Mol Biol Rep DOI 10.1007/s11033-014-3228-0
Susceptibility to Helicobacter pylori infection: results of an epidemiological investigation among gastric cancer patients Nikola Panic • Elena Mastrostefano • Emanuele Leoncini • Roberto Persiani • Dario Arzani Rosarita Amore • Riccardo Ricci • Federico Sicoli • Stefano Sioletic • Milutin Bulajic • Domenico D’ Ugo • Walter Ricciardi • Stefania Boccia
•
Received: 14 May 2013 / Accepted: 3 February 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The aim of this study was to identify the clinical, demographic, lifestyle factors and selected genetic polymorphisms that affect the susceptibility towards Helicobacter pylori (H. pylori) infection in gastric cancer patients. Histological confirmed gastric adenocarcinoma cases that underwent curative gastrectomy between 2002 and 2012 were included. Gastric biopsy samples were obtained to determine the H. pylori status, and further cagA status and vacA m and s genotypes by polymerase chain reaction. Patients were interviewed with structured questionnaires, and blood samples were collected for EPHX1, GSTM1, GSTT1, IL1B, IL1RN, MTHFR and p53 genotyping. Proportions were compared in univariate analysis, while the relation between putative risk factors and H. pylori status and genotype were measured using logistic regression analysis. One hundred forty-nine gastric cancer patients were included, of which 78.5 % were H. pylori positive. Among positive patients
50 % were cagA?, 72.5 % vacA m1 and 80.7 % vacA s1. The presence of cagA was less frequent among vacA m1 (p = 0.031) and vacA s1 (p = 0.052) subtypes. The presence of father history for any cancer was a significant risk factor for H. pylori infection [adjusted odds ratio (OR) = 8.18, 95 % confidence interval (CI) 1.04–64.55]. EPHX1 exon 3 T [ C (OR = 0.35, CI 95 % 0.13–0.94), IL1B-511 T [ C (OR = 0.38, CI 95 % 0.15–0.97) and IL1-RN VNTR (OR = 0.19, CI 95 % 0.06–0.58) polymorphisms were protective towards H. pylori infection in the univariate analysis. Wine consumption was associated with higher risk of carrying the H. pylori vacA m1 virulent subtype (p = 0.034). Lastly, cardiovascular diseases were less common among cagA positive subjects (p = 0.023). Father history of any cancer is a risk factor for H. pylori infection. Polymorphisms in IL1B-511, IL1-RN and EPHX1 exon 3 genes might be protective towards H. pylori infection.
N. Panic E. Mastrostefano E. Leoncini D. Arzani R. Amore W. Ricciardi S. Boccia (&) Institute of Public Health-Section of Hygiene, Universita` Cattolica del Sacro Cuore, Rome, Italy e-mail: [email protected]
S. Sioletic The Ludwig Center at Dana-Farber/Harvard Cancer Center, Boston, MA, USA
N. Panic M. Bulajic Faculty of Medicine, University of Belgrade, Belgrade, Serbia N. Panic M. Bulajic University Clinical-Hospital Center ‘‘Dr DragisaMisovicDedinje’’, Belgrade, Serbia
M. Bulajic University Clinical Hospital ‘‘Santa Maria della Misericordia’’, Udine, Italy S. Boccia IRCCS San Raffaele, Pisana, Rome, Italy
R. Persiani F. Sicoli D. D. Ugo Department of Surgery, Universita` Cattolica del Sacro Cuore, Rome, Italy R. Ricci Department of Pathology, Universita` Cattolica del Sacro Cuore, Rome, Italy
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Mol Biol Rep
Keywords Helicobacter pylori Gastric cancer Genetic polymorphism Family history
Introduction Gastric cancer accounts for about 10 % of all cancers worldwide, being the fourth most common cancer and the second cause of cancer death [1]. Incidence and mortality are high in Japan, China, Russia and most central and eastern Europe, where the prevalence of Helicobacter pylori (H. pylori) is relatively high compared to western Europe and the USA (60–90 % vs. 30–40 % of the population) [2]. Gastric carcinogenesis is the result of a complex interaction between environmental and genetic factors. Among the environmental, H. pylori infection, tobacco smoking, low fruit and vegetable intake, salt or salty food intake and the lack of food refrigeration plays a major role, while for the genetic factors accumulating evidences report that the individual genetic susceptibility to gastric cancer involves several