; vacA: Vacuolating cytotoxin A.

Table 9 Stage-associated transcriptional signature validation and correlation analysis (n = 159)

Parameter/variable	Spearman (ρ)	P value	VIF	ROC-AUC (95%CI)	Directional trend/validation
babA	-0.467	< 0.001b	1.05	0.702 (0.632-0.795)	Downregulation
iceA	-0.133	0.155	1.00	0.584 (0.540-0.717)	Variable
cagA	0.122	0.126	1.05	0.612 (0.453-0.645)	Upregulation
vacA	-0.010	0.210	1.10	0.528 (0.457-0.647)	Neutral
Combined model (apparent, full-cohort)	ρ = 0.494	< 0.001b	1.02	0.769	Linear fit
Combined model (Nested 5-fold CV)	-	-	-	0.747 (0.664-0.823)	Calibration: Hosmer-Lemeshow χ2 = 5.53, df = 3, P > 0.05
5-fold cross-validation (Cauchit)	-	-	-	-	Accuracy: 0.535; Kappa: 0.325
CART - decision tree (apparent, full-cohort)	-	-	-	0.867	Sensitivity: 74.5%; specificity: 81.7%
CART - decision tree (Nested 5-fold CV)	-	-	-	0.68 (95%CI: 0.59-0.77)	Sensitivity: 54.5%; specificity: 75.0%

^bP < 0.01.

cagA: Cytotoxin-associated gene A; vacA: Vacuolating cytotoxin A; babA: Blood group antigen-binding adhesin A; iceA: Induced by contact with epithelium gene A; VIF: Variance inflation factor; ROC: Receiver operating characteristic; AUC: Area under the curve; CART: Classification and regression tree; CV: Cross-validation.