Although the most extensive studies revealed the role of H. pylori VacA and CagA toxins in the development of gastric adenocarcinoma, the magnitude of this association and the correlations of vacA mosaicism and cagA status with cardia gastric adenocarcinoma (CGA) still remain controversial.
OBJECTIVE:
We aimed to examine the linkage of H. pylori highly cytotoxic genotypes to CGA in Iranian populations as a model.
METHODS:
A total of 601 Iranian patients were enrolled. Biopsies were cultured, genotyped, and anatomically and histologically classified.
RESULTS:
The vacA c1 genotype, but not cagA status, showed a strong association with the risk of both CGA and non-cardia adenocarcinoma (NCGA), whether the controls were non-tumors, as those with either non-atrophic gastritis or peptic ulcerations, (the OR (95%CI) was 14.11 (4.91–40.52) and 9.59 (4.06–22.65), respectively) or those with NAG (the OR (95%CI) was 10.71 (3.49–32.82) and 8.11 (3.26–20.16), respectively). The vacA c1/cagA genotype was significantly associated with an increased risk of NCGA, whether the controls were non-tumors or those with NAG; the adjusted risk was 4.706 (1.41–15.67) and 4.85 (1.42–16.51), respectively.
CONCLUSIONS:
The H. pylori vacA c1 genotype, but not cagA status, might be the first important bacterial biomarker for predicting the cardia adenocarcinoma risk in male patients aged 55 in Iran.
Although the incidence of stomach cancer is continually decreasing [1]; however, it remains as the fifth common cancer worldwide (constitutes 6.8% of the global cancer in the world) and the third cancer-related mortality (8.8%) [2]. Gastric adenocarcinoma (GA) accounts for approximately 90% of all stomach cancers that can be classified based on the anatomic site and histologic type of the tumor. Anatomically, there are two subtypes of GA, cardia (proximal: the upper part of the stomach adjoining the esophagus) gastric adenocarcinoma (CGA) and non-cardia (distal: the middle and distal parts of the stomach) gastric adenocarcinoma (NCGA). According to the Laurén classification, GA consists of two main histologic variants, intestinal and diffuse [3].
Several reports indicate that the incidence of CGA has steadily increased in the last several decades, especially in the Western countries [4, 5, 6]. This is in contrast to the decline in the incidence of NCGA worldwide [4, 7, 8]. NCGA is seen in higher rates in high-risk geographic regions, such as Eastern Europe, East Asia, Central, and South America [9], whereas the incidence of CGA is much higher in low risk populations [10], such as western populations [7, 8]. Intestinal-type gastric adenocarcinoma (IGA) is more common in high-risk areas, such as East Asia, East Europe, and South and Central America [11], whereas diffuse-type gastric adenocarcinoma (DGA) is uniformly distributed all over the world [12]. This difference in the incidence of GA rates based on the anatomic sub-site and histologic features may be due to differences in the prevalence of Helicobacter pylori (H. pylori) infection and differences in the genetic diversity of H. pylori strains. Many clinical and epidemiologic studies have shown that the colonization of the human stomach with H. pylori is as an important risk factor for NCGA development [13, 14] such that 77% of the cases of NCGA are caused by this bacterial infection [13]. In contrast, CGA has negative [14], positive [15] or no association with H. pylori infection [13, 16, 17].
Interestingly, in a meta-analysis study, the presence of H. pylori was related to an increased CGA risk in high-risk settings, but an inverse association existed in low-risk settings [18]. For example, several studies of Asian countries have shown a high positive correlation between H. pylori infection and CGA, whereas some studies of Western countries have found no association or an inverse association [13, 19]. It has been reported that H. pylori colonization is linked to both types, IGA and DGA [13, 20, 21], but in some studies have not found such a relationship between DGA and H. pylori infection; and more often associated with genetic abnormalities. Heterogeneity among H. pylori virulence genes may be an important factor in creation of the different types of GA. H. pylori heterogeneity has been strongly investigated the past two decades to identify possible virulence factors associated with the GA.
Polymorphic variations in H. pylorivacA gene result in differences in the levels of the cytotoxicity and pathogenicity. Variations in the signal (s) region (encodes a part of the N-terminal region of the 88-kDa mature protein), the middle (m) region (encodes a part of 55-kDa C-terminal (p) domain), the intermediate (i) region (situated between s and m regions, which has an important role in vacuolating activities), and the deletion (d) region (located between m and i regions) of vacA play a major role in increasing the risk of gastric diseases [22, 23, 24, 25, 26]. Recently, we have identified the fifth polymorphic site located at the 3’-end region of the H. pylori vacA gene and termed c-region. Two allelic variants of this region were denoted c1 (with 15 bp deletion) and c2 (no deletion). The c1-type association was independent of, and larger than, the associations of the m-, i-, and d-type of vacA or cagA status with GC [22]. cagA, which encodes an oncogenic CagA, is considered a virulence marker [27]. cagA-positive strains have strong association with GA risk [22, 28]. Several studies have shown that, compared to cagA strains, infection with strains carrying cagA gene is linked to a higher risk of NCGC [28, 29, 30, 31, 32, 33]. Other studies revealed a significant negative correlation between the presence of cagA-positive strains and the risk of CGC [14, 17, 34]. Moreover, some studies showed no correlation between the presence of cagA-positive strains and the risk of CGC or their protective effect on the incidence of CGC [32, 34, 35]. There was a significant relationship between the attendance of vacA and the risk of NCGA, but not CGA [17, 36]. It has been reported that the risk variation in the incidence of histologic sub-type of GA may also reflect differences between cagA and cagAH. pylori strains [37, 38, 39]. CagA and VacA were also associated with an increased risk of both IGA and DGA [36]. Although the most extensive studies revealed the role of H. pylori VacA and CagA toxins in the development of GC [17, 22, 23, 24, 40, 41, 42, 43], the magnitude of this association and the correlations of vacA genotypes and cagA status with the anatomic origin and histologic features of the tumor still remain controversial. Iran is a country with the higher rate of the outbreak of H. pylori infection (69%) [44]; and with a high incidence of gastric adenocarcinoma, ranked fourth in Asia [45, 46]. The highest rates of CGC have occurred in Central Asia countries, such as Iran [47]. Moreover, the role of bacterial genotypes in the incidence of GC has been much stressed in Iran [22, 24, 42, 48]. Therefore, we aimed to examine the linkage of H. pylori highly cytotoxic genotypes to cardia adenocarcinoma in Iranian populations as a model.