genes with small effects [3–9]. H. pylori is proven to be group 1 carcinogen for gastric cancer [10], as it induces chronic inflammation of the gastric mucosa [11]. Some reports suggest that up to 85 % of noncardia gastric cancers are H. pylori related [12]. The virulence of H. pylori is determined by polymorphisms of the cagA and vacA genes, coding for certain bacteria virulence factors. CagA codes for a protein that alters numerous signaling pathways associated with malignant transformation of cells [13], while VacA is a secreted pore-forming toxin that causes cell vacuolation, apoptosis, and inhibition of T cell activation and proliferation [14]. CagA is present in 60–70 % of H. pylori strains in Western population and almost 100 % in East-Asia [15], while vacA is present in literally every H. pylori strain. However there is a variation in vacA vacuolation activity depending on differences in middle (m1/m2) and signal (s1/s2) regions [16]. The infection with virulent strains of H. pylori, carrying cagA [17] and the more active forms of vacA (s1/m1 forms) [16, 18], is reported to increase the risk of developing gastric cancer respect to the less virulent forms. Though not being a necessary component cause [8], H. pylori infection represents certainly a key step in gastric carcinogenesis, as testified by the observation that the eradication of H. pylori decreases the risk of precancerous lesions [19] and gastric cancer [20]. In this sense, the identification of the lifestyle and host genetic factors that influence the susceptibility to H. pylori infection have the potential to lead to more specific prevention strategies towards gastric cancer. The aim of our study was to identify the factors influencing the infection of H. pylori among a set of gastric
123
cancer patients. The demographics, lifestyle, family history characteristics, and selected genetic polymorphisms were investigated in association with H. pylori infection status, and with the presence of cagA and vacA m1 and s1 virulent strains.
Methods Study population Consecutive primary gastric adenocarcinoma patients with histological confirmation, who underwent a curative gastrectomy in the ‘‘A. Gemelli’’ teaching hospital during the period 2002–2012, were asked to participate in the study [21–24]. Gastric cancer cases were classified as intestinal and diffuse according to Lauren classification [25]. Clinical data (tumor site, lymphnode involvement, grade and stage) were retrieved from medical charts. Written informed consent was obtained from all study subjects. This study was performed according to the Declaration of Helsinki and was approved by the ethics committee of the Universita` Cattolica del Sacro Cuore. Data collection Patients were interviewed by trained medical doctors using a structured questionnaire to collect information on demographic data, lifestyle habits (smoking history, alcohol consumption, history of previous illness, dietary habits), comorbidities (cardiovascular diseases, diabetes, respiratory diseases) and family history of cancer (1st and 2nd degree). Questionnaires on lifestyle habits referred to 1 year prior the diagnosis of gastric cancer for cases. Cigarette smoking status was categorized as never and ever-smokers (including both current and former smokers). The amount of smoking was evaluated as pack-years. A pack-year is defined as the product of packs smoked per day and the total years of smoking. The study categorized drinkers in three categories-heavy drinkers (who consumed 24 g per day or more), moderate drinkers (12–23 g per day) and light drinkers (\12 g a day). A standard drink is equal to 12.0 g of pure alcohol. Gastric cancer cases and H. pylori genotyping A venous blood sample was drawn from each participant, collected into EDTA-coated tubes from which DNA was isolated from peripheral blood lymphocytes by standard methods. Identification of the microsomal epoxide hydrolase (EPHX1) exon3 and exon4 polymorphisms, EPHX1 exon3 T [ C (rs1651740) and EPHX1 exon4 A [ G (rs2234922), was performed using a polymerase chain
Mol Biol Rep