Oligonucleotide primers used for PCR
Genes
Primers
Sequences (5’3’)
Size of PCR products (bp)
Optimized annealing
temperature (C)
16 S rDNA
HP1
GCA ATC AGC GTC AGT AAT GTT C
519
56
HP2
GCT AAG AGA TCA GCC TAT GTC C
vacA
c1/-c2
c1-F
ATC ATY SGT TAT GRH AAT GTT TCT
c1: 600–700
55
R-nd
TTA TGC TCT AAA CTG GCT A
c2-F
ATT ATA ATT TAG TAG GAG TGC AAG G
c2: 600–700
55
R-nd
TTA TGC TCT AAA CTG GCT A
cagA
CAG1
ACC CTAGTC GGT AAT GGG TTA
591–856
50
CAG2
GTA ATT GTC TAG TTT CGC
cag PAI empty site
Cp1
ACTTTCACGCCCTTTCCCTCC
593
51
Cp2
TTGCATGCGTTATTATTTCAC
Materials and methods
Subjects
A total of 601 patients from different regions of Iran participated in this study. They were referred to endoscopy units in hospitals and physicians’ offices across the country. Gastric biopsy samples were obtained from patients with non-atrophic gastritis (NAG), peptic ulcerations (PUs) and GA. The study was approved by the research Ethics Committee of GCRC. All patients signed written informed consent.
Endoscopy and biopsy sampling and cultivation
Gastric biopsies were taken from the antrum and/or the corpus, of which one was used for rapid urease test and another for cultivation. Tumor biopsies were also taken and examined histologically. The tumors originated from the lower one-third of the esophagus, above the -line, were considered as esophageal adenocarcinoma, but not CGA, and excluded from the analysis. When determining the anatomic origin of a tumor was impossible by endoscopist, it was categorized as unspecified. The biopsies were then cultured on selective Brucella agar plates under micro-aerobic conditions. Brucella agar plates (Merck, Germany) contained 10% blood, vancomycin (10 mg/mL; Zakaria, Iran), trimethoprim (5 mg/mL; MP Biomedicals, France), and amphotericin B (4 mg/mL; Bristol-Myers Squibb, USA). The bacterial colonies were identified based on Gram’s staining, typical cell morphology, and positive reactions to catalase, oxidase, and urease, as well as PCR amplification of H. pylori-specific 16S rDNA.
Histologic examination
One of the biopsy specimens was fixed in 10% formalin and embedded in paraffin, and then the tissue sections for histopathologic examinations were prepared. The Lauren’s classification was performed based on morphology and mucin staining, and the histo-morphologic architecture of the tumors was expressed as intestinal-type or diffuse-type gastric adenocarcinomas. Histopathologic assessments were also applied regarding the updated Sydney classification system [49].
DNA extraction, primers and PCR conditions and genotyping
Total DNAs were extracted from H. pylori strains with the Genomic DNA purification kit (Fermentas, UK) based on the manufacturer’s protocol. PCR amplification was performed in 30 L reaction volume, containing 3 L of 10X PCR buffer (CinnaGen, Iran), 1 mM/L MgCl2, 2U of Taq DNA polymerase (CinnaGen Co., Iran), 200 M/L dNTP (CinnaGen Co., Iran), 0.5 M of each primer, and 25 ng of bacterial DNA. The utilized primers for PCR are summarized in Table 1. The PCR conditions were at 96C for 180 s; then 35 cycles of 96C for 40 s (denaturation), an optimized annealing temperature for each allele (Summarized in Table 1) for 40 s, and 72C for 40 s (extension), and a final incubation cycle at 72C for 7 min (final extension). Finally, PCR products were separated by gel electrophoresis, stained with ethidium bromide, and visualized using a UV transilluminator. The band sizes corresponding to each gene and allele are listed in Table 1. Escherichia DH5 strain and deionized water were used as negative controls.
Characteristics of patients enrolled in this study
Characteristics
No. of patients (%)
Age groups
55
123/282 (43.6)
55
157/282 (55.7)
No data
2/282 (0.7)
Sex groups
Males
178/282 (63.1)
Females
104/282 (36.9)
Types of gastroduodenal diseases
Control
194/282 (68.8)
Non-atrophic gastritis
136/194 (70.1)
Peptic ulcer
58/194 (29.9)
Case
88/282 (31.2)
Cardia gastric adenocarcinoma
38/88 (43.2)
Non-cardia gastric adenocarcinoma
46/88 (52.3)
Unspecified
4/88 (4.5)
Intestinal-type adenocarcinoma
57/88 (64.8)
Diffuse-type adenocarcinoma
25/88 (28.4)
Mucin producing-type adenocarcinoma
2/88 (2.3)
Signet ring-type adenocarcinoma
2/88 (2.3)
Adenocarcinoma, poorly differentiated
1/88 (1.1)
Adenocarcinoma, moderate differentiation
1/88 (1.1)
Total
282/282 (100)
For confirmatory purposes, the amplified fragments of each gene/allele from thirteen strains were purified and sequenced with both forward and reverse primers using a BigDye technology on an ABI3700XL DNA sequencer (Applied Biosystems). The BLAST program (http://www.ncbi.nlm.nih.gov) also was utilized to compare the nucleotide sequences with those in GenBank.
Statistical analysis
The Fisher’s exact and Chi-square tests were used to evaluate significant differences between the frequencies of each gene/allele and genotype combination and anatomic site and histologic variants of the tumor. To determine the effect of each genotype on the risk of CGA, NCGA, and the different histologic types of GA, we performed simple logistic regression analysis by the Enter method and multiple logistic regression analysis by the Forward Stepwise LR (Likelihood Ratio) method with adjustment for a threshold age of 55 years and sex. All the -values were two-sided, and -values 0.05 were indicated as statistically significant. The SPSS version 19 was used for data analysis. The proportions of associations might be incorrectly classified as significant due to multiple testing. We therefore estimated the false discovery rate (FDR) and its analog the -value among the associations tested by using the -value package in version 3.1.1.