reaction (PCR) followed by restriction-fragment length polymorphism analysis (RFLP) [26]. Gluthatione S-transferase M1 (GSTM1) and T1 (GSTT1) alleles were identified using a known multiplex PCR based method [27]. Corresponding primers and PCR–RFLP conditions to determine interleukin 1B (IL1B) polymorphisms, IL1B-31 C [ T (rs1143627) and IL1B-511 T [ C (rs16944), and the variable number of tandem repeats in interleukin 1 receptor antagonist (IL1-RN VNTR) polymorphism have been previously described by Ruzzo et al. [28]. Analysis of interleukin 1B exon 5 polymorphism (IL1B ? 3954 C [ T, rs1143634) was described by El Omar et al. [29]. Genotyping for methylenetetrahydrofolate reductase (MTHFR) polymorphisms, MTHFR 677C [ T (rs1801133) and MTHFR1298 A [ C (rs1801131) was performed by PCR– RFLP [30]. Genotyping for p53 exon 4 [rs1042522], p53 intron 3 [rs17883323] and p53 intron 6 [rs1625895] polymorphisms was performed as described by Wu et al. [31]. Genotyping of p73 exon 2 G4C14-to-A4T14 [rs227353/ rs1801173] was performed using PCR [32]. The polymorphic site at nucleotide 638 in exon 7 (Arg213His) (rs9282861) of the sulfotransferase gene (SULT 1A1) were genotyped by PCR–RFLP analysis [33]. Tumor necrosis factor alpha (TNFA) polymorphism TNF-308G [ A (rs1800629) was determined as described by Lee et al. [34]. Fresh gastric biopsies were obtained by each patient after gastrectomy, and DNA was extracted using standard methods. H. pylori positivity was determined as described by Mapstone et al. [35], while cagA status and vacA genotypes (m- and s-region) were identified by multiplex PCR as previously reported by Chattopadhyay et al. [36]. Ten percent of the total samples were randomly selected and reanalyzed with 100 % of concordance. Statistical analysis Descriptive analysis using proportion and mean ± SD was computed for categorical and quantitative variables, respectively. Differences between groups were calculated using a Chi squared, two-sample t-tests or Fisher’s exact tests, as appropriate. The relationship between H. pylori positivity, cagA status, vacA regions and putative risk factors were measured using the adjusted odds ratios (ORs) and their 95 % confidence interval (CI) from logistic regression analysis. Variables with a p value of \0.10 from the univariate analysis were considered candidates for the multivariate logistic regression model using a backward elimination procedure. The Stata software, version 11.0 was used (StataCorp 2007; Stata Statistical Software, College Station, TX, US).
Table 1 Clinical features and Helicobacter pylori status of gastric cancer patients (n = 149) n
%
Gender Male
78
52.3
Female
71
47.7
Age (years) \50
13
8.8
50–59
22
15.0
60–69 70–79
39 56
26.5 38.1
[80
17
11.6
Elementary school
52
47.7
High-school
29
26.6
Secondary education
28
25.7
Educational level
Tumor gradinga 1
2
2.3
2
25
28.4
3
61
69.3
No
38
31.4
Yes
83
68.6
1 2
28 33
23.7 28.0
3
34
28.8
4
23
19.5
Intestinal
76
58.0
Diffuse
55
42.0
Positive
117
78.5
Negative
32
21.5
cagA (-)i
57
50.0
cagA (?)
57
50.0
vacA (m2)i
25
27.5
vacA (m1, m1/m2)
66
72.5
Lymph nodes involvement
Tumor stage
Tumor histotype
Helicobacter pylori
Helicobacter pylori positive cases*
a
vacA (s2)i
22
19.3
vacA (s1, s1/s2) cagA (?), vacA (m1, m1/m2)
92 41
80.7 46.1
cagA (?), vacA (s1, s1/s2)
48
43.2
vacA (m1, m1/m2), vacA (s1, s1/s2)
55
61.1
cagA (?), vacA (m1, m1/m2), vacA (s1, s1/s2)
37
42.0
Missing value = 41 %
* Missing values: cagA = 3 %, vacA m = 22 %, vacA s = 3 % i
cagA: cytotoxin associated gene A
vacA: vacuolating cytotoxin A
123
123
12–23
\12
Alcohol intake (g/day)
Ever drinkers
Never
Spirits
Ever drinkers
Never
Beer
Current drinker
Wine Never
12
57.1 %
19.1 %
12.5 %
4 4
87.5 %
9.4 %
90.6 %
68.8 %
31.2 %
28.1 %
28.1 %
15.6 %
56.3 %
31.8 %
18.2 %
50.0 %
±12.52
40.6 %
64.3 % 35.7 %
75.9 %
21.8 %
25.0 %
28
3
29
22
10
9 9
Current smokers
[20 pack years
Alcohol
5
Ex-smokers
Never
18
7
Secondary education
Cigarette smoking
4
Middle school
Elementary education
11
68.4
Educational level
13
Age (years)
18 10
Male sex
Demographic
1–2 3–4
Stage
22
12
Diffuse
Lymph nodes involvement
19
Intestinal
Histotype
Clinical characteristics
32
15
14
101
23
92
77
38
44
23
34
58
21
25
41
66.8
65
43 47
61
43
57
n = 117
n = 32
%
HP (?)
HP (-)
41.0 %
19.2 %
12.2 %
87.8 %
20.0 %
80.0 %
67.0 %
33.0 %
37.6 %
20.0 %
29.6 %
50.4 %
24.1 %
28.7 %
47.1 %
±11.13
56.5 %
47.8 % 52.2 %
66.3 %
78.2 %
75 %
%
0.346
1.000
0.164
0.848
0.321
0.252
0.557
0.490
0.111
0.127
0.334
0.672
p value
17
7
7
50
13
44
38
19
19
13
15
29
13
12
20
67.3
34
24 19
29
17
31
n = 57
cagAi (-)
HP (?)
42.5 %
17.5 %
12.3 %
87.7 %
22.8 %
77.2 %
66.7 %
33.3 %
33.3 %
22.8 %
26.3 %
50.9 %
28.9 %
26.7 %
44.4 %
±10.70
59.6 %
55.8 % 44.2 %
65.9 %
39.5 %
56.4 %
%
14
8
7
48
10
45
38
17
23
9
18
28
8
12
19
66.3
3
19 28
32
26
24
n = 57
cagA (?)
37.8 %
21.6 %
12.7 %
87.3 %
18.2 %
81.8 %
69.1 %
30.9 %
40.4 %
16.4 %
32.7 %
50.9 %
20.5 %
30.8 %
48.7 %
±11.88
54.6 %
40.4 % 59.6 %
66.7 %
60.5 %
43.6 %
%
Table 2 Demographics and lifestyle characteristics of 149 gastric cancer patients according to Helicobacter pylori status, and cagA status and vacA m and s-regions
0.873
0.943
0.545
0.784
0.437
0.612
0.673
0.639
0.585
0.144
0.939
0.098
p value
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[9 times/week Add salt to meals
%
25.0 %
Male sex
Demographic
3–4
1–2
13
10
18
22
12
Diffuse
Lymph nodes involvement Stage
19
Intestinal
Histotype
40.6 %
35.7 %
64.3 %
75.9 %
21.8 %
65
47
43
61
43
57
n = 117
n = 32
Clinical characteristics
HP (?)
HP (-)
3.6 % 3.3 %
1 1
Gastric cancer (first degree)
33.3 %
6.3 %
18.8 %
59.4 %
28.1 % 21.9 %
56.3 %
Any cancer (father)
Any cancer (first degree)
10
2
Respiratory diseases
Cancer family history
6
Diabetes
Cardiovascular diseases
19
9 7
5/9 times/week
Comorbidities
5 18
\5 times/week 15.6 %
40.6 %
13
Fresh vegetables
[9 times/week
53.1 %
2 17
6.3 %
23.8 %
5/9 times/week
5
%
56.5 %
52.2 %
47.8 %
66.3 %
78.2 %
75 %
10
24
41
14
18
54
39 23
52
20
61
44
7
31
0.111
0.127
0.334
0.672
p value
n = 117
n = 32
%
HP (?)
HP (-)
\ 5 times/week
Fruit
Dietary Habits
C24
Table 2 continued
13
11
6
14
6
14
n = 25
%
vacA (m2)i
HP (?)
9.0 %
23.1 %
36.9 %
12.1 %
15.5 %
46.9 %
35.1 % 20.0 %
46.8 %
18.0 %
54.5 %
39.3 %
6.3 %
39.8 %
%
32.6 %
54.2 %
64.7 %
35.3 %
77.8 %
17.6 %
3
10
19
8
9
33
19 10
23
13
30
22
4
16
n = 57
cagAi (-)
HP (?)