Results
Classification of patients
The present study is an Iranian population-based case-control study. Of the 111 case patients, 56 had NCGA, 41 CGA, and 6 both the types of CGA and NCGA, and 8 with a pathologic diagnosis of “no-tumor”, “MALT lymphoma” or “invasive squamous cell-type carcinoma” were excluded. Control subjects included 490 with NAG and PUs. Overall, 390/593 patients (65.8%) were positive for H. pylori infection by rapid urease test. Due to some severe contaminations in the stomach of patients, only 282 H. pylori strains were successfully obtained from the biopsy cultures of gastric antrum and body mucosa, and were genotyped. They included 88 cases (38 with CGA, 46 with NCGA, and 4 with both the types of CGA and NCGA) and 194 controls (136 with NAG and 58 with PUs). The characteristics of patients enrolled in this study are summarized in Table 2.
Prevalence of H. pylori vacA c-region genotypes and cagA gene
In general, the vacA c-region genotypes, and the status of cagA gene were determined in 98.2% (277) and 98.6% (278) of strains, respectively. Neither vacA c-regions nor cagA gene could be amplified in five (1.8%) and four (1.4%) strains, respectively. The frequency of vacA c1 was 99/282 (35.1%), c2 163/282 (57.8%), c1c2 15/282 (5.3%) (6 controls, 7 those with CGA, and 2 those with NCGA), cagA178/282 (63.1%), and cagA100/282 (35.5%) (Table 3). The samples which had mixed infection with multiple strains in the same patient and showed the vacA c1c2 genotypes were excluded from the final analysis.
Frequencies (%) of H. pylori vacA c-region and cagA statues
Genotypes
Frequency (N)
Percent (%)
vacA c region
277/282
98.2
c1
99/282
35.1
c2
163/282
57.8
c1 and c2
15/282
5.3
Undetectable
5/282
1.8
cagA status
278/282
98.6
cagA
178/282
63.1
cagA
100/282
35.5
Undetectable
4/282
1.4
Association between age and sex and the anatomic origin and histologic type of the tumors
In GC group, more than 80% of patients were male and had 55 yr. of age and older. Statistical analysis showed also a significant correlation between sex or age and NCGA and CGA as well as IGA and DGA, whether the controls were non-tumors or those with NAG ( 0.05; Table 4). Non-tumors involved the patients with either NAG or PUs, and those with benign tumors were not welcome. The association of age 55 with DGA was much larger than its association with IGA, whether the controls were non-tumors (ORs 29.8 vs. 8.37, 0) or those with NAG (ORs 31.9 vs. 8.97, 0; Table 4).
Associations of H. pylori vacA c-region genotypes and cagA status with the risk of NCGA and CGA as well as IGA and DGA
Table 6 describes the risk of GA with respect to H. pylori genotypes by simple logistic regression analysis, where the controls were non-tumors. The frequency of the c1-type of vacA was higher in patients with CGA (73.3%) and NCGA (68.2%) than in non-tumors (23.4%). The analysis confirmed the associations of this genotype with an increased risk of both CGA and the NCGA; the OR (95% CI) was 9.01 (3.74–21.70) and 7.02 (3.41–14.44), respectively. There was no significant difference in the prevalence of the strains carrying cagA gene between the patients with either CGA or NCGA and control groups (non-tumors). The vacA c1/cagA genotype was significantly associated with an increased risk of NCGA; the OR (95% CI) was 5.27 (1.79–15.50). As illustrated in Table 6, the results of simple logistic regression analysis for the risk of GA, where the controls were those with NAG, demonstrated that the vacA c1 and vacA c1/cagA genotypeswere remarkably associated with an increased risk of both CGA and the NCGA; the OR (95% CI) was 8.50 (3.45–20.96) and 6.62 (3.13–14.02), respectively, for c1 and 3.57 (1.20–10.55) and 6.07 (2.02–18.22), respectively, for vacA c1/cagA. No significant correlation was found between cagA statues and the risk of CGA or NCGA.
Following further analysis was conducted based on the histologic features of the tumor expressed as IGA and DGA. The frequency of the vacA c1 genotype in patients with IGA (70.6%) and DGA (77.3%) was significantly higher than in those with non-tumors (23.4%) (Table 6). It was significantly associated with the risk of IGA and DGA by simple logistic regression analysis; the OR (95% CI) was 7.87 (3.93–15.72), and 11.14 (3.88–31.98), respectively. The cagA genotype was not independently associated with the risk of IGA and DGA (Table 6). The presence of both the vacA c1 and cagA genotypes further increased the risk of DGA (OR 13.97; 95% CI, 1.70–114.77), but not IGA (OR 4.07, 95% CI, 1.63–10.13). The results of simple logistic regression analysis for the risk of GA, where the controls were those with NAG, demonstrated that the vacA c1 and vacA c1/cagA genotypes were associated with an increased risk of IGA and DGA (Table 6); the OR (95% CI) was 7.42 (3.60–15.28) and 10.15 (3.59–30.7), respectively for vacA c1 and 4.68 (1.83–11.95) and 16.07 (1.93–133.51), respectively for vacA c1/cagA. There was not a significant association between the cagA statues and the risk of both IGA and DGA.
Finally, the multiple logistic regression analysis revealed that the vacA c1 genotype was independently and significantly associated with the age- and sex-adjusted risk for CGA, NCGA, IGA, and DGA, whether the controls were non-tumors (the OR (95% CI) was 14.11 (4.91–40.52), 9.59 (4.06–22.65), 11.91 (4.99–28.45), and 16.93 (4.97–57.68), respectively) or those with NAG (the OR (95% CI) was 10.71 (3.49–32.82), 8.11 (3.26–20.16), 9.56 (3.86–23.63), and 11.22 (3.077–40.94), respectively) (Table 7). The multiple logistic regression analysis also showed that the vacA c1/cagA genotype was significantly correlated with the age- and sex-adjusted risk for NCGA, IGA, and DGA, where the controls were non-tumors (the OR (95% CI) was 4.706 (1.41–15.67), 3.92 (1.32–11.64), and 12.37 (1.29–118.33), respectively) and with the risk for the NCGA and the IGA, where the controls were those with NAG (the OR (95% CI) was 4.85 (1.42–16.51), and 3.93 (1.28–12.03), respectively) (Table 7).