36
28
25
35
28
29
n = 66
%
57.1 %
52.8 %
47.2 %
64.8 %
82.5 %
67.4 %
vacA (m1, m1/m2)
0.457
0.019
0.716
0.280
0.660
0.214
0.816
0.640
0.376
p value
0.802
0.391
0.307
0.138
p value
11
13
8
14
7
13
n = 22
50.0 %
61.9 %
38.1 %
66.7 %
16.7
22.6 %
7
14
21
6
9
21
18 13
28
7
30
21
2
15
n = 57
%
vacA (s2)i
5.6 %
20.0 %
35.2 %
14.0 %
15.8 %
53.9 %
34.5 % 17.5 %
41.8 %
23.6 %
53.6 %
39.3 %
7.1 %
40.0 %
%
cagA (?)
53
33
33
46
35
42
n = 92
%
58.9 %
50.0 %
50.0 %
67.6 %
83.3 %
76.4 %
vacA (s1, s1/s2)
13.0 %
27.4 %
38.9 %
10.7 %
16.1 %
37.5 %
34.0 % 23.6 %
52.8 %
13.2 %
56.6 %
39.6 %
3.8 %
40.5 %
%
0.450
0.341
0.933
0.401
p value
0.320
0.379
0.690
0.592
0.967
0.023
0.425
0.320
0.856
p value
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123
123
7
Secondary education
5
9
9
Ex-smokers
Current smokers
[20 pack years Alcohol
22
Current drinker
3
Ever drinkers
4
Ever drinkers
5
18
9
\5 times/week
5/9 times/week
[9 times/week
Fresh vegetables
28.1 %
56.3 %
15.6 %
40.6 %
13
5/9 times/week
[9 times/week
53.1 %
2
17
\ 5 times/week 6.3 %
23.8 %
5
Dietary Habits Fruit
C24
57.1 %
4
12
12–23
19.1 %
12.5 %
87.5 %
9.4 %
90.6 %
68.8 %
31.2 %
28.1 %
28.1 %
15.6 %
56.3 %
31.8 %
18.2 %
50.0 %
±12.52
\12
Alcohol intake (g/day)
28
Never
Spirits
29
Never
Beer
10
Never
Wine
18
Never
Cigarette smoking
4
11
68.4
39
52
20
61
44
7
31
32
15
14
101
23
92
77
38
44
23
34
58
21
25
41
66.8
n = 117
n = 32
%
HP (?)
HP (-)
Middle school
Elementary education
Educational level
Age (years)
Table 2 continued
35.1 %
46.8 %
18.0 %
54.5 %
39.3 %
6.3 %
39.8 %
41.0 %
19.2 %
12.2 %
87.8 %
20.0 %
80.0 %
67.0 %
33.0 %
37.6 %
20.0 %
29.6 %
50.4 %
24.1 %
28.7 %
47.1 %
±11.13
%
0.640
0.376
0.346
1.000
0.164
0.848
0.321
0.252
0.557
0.490
p value
8
12
3
12
12
0
5
5
3
4
21
4
21
12
13
6
3
9
12
5
5
8
68.8
n = 25
34.8 %
52.2 %
13.0 %
50.0 %
50.0 %
–
38.5 %
38.5 %
23.1 %
16.0 %
84.0 %
16.0 %
84.0 %
48.0 %
52.0 %
25.0 %
12.5 %
37.5 %
50.0 %
27.8 %
27.8 %
44.4 %
±12.56
%
vacA (m2)i
HP (?)