Association between age and sex and the anatomic origin and histologic type of the tumors
Genotypes
Cardia gastric adenocarcinoma
Non-cardia gastric adenocarcinoma
Intestinal type adenocarcinoma
Diffuse type adenocarcinoma
Control No.(%)
Case No.(%)
P value
OR
95% CI
Total
Case No.(%)
P value
OR
95% CI
Total
Case No.(%)
P value
OR
95% CI
Total
Case No.(%)
P value
OR
95% CI
Total
Gastric adenocarcinomas vs. non-tumors
Sex
Male
111(56.9)
31(81.6)
0.006
3.35
1.40–7.98
142(60.9)
35(76.1)
0.019
2.40
1.15–5.018
146(60.6)
42(75.0)
0.016
2.27
1.16–4.42
153(61.0)
21(84.0)
0.015
3.97
1.31–12.00
132(60.0)
Female
84(43.1)
7(18.4)
1 (ref)
1 (ref)
1 (ref)
91(39.1)
11(23.9)
1 (ref)
1 (ref)
1 (ref)
95(39.4)
14(25.0)
1 (ref)
1 (ref)
1 (ref)
98(39.0)
4(16.0)
1 (ref)
1 (ref)
1 (ref)
88(40.0)
Age, y
55
54(27.8)
32(84.2)
0.00
13.82
5.47–34.93
86(37.1)
34(75.6)
0.00
8.01
3.79–16.94
88(36.8)
42(76.4)
0.00
8.37
4.17–16.81
96(38.6)
23(92.0)
0.00
29.81
6.79–130.79
77(35.2)
55
140(72.2)
6(15.8)
1 (ref)
1 (ref)
1 (ref)
146(62.9)
11(24.4)
1 (ref)
1 (ref)
1 (ref)
151(63.2)
13(23.6)
1 (ref)
1 (ref)
1 (ref)
153(61.4)
2(8.0)
1 (ref)
1 (ref)
1 (ref)
142(64.8)
Gastric adenocarcinomas vs. non-atrophic gastritis
Risk estimates for CGA, NCGA, IGA, and DGA in relation to H. pylori vacA c-region genotypes and cagA status in a simple logistic regression analysis, where the controls were non-tumors
Genotypes
Cardia gastric adenocarcinoma
Non-cardia gastric adenocarcinoma
Intestinal-type adenocarcinoma
Diffuse-type adenocarcinoma
Control No.(%)
Case No.(%)
P-value
OR
95% CI
Q-value
Case No.(%)
P-value
OR
95% CI
Q-value
Case No.(%)
P-value
OR
95% CI
Q-value
Case No.(%)
P-value
OR
95% CI
Q-value
vacA c-region
c1
43(23.4)
22(73.3)
9.21e-07
9.01
3.74–21.70
2.76e-06
30(68.2)
1.13e-07
7.02
3.41–14.44
3.39e-07
36(70.6)
5.22e-09
7.87
3.93–15.72
1.56e-08
17(77.3)
7.32e-06
11.14
3.88–31.98
2.19e-05
c2
141(76.6)
8(26.7)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
14(31.8)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
15(29.4)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
5(22.7)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
cagA status
cagA
127(65.5)
20(55.6)
2.57e-01
0.659
0.32–1.35
2.57e-01
28(63.6)
8.18e-01
0.923
0.46–1.82
8.18e-01
33(61.1)
5.54e-01
0.82
0.44–1.54
5.54e-01
16(64.0)
8.84e-01
0.93
0.33–2.23
8.84e-01
cagA
67(34.5)
16(44.4)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
16(36.4)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
21(38.9)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
9(36.0)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
Genotype combination
vacA c1/ cagA
38(39.2)
12(66.7)
3.64e-02
3.105
1.07–8.97
5.46e-02
17(77.3)
2.46e-03
5.27
1.79–15.50
3.70e-03
21(72.4)
2.49e-03
4.07
1.63–10.13
3.74e-03
9(90.0)
1.41e-02
13.97
1.70–114.77
2.11e-02
vacA c2/ cagA
59(60.8)
6(33.3)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
5(22.7)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
8(27.6)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
1(10.0)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
Risk estimates for CGA, NCGA, IGA, and DGA in relation to H. pylori vacA c-region genotypes and cagA status in a simple logistic regression analysis, where the controls were those with non-atrophic gastritis
Genotypes
Cardia gastric adenocarcinoma
Non-cardia gastric adenocarcinoma
Intestinal type adenocarcinoma
Diffuse type adenocarcinoma
Control No.(%)
Case No.(%)
P-value
OR
95% CI
Q-value
Case No.(%)
P-value
OR
95% CI
Q-value
Case No.(%)
P-value
OR
95% CI
Q-value
Case No.(%)
P-value
OR
95% CI
Q-value
vacA c-region
c1
32(24.4)
22(73.3)
3.28e-06
8.50
3.45–20.96
9.85e-06
30(68.2)
7.48e-07
6.62
3.13–14.02
2.24e-07
36(70.6)
5.31e-08
7.42
3.60–15.28
1.59e-07
17(77.3)
1.74e-05
10.15
3.59–30.78
5.24e-05
c2
99(75.6)
8(26.7)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
14(31.8)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
15(29.4)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
5(22.7)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
cagA status
cagA
80(58.8)
20(55.6)
7.23e-01
0.875
0.41–1.83
7.23e-01
28(63.6)
5.71e-01
1.22
0.60–2.47
5.71e-01
33(61.1)
7.72e-01
1.10
0.57–2.09
7.72e-01
16(64.0)
8.18e-01
1.24
0.51–3.01
8.18e-01
cagA
56(41.2)
16(44.4)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
16(36.4)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
21(38.9)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
9(36.0)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
Genotype combination
vacA c1/ cagA
28(35.9)
12(66.7)
2.13e-02
3.57
1.20–10.55
3.19e-02
17(77.3)
1.30e-03
6.07
2.02–18.22
1.95e-03
21(72.4)
1.22e-03
4.68
1.83–11.95
1.83e-03
9(90.0)
2.46e-03
16.07
1.93–133.51
3.70e-03
vacA c2/ cagA
50(64.1)
6(33.3)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
5(22.7)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
8(27.6)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
1(10.0)
1 (ref)
1 (ref)
1 (ref)
1 (ref)
Age- and sex-adjusted risk for CGA, NCGA, IGA, and DGA in relation to H. pylori vacA c-region genotypes and cagA status in a multiple logistic regression analysis
Cardia gastric adenocarcinoma
Non-cardia gastric adenocarcinoma
Intestinal-type adenocarcinoma
Diffuse-type adenocarcinoma
Genotypes
P-value
OR
95% CI
Q-value
P-value
OR
95% CI
Q-value
P-value
OR
95% CI
Q-value
P-value
OR
95% CI
Q-value
Gastric adenocarcinoma vs. non-tumors
vacA c1 vs. vacA c2
8.73e-07
14.11
4.91–40.52
2.62e-06
2.52e-07
9.59
4.06–22.65
7.56e-07
2.41e-08
11.91
4.99–28.45
7.24e08
6.04e-06
16.93
4.97–57.68
1.81e-05
cagA vs. cagA
3.63e-02
0.39
0.16–0.94
5.44e-02
1.81e-01
–
–
1.81e-01
8.40e-02
–
–
8.40e-02
1.86e-01
–
–
1.86e-01
vacA c1/cagA vs. vacA c2/cagA
9.52e-02
–
–
9.52e-02
1.16e-02
4.706
1.41–15.67
1.74e-02
1.37e-02
3.92
1.32–11.64
2.06e-02
2.89e-02
12.37
1.29–118.33
4.34e-02
Gastric adenocarcinoma vs. non-atrophic gastritis
vacA c1 vs. vacA c2
3.30e-05
10.71
3.49–32.82
9.90e-05
6.61e-06
8.11
3.26–20.16
1.98e-05
1.00e-06
9.56
3.86–23.63
3.01e-06
2.50e-04
11.22
3.077–40.94
7.50e-04
cagA vs. cagA
1.32e-01
–
–
1.32e-01
5.24e-01
–
–
5.24e-01
2.98e-01
–
–
2.98e-01
3.70e-01
–
–
3.70e-01
vacA c1/cagA vs. vacA c2/cagA
1.30e-01
–
–
1.32e-01
1.15e-02
4.85
1.42–16.51
1.72e-02
1.61e-02
3.93
1.28–12.03
2.41e-02
3.61e-02
11.57
1.172–114.26
5.41e-02
a: Odds ratio; b: Confidence interval; c False discovery rate-adjusted P-value; d: Boldface data indicate statistically significant results.
Discussion
In the present study, 65.8% of patients were positive for H. pylori infection. The frequency of the c1-type of vacA was higher in patients with CGA (73.3%) and NCGA (68.2%) than in either non-tumors (23.4%) or those with NAG (24.4%). Eventually, after being adjusted for confounding factors, the multiple logistic regression analysis revealed that the c1 genotype had a strong correlation with an enhanced risk of CGA and NCGA, whether the controls were non-tumors (ORs 14.11 and 9.59, respectively) or those with NAG (ORs 10.71 and 8.11, respectively). These findings provide the first evidence for the determinant role of the H. pylorivacA c1 genotype in the causing of both cardia and non-cardia adenocarcinomas in male patients aged 55 in Iran. Recently, we have proposed that the H. pylorivacA i1 (OR 37.52) and d1 (OR 7.17) genotypes might be important determinants of non-cardia cancer risk in Ardabil, a very high-risk area in Northwestern Iran. The m1, not independently, but in combination might further define GC risk [42]. Furthermore, Shakeri et al. conducted a study on 272 cases of GA and 524 controls in Northeastern Iran, to assess the associations of seropositivity to H. pylori antigens with GA. They showed that VacA was related to an increased risk of NCGA (OR 2.8), but not CGA [17]. In one study, da Costa et al. observed that the frequencies of vacA s1, vacA m1, vacA s1m1, vacA s2m2, and cagA were 85.5%, 70.1%, 66.6%, 11.1%, and 64.9%, respectively in patients with GC. The distribution of these genotypes was similar in tumors from both regions cardia and non-cardia; so no relationship was found [50]. In the current study, the vacA c1 genotype also was linked to an elevated risk of both IGA and DGA, whether the controls were non-tumors (adjusted ORs 11.91 and 16.93, respectively) or those with NAG (adjusted ORs 9.56 and 11.22, respectively). Similar results were obtained in Ardabil, where the i1- and d1-types of vacA were linked to increased risks of intestinal- (OR 14.04) and diffuse-type (OR 7.71) adenocarcinomas, respectively [42] In a recent study, Figura et al. examined 226 H. pylori colonies from 15 patients with IGC and 13 patients with DGC; and tested the associations of polymorphisms of pathogenic cagA and vacA with histopathologic variables. They reported that 80.95% of vacA s1/m2-carrying strains were significantly isolated from DGC cases ( 0.001) [51]. These findings reflect the fact that some H. pylori genotypes might also be important for the development of the histologic types of GC.
Our study showed no significant statistical correlation between the cagA genotype and the risk of CGA, whether the controls were non-tumors (OR 0.659) and or those with NAG (OR 0.875). These results correspond with previous studies [32, 36, 52, 53]. For example, Bornschein et al. study on 152 patients with GC (73 with CGC and 79 with NCGC) showed that the prevalence of CagA status was similar in proximal and distal GC (77.2 vs. 84.6%) as well as in intestinal- and diffuse-type GC (82.6 vs. 79.2%) by serologic assessment [53]. Another study from Sweden also showed no association between CagA antibodies and cardia cancer (OR 1.0) [16] However, other studies demonstrated a significant inverse relationship between cagA strains and the development of CGA, which reduces the risk for this type of tumor [14, 34]. In contrast, in Shakeri et al. study, CagA seropositivity was associated with an increased risk of both CGA (OR 1.9) and NCGA (OR 3.4) [17].
In the present study, no significant difference was found in the prevalence of strains carrying cagA gene between patients with NCGA (63.6%) and either non-tumors (65.5%) or those with NAG (58.8%). In some studies, infection with cagA compared with cagA strains was related to an increased risk of non-cardia cancer [28, 29, 30, 31]. For example, in a case-control study on Swedish community, there was a significant correlation between the presence of CagA (OR 9.2) and VacA (OR 3.5) and the risk of NCGA, but not CGA [36]. In a study on 41 cardia and 339 non-cardia cancer cases, and 380 controls, a positive association was not found for cagA strains with cardia cancer, but cagA strains was related to an increased risk of non-cardia cancer (OR 1.60) [32]. Wang et al., in a case-control study (257 cases with non-cardiac cancer and 514 controls) in Xi’an, China showed that the cagA strains of H. pylori had a strong correlation with non-cardiac cancer [33]. In contrast, in a study from New Jersey and western Washington, infection with cagA strains did not show a significant association with non-cardia cancer (OR 1.4), but an inverse association with the risk of esophageal and gastric cardia adenocarcinoma was found (OR 0.4) [34].