20
29
12
32
26
4
18
18
9
6
58
13
51
46
18
30
17
15
31
10
15
23
65.4
n = 66
32.8 %
47.5 %
19.7 %
51.6 %
41.9 %
6.5 %
40.0 %
40.0 %
20.0 %
9.4 %
90.6 %
20.3 %
79.7 %
71.9 %
28.1 %
46.2 %
27.0 %
23.8 %
49.2 %
20.8 %
31.3 %
47.9 %
±10.79
%
vacA (m1, m1/m2)
0.852
0.613
0.971
0.458
0.770
0.034
0.071
0.250
0.877
0.213
p value
11
8
3
12
10
0
6
6
3
4
18
7
15
13
9
7
5
6
11
5
3
9
67.6
n = 22
50.0 %
36.4 %
13.6 %
54.5 %
45.5 %
–
40.0 %
40.0 %
20.0 %
18.2 %
81.8 %
31.8 %
68.2 %
59.1 %
40.9 %
31.8 %
22.7 %
27.3 %
50.0 %
29.4 %
17.6 %
52.9 %
±12.76
%
vacA (s2)i
26
40
17
47
31
6
25
26
10
10
80
15
75
63
27
35
17
27
43
15
22
30
66.6
n = 92
31.3 %
48.2 %
20.5 %
56.0 %
36.9 %
7.1 %
41.0 %
42.6 %
16.4 %
11.1 %
88.9 %
16.7 %
83.3 %
70.0 %
30.0 %
39.3 %
19.5 %
31.0 %
49.4 %
22.4 %
32.8 %
44.8 %
±10.87
%
vacA (s1, s1/s2)
0.277
0.497
0.945
0.470
0.135
0.326
0.347
0.918
0.506
0.721
p value
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HP Helicobacter pylori, icagA cytotoxin associated gene A, vacA vacuolating cytotoxin A
1.000
0.683 9.3 %
21.7 % 18
8 4.6 %
21.1 % 4
1 0.667
0.654 21.4 %
9.8 % 6
12
Results
4.2 %
26.1 % 6
1 0.457
0.019 23.1 %
9.0 % 10
24
1 Gastric cancer (first degree)
3.3 %
1 Any cancer (father)
3.6 %
0.897 34.9 % 30 36.4 % 8 0.812 36.1 % 22 33.3 % 8 0.716 36.9 % 41 33.3 % 10 Any cancer (first degree)
Cancer family history
0.303
0.459 13.2 % 12 9.1 % 2 0.292 15.4 % 10 8.0 % 2 0.280 12.1 % 14 2 Respiratory diseases
6.3 %
0.472 49.4 %
18.2 % 16
45 40.9 %
9.1 % 2
9 0.819
0.821 18.8 %
45.3 % 29
12 16.7 %
48.0 % 12
4 0.660
0.214 46.9 %
15.5 % 18
54
6 Diabetes
59.4 % 19 Cardiovascular diseases
18.8 %
0.739 19.5 % 17 22.7 % 0.798 19.1 % 12 16.7 % 4 0.816 20.0 % 23 21.9 % 7 Add salt to meals
Comorbidities
n = 117 n = 32
% HP (?) HP (-)
% Table 2 continued
5
% n = 92 % n = 22 % n = 66 % n = 25
vacA (m2)i
p value
HP (?)
vacA (m1, m1/m2)
p value
vacA (s2)i
vacA (s1, s1/s2)
p value
Mol Biol Rep
The clinical and demographic features of 149 gastric cancer patients included in study are reported in Table 1. Fifty-two percent of the cases were male, more than 70 % were aged C60 years old and 47.4 % had only elementary school education. Most cases were high grades of gastric cancer (grade 3: 69.3 %) and had lymph nodes involvement (68.6 %). Fifty-eight percent were intestinal histotype, and overall 78.5 % of the cases were H. pylori positive. Among the positive H. pylori cases, 50 % were cagA?, 72.5 % vacA m1 subtype and 80.7 % were vacA s1 subtype. Forty-six percent and 43.2 % were simultaneously cagA and, respectively, vacA m1 or vacA s1 positive, while 61.1 % were at the same time vacA m1 and s1 subtype. CagA?, vacA m1 and vacA s1 genotype was simultaneously present in 42 % of strains. Overall, cagA? strains were not associated with the co-presence of vacA m1 (p = 0.031) or vacA s1 (p = 0.052) subtype (data not shown). Data on demographics, lifestyle habits, comorbidities and cancer family history according to H. pylori status and H. pylori cagA status and vacA genotypes are reported in Table 2. CagA positive strains were more common among the diffuse histotype