A simple logistic regression analysis in our study also showed that the cagA genotype was not associated with a statistically significant increased risk for IGA, and DGA. Our results were in compliance with previous studies from India andLatin America countries, so that a study on Indian patients indicated that 34% of the control gastric tissues were cagA, however only 26.2% of the gastric cancer groups (29.5% in DGA and 19.0% in IGA) were harboring the cagA gene. Statistical analysis showed no association between this genotype and either histologic variety of GA [54]. Cardenas-Mondragon et al. in a case-control study of patients with gastric disease in Latin America observed the frequency of cagA decreased in intestinal-type 28/50 (56%) but it was increased in the diffuse-type of GC 50/64 (78.1%) compared with NAG 157/225 (69.8%). However, these differences were not statistically meaningful [55]. These were not in agreement with the results of a recent case-control study in Sweden, where CagA and VacA were also associated with a heightened risk of both intestinal (ORs 6.0 and 3.7, respectively) and diffuse (ORs 20.6 and 3.9, respectively) histologic subtypes [36]. Furthermore, in Figura et al. study, a total of 214 strains were cagA-positive that were isolated from both IGC and DGC patients; and 12 strains were cagA-negative, all of which were isolated from DGC patients ( 0.001) [51]. In our study, the presence of the cagA genotype in combination with the vacA c1 genotype was associated with NCGA, IGA, and DGA, while the cagA genotype alone was not associated with these diseases. These findings might reveal a significant concordance between the vacA c1 and cagA genotypes; however, it still remains obscure.
Our study has some strengths. We enrolled biopsy-proven gastric adenocarcinomas and examined directly the genetics of the active H. pylori strains isolated from gastric biopsies of all case and control individuals. Furthermore, we adopted two control groups; non-tumors and those with NAG. This, in turn, increased the reliability of association analyses because, in addition to NAG, PUs also was included in the first group. Most of the above studies, however, tested VacA or CagA seropositivity among H. pylori-infected subjects, and some of them did not even include information on prior attempts at H. pylori eradication in individuals. Our study has also some limitations. We did not find a significant association between the cagA genotype alone and the risk of NCGA and the different histologic types of GA. It also showed no effect on cardia adenocarcinoma risk, even in combination with the c1-type of vacA. One reason might be that we did not assess the diversity of the carboxyl-terminal region of CagA and its EPIYA motifs. It has been shown that the strains carrying CagA with a large number of EPIYA-C motifs are more likely linked to gastric precancerous lesions and GC [56, 57]. As previously shown, all the Iranian H. pylori strains are Western-type, representing type-C phosphoryaltion motif [58, 59]. Therefore, determining the number of EPIYA-C motifs and recruiting a larger sample size might be important for predicting the risk of cardia and non-cardia as well as intestinal- and diffuse-type adenocarcinomas in relation to the cagA genotype.
In sum, the H. pylori vacA c1 genotype, but not cagA status, might be the first important bacterial biomarker for predicting the cardia adenocarcinoma risk in male patients aged 55 in Iran. c1, whether independently or in combination with cagA, might also predict the risk of non-cardia and intestinal- or diffuse-type gastric adenocarcinomas. Further studies from other parts of the world are needed to assess the strength of these findings.
Footnotes
Acknowledgments
This study was supported by the Research Council of the University of Mohaghegh Ardabili grant 95/D/13/14100. The supporter had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study. Parts of the study have been presented at the 23 United European Gastroenterology Week, Barcelona, Spain, October 24–28, 2015. The authors declare no conflicts of interest.
References
1.
HowsonC.P.HiyamaT. and WynderE.L., The decline in gastric cancer: epidemiology of an unplanned triumph, Epidemiol Rev8 (1986), 1–27.
2.
FerlayJ.SoerjomataramI.DikshitR.EserS.MathersC.RebeloM.ParkinD.M.FormanD. and BrayF., Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012, Int J Cancer136 (2015), E359–86.
3.
LaurenP., The Two Histological Main Types of Gastric Carcinoma: Diffuse and So-called Intestinal-type Carcinoma. An Attempt at a Histo-clinical Classification, Acta Pathol Microbiol Scand64 (1965), 31–49.
4.
BotterweckA.A.SchoutenL.J.VolovicsA.DorantE. and van Den BrandtP.A., Trends in incidence of adenocarcinoma of the oesophagus and gastric cardia in ten European countries, Int J Epidemiol29 (2000), 645–654.
5.
DevesaS.S.BlotW.J. and FraumeniJ.F., Jr., Changing patterns in the incidence of esophageal and gastric carcinoma in the United States, Cancer83 (1998), 2049–2053.
6.
CarrJ.S.ZafarS.F.SabaN.KhuriF.R. and El-RayesB.F., Risk factors for rising incidence of esophageal and gastric cardia adenocarcinoma, J Gastrointest Cancer44 (2013), 143–151.
7.
BlotW.J.DevesaS.S.KnellerR.W. and FraumeniJ.F., Jr., Rising incidence of adenocarcinoma of the esophagus and gastric cardia, Jama265 (1991), 1287–1289.
8.
BrownL.M. and DevesaS.S., Epidemiologic trends in esophageal and gastric cancer in the United States, Surg Oncol Clin N Am11 (2002), 235–256.
9.
ParkinD.M. and MuirC.S., Cancer Incidence in Five Continents. Comparability and quality of data, IARC Sci Publ (1992), 45–173.
10.
de MartelC.FormanD. and PlummerM., Gastric cancer: epidemiology and risk factors, Gastroenterol Clin North Am42 (2013), 219–240.
11.
S. European Organization for Cooperation in Cancer Prevention StudiesReedP.I. and HillM.J., Gastric Carcinogenesis: Proceedings of the 6th Annual Symposium of the European Organization for Cooperation in Cancer Prevention Studies (ECP), London, UK, 7-8 March 1988, Excerpta Medica, 1988.
12.
MunozN.CorreaP.CuelloC. and DuqueE., Histologic types of gastric carcinoma in high- and low-risk areas, Int J Cancer3 (1968), 809–818.
13.
GroupH.A.C.C., Gastric cancer and Helicobacter pylori: a combined analysis of 12 case control studies nested within prospective cohorts, Gut49 (2001), 347–353.
14.
HansenS.MelbyK.K.AaseS.JellumE. and VollsetS.E., Helicobacter pylori infection and risk of cardia cancer and non-cardia gastric cancer. A nested case-control study, Scand J Gastroenterol34 (1999), 353–360.
15.
EgiY.ItoM.TanakaS.ImagawaS.TakataS.YoshiharaM.HarumaK. and ChayamaK., Role of Helicobacter pylori infection and chronic inflammation in gastric cancer in the cardia, Jpn J Clin Oncol37 (2007), 365–369.
16.
YeW.HeldM.LagergrenJ.EngstrandL.BlotW.J.McLaughlinJ.K. and NyrenO., Helicobacter pylori infection and gastric atrophy: risk of adenocarcinoma and squamous-cell carcinoma of the esophagus and adenocarcinoma of the gastric cardia, J Natl Cancer Inst96 (2004), 388–396.
17.
ShakeriR.MalekzadehR.NasrollahzadehD.PawlitaM.MurphyG.IslamiF.SotoudehM.MichelA.EtemadiA.WaterboerT.PoustchiH.BrennanP.BoffettaP.DawseyS.M.KamangarF. and AbnetC.C., Multiplex pylori Serology and Risk of Gastric Cardia and Noncardia Adenocarcinomas, Cancer Res75 (2015), 4876–4883.
18.
Cavaleiro-PintoM.PeleteiroB.LunetN. and BarrosH., Helicobacter pylori infection and gastric cardia cancer: systematic review and meta-analysis, Cancer Causes Control22 (2011), 375–387.
19.
DawseyS.M.MarkS.D.TaylorP.R. and LimburgP.J., Gastric cancer and H pylori, Gut51 (2002), 457–458.
20.
HarumaK.KomotoK.KamadaT.ItoM.KitadaiY.YoshiharaM.SumiiK. and KajiyamaG., Helicobacter pylori infection is a major risk factor for gastric carcinoma in young patients, Scand J Gastroenterol35 (2000), 255–259.
21.
KomotoK.HarumaK.KamadaT.TanakaS.YoshiharaM.SumiiK.KajiyamaG. and TalleyN.J., Helicobacter pylori infection and gastric neoplasia: correlations with histological gastritis and tumor histology, Am J Gastroenterol93 (1998), 1271–1276.
22.
BakhtiS.Z.Latifi-NavidS.MohammadiS.ZahriS.BakhtiF.S.FeiziF.YazdanbodA. and SiavoshiF., Relevance of Helicobacter pylori vacA 3’-end Region Polymorphism to Gastric Cancer, Helicobacter21 (2016), 305–316.
23.
OgiwaraH.SugimotoM.OhnoT.VilaichoneR.K.MahachaiV.GrahamD.Y. and YamaokaY., Role of deletion located between the intermediate and middle regions of the Helicobacter pylori vacA gene in cases of gastroduodenal diseases, J Clin Microbiol47 (2009), 3493–3500.
24.
RheadJ.L.LetleyD.P.MohammadiM.HusseinN.MohagheghiM.A.Eshagh HosseiniM. and AthertonJ.C., A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate region, is associated with gastric cancer, Gastroenterology133 (2007), 926–936.
25.
BasiriZ.SafaralizadehR.BonyadiM.J.SomiM.H.MahdaviM. and Latifi-NavidS., Helicobacter pylori vacA d1 genotype predicts risk of gastric adenocarcinoma and peptic ulcers in northwestern Iran, Asian Pac J Cancer Prev15 (2014), 1575–1579.
26.
MottaghiB.SafaralizadehR.BonyadiM.Latifi-NavidS. and SomiM.H., Helicobacter pylori vacA i region polymorphism but not babA2 status associated to gastric cancer risk in northwestern Iran, Clin Exp Med16 (2016), 57–63.
27.
WroblewskiL.E. and PeekR.M., Jr., Helicobacter pylori in gastric carcinogenesis: mechanisms, Gastroenterol Clin North Am42 (2013), 285–298.
28.
HuangJ.Q.ZhengG.F.SumanacK.IrvineE.J. and HuntR.H., Meta-analysis of the relationship between cagA seropositivity and gastric cancer, Gastroenterology125 (2003), 1636–1644.
29.
ParsonnetJ.FriedmanG.D.OrentreichN. and VogelmanH., Risk for gastric cancer in people with CagA positive or CagA negative Helicobacter pylori infection, Gut40 (1997), 297–301.
30.
BlaserM.J.Perez-PerezG.I.KleanthousH.CoverT.L.PeekR.M.ChyouP.H.StemmermannG.N. and NomuraA., Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach, Cancer Res55 (1995), 2111–2115.
31.
KuipersE.J.Perez-PerezG.I.MeuwissenS.G. and BlaserM.J., Helicobacter pylori and atrophic gastritis: importance of the cagA status, J Natl Cancer Inst87 (1995), 1777–1780.
32.
PeleteiroB.Cavaleiro-PintoM.BarrosR.BarrosH. and LunetN., Is cardia cancer aetiologically different from distal stomach cancer? Eur J Cancer Prev20 (2011), 96–101.
33.
WangX.Q.YanH.TerryP.D.WangJ.S.ChengL.WuW.A. and HuS.K., Interactions between CagA and smoking in gastric cancer, World J Gastroenterol17 (2011), 3330–3334.
34.
ChowW.H.BlaserM.J.BlotW.J.GammonM.D.VaughanT.L.RischH.A.Perez-PerezG.I.SchoenbergJ.B.StanfordJ.L.RotterdamH.WestA.B. and FraumeniJ.F., Jr., An inverse relation between cagA+ strains of Helicobacter pylori infection and risk of esophageal and gastric cardia adenocarcinoma, Cancer Res58 (1998), 588–590.
35.
WuA.H.CrabtreeJ.E.BernsteinL.HawtinP.CockburnM.TsengC.C. and FormanD., Role of Helicobacter pylori CagA+ strains and risk of adenocarcinoma of the stomach and esophagus, Int J Cancer103 (2003), 815–821.
36.
SongH.MichelA.NyrenO.EkstromA.M.PawlitaM. and YeW., A CagA-independent cluster of antigens related to the risk of noncardia gastric cancer: associations between Helicobacter pylori antibodies and gastric adenocarcinoma explored by multiplex serology, Int J Cancer134 (2014), 2942–2950.
37.
BrennerH.ArndtV.StegmaierC.ZieglerH. and RothenbacherD., Is Helicobacter pylori infection a necessary condition for noncardia gastric cancer? Am J Epidemiol159 (2004), 252–258.
38.
HatakeyamaM., Oncogenic mechanisms of the Helicobacter pylori CagA protein, Nat Rev Cancer4 (2004), 688–694.
39.
BlaserM.J. and BergD.E., Helicobacter pylori genetic diversity and risk of human disease, J Clin Invest107 (2001), 767–773.
40.
AbdiE.Latifi-NavidS.Latifi-NavidH. and SafarnejadB., Helicobacter pylori vacuolating cytotoxin genotypes and preneoplastic lesions or gastric cancer risk: a meta-analysis, J Gastroenterol Hepatol31 (2016), 734–744.
41.
MatosJ.I.de SousaH.A.Marcos-PintoR. and Dinis-RibeiroM., Helicobacter pylori CagA and VacA genotypes and gastric phenotype: a meta-analysis, Eur J Gastroenterol Hepatol25 (2013), 1431–1441.
42.
AbdiE.Latifi-NavidS.ZahriS.YazdanbodA. and SafaralizadehR., Helicobacter pylori genotypes determine risk of non-cardia gastric cancer and intestinal- or diffuse-type GC in Ardabil: A very high-risk area in Northwestern Iran, Microb Pathog107 (2017), 287–292.
43.
KiM.R.HwangM.KimA.Y.LeeE.M.LeeE.J.LeeM.M.SungS.E.KimS.H.LeeH.S. and JeongK.S., Role of vacuolating cytotoxin VacA and cytotoxin-associated antigen CagA of Helicobacter pylori in the progression of gastric cancer, Mol Cell Biochem396 (2014), 23–32.
44.
NouraieM.Latifi-NavidS.RezvanH.RadmardA.R.MaghsudluM.Zaer-RezaiiH.AminiS.SiavoshiF. and MalekzadehR., Childhood hygienic practice and family education status determine the prevalence of Helicobacter pylori infection in Iran, Helicobacter14 (2009), 40–46.
45.
AlizadehA.H.AnsariS.RanjbarM.ShalmaniH.M.HabibiI.FirouziM. and ZaliM.R., Seroprevalence of Helicobacter pylori in Nahavand: a population-based study, East Mediterr Health J15 (2009), 129–135.
46.
DerakhshanM.H.YazdanbodA.SadjadiA.R.ShokoohiB.McCollK.E. and MalekzadehR., High incidence of adenocarcinoma arising from the right side of the gastric cardia in NW Iran, Gut53 (2004), 1262–1266.
47.
ColquhounA.ArnoldM.FerlayJ.GoodmanK.J.FormanD. and SoerjomataramI., Global patterns of cardia and non-cardia gastric cancer incidence in 2012, Gut64 (2015), 1881–1888.
48.
GholizadeTobnaghS.BakhtiS.Z.Latifi NavidS.ZahriS. and Sadat BakhtiF., Role of Plasticity Region Genes and cagE gene of cagPAI of Helicobacter pylori in Development of Gastrointestinal (GI) Diseases, Asian Pac J Cancer Prev18 (2017), 43–49.
49.
DixonM.F.GentaR.M.YardleyJ.H. and CorreaP., Classification and grading of gastritis. The updated Sydney System. International Workshop on the Histopathology of Gastritis, Houston 1994, Am J Surg Pathol20 (1996), 1161–1181.
50.
da CostaD.M.Dos Santos PereiraE.de Lima Silva-FernandesI.J.FerreiraM.V. and RabenhorstS.H., Characterization of Gastric Cardia Tumors: Differences in Helicobacter pylori Strains and Genetic Polymorphisms, Dig Dis Sci60 (2015), 2712–2717.
51.
FiguraN.ValassinaM.MorettiE.VindigniC.CollodelG.IacoponiF.GiordanoN.RovielloF. and MarrelliD., Histological variety of gastric carcinoma and Helicobacter pylori cagA and vacA polymorphism, Eur J Gastroenterol Hepatol27 (2015), 1017–1021.
52.
SimanJ.H.EngstrandL.BerglundG.ForsgrenA. and FlorenC.H., Helicobacter pylori and CagA seropositivity and its association with gastric and oesophageal carcinoma, Scand J Gastroenterol42 (2007), 933–940.
53.
BornscheinJ.SelgradM.WarneckeM.KuesterD.WexT. and MalfertheinerP.,
pylori infection is a key risk factor for proximal gastric cancer, Dig Dis Sci55 (2010), 3124–2131.
54.
PandeyA.TripathiS.C.MahataS.VishnoiK.ShuklaS.MisraS.P.MisraV.HedauS.MehrotraR.DwivediM. and BhartiA.C., Carcinogenic Helicobacter pylori in gastric pre-cancer and cancer lesions: association with tobacco-chewing, World J Gastroenterol20 (2014), 6860–6868.
55.
Cardenas-MondragonM.G.TorresJ.Flores-LunaL.Camorlinga-PonceM.Carreon-TalaveraR.Gomez-DelgadoA.KasamatsuE. and Fuentes-PananaE.M., Case-control study of Epstein-Barr virus and Helicobacter pylori serology in Latin American patients with gastric disease, Br J Cancer112 (2015), 1866–1873.
56.
ArgentR.H.KiddM.OwenR.J.ThomasR.J.LimbM.C. and AthertonJ.C., Determinants and consequences of different levels of CagA phosphorylation for clinical isolates of Helicobacter pylori, Gastroenterology127 (2004), 514–523.
57.
SicinschiL.A.CorreaP.PeekR.M.CamargoM.C.PiazueloM.B.Romero-GalloJ.HobbsS.S.KrishnaU.DelgadoA.MeraR.BravoL.E. and SchneiderB.G., CagA C-terminal variations in Helicobacter pylori strains from Colombian patients with gastric precancerous lesions, Clin Microbiol Infect16 (2010), 369–378.
58.
Honarmand-JahromyS.SiavoshiF.MalekzadehR.SattariT.N. and Latifi-NavidS., Multiple repeats of Helicobacter pylori CagA EPIYA-C phosphorylation sites predict risk of gastric ulcer in Iran, Microb Pathog89 (2015), 87–92.
59.
VaziriF.Najar PeerayehS.AlebouyehM.MolaeiM.MaghsoudiN. and ZaliM.R., Determination of Helicobacter pylori CagA EPIYA types in Iranian isolates with different gastroduodenal disorders, Infect Genet Evol17 (2013), 101–105.