of gastric cancer respect to the intestinal, even though not significantly different. The proportion of current wine drinkers was higher among vacA m1 or m1/m2 strains (71.9 %) respect to vacA m2 (48.0 %) (p = 0.034). The consumption of more than 20 pack-years of cigarettes was more common in patients infected by vacA m1 or m1/m2 than vacA m2, although not significantly different (p = 0.071). Cardiovascular diseases (CVDs) were more frequent in patients infected by cagA negative strains than in cagA positive strains (p = 0.023). H. pylori positive patients were more likely to have a father with a history of any cancer compared with the H. pylori negative patients (23.1 vs. 3.6 %, respectively; p = 0.019). Table 3 reports the distribution of the studied polymorphisms in our gastric cancer patients. The variant genotypes of EPHX1 exon 3 (p = 0.032), IL1B-511 (p = 0.039) and IL1-RN (p = 0.002) gene were more common among H. pylori negative than positive patients. Concerning the H. pylori strains, IL1-RN VNTR (p = 0.051) and SULT1A1 (p = 0.047) variant genotypes were borderline more common among cagA positive cases. Lastly, MTHFR 677 variant allele was borderline more frequent among those infected with carriers of vacA m1 subtype (p = 0.072) than in m2, while p53 exon 4 homozygote variant genotype was less common in patients infected with vacA s1 subtype than in s2 (p = 0.050). The ORs for H. pylori positivity based on univariate and multivariate analyses are reported in Table 4. We identified EPHX1 exon 3 (OR = 0.35, CI 95 % 0.13–0.94), IL1B-
123
123 78.1
8
CG/GG
29
17
AC/CC
p53 Ex4 ?119C [ G (rs1042522)
14
AA
MTHFR 1298A [ C (rs1801131)
90.6
54.8
45.2
25.0
14
TT
43.8
10
31.2
25.0
75.0
28.1
71.9
3.1
96.9
21.9
CT
8
24
9
23
1
31
7
40.6
59.4
CC
MTHFR 677C [ T (rs1801133)
22
2L/LL
TT IL1-RN VNTR
CC/TC
IL1B-511 T [ C (rs16944)
TT
CC/CT
IL1B ? 3954C [T (rs1143634)
CC
TT/CT
25
13
IL1B -31C [ T (rs1143627)
19
68.8
22
null
31.3
10
65.6 34.4
25.0
8 21 11
75.0
24
wt
GSTT1 Ex5-49 1>-
null
wt
GSTM1Ex4 1 10 1>-
AA AG/GG
EPHX1 exon 4 A [ G H139R (rs2234922)
CC
TT/TC
EPHX1 exon3 T [ C Y113H (rs1651740)
110
53
56
17
56
44
7
109
15
101
10
106
13
104
39
75
62
52
74 41
12
104
n = 117
n = 32
%
HP (?)
*HP (-)
94.0
48.6
51.4
14.5
47.9
37.6
6.8
93.2
12.9
87.1
8.6
91.4
11.1
88.9
34.2
65.8
54.4
45.6
64.4 35.7
10.3
89.7
%
0.497
0.541
0.366
0.002
0.039
0.294
0.113
0.503
0.146
0.894
0.032
p value
52
28
25
8
31
18
1
56
6
51
7
50
5
52
19
35
31
23
38 19
5
52
n = 57
cagA (-)i
HP (?)
91.2
52.8
47.2
14.0
54.4
31.6
1.8
98.2
10.5
89.5
12.3
87.7
8.8
91.2
35.2
64.8
57.4
42.6
66.7 33.3
8.8
91.2
%
55
24
31
9
24
24
7
50
8
48
3
53
7
50
20
37
29
28
34 21
7
49
n = 57
cagA (?)
96.5
43.6
56.4
15.8
42.1
42.1
12.3
87.7
14.3
85.7
5.4
94.6
12.3
87.7
35.1
64.9
50.9
49.1
61.8 38.2
12.5
87.5
%
Table 3 Distribution of the selected gene polymorphisms of 149 gastric cancer patients according to Helicobacter pylori status, and cagA status and vacA m- and s-regions
0.219
0.339
0.405
0.051
0.544
0.168
0.381
0.991
0.490
0.592
0.368
p value
Mol Biol Rep
%
15.6
5
68.8
10 22
wt
19 13
wt
null
GSTT1 Ex5-49 1>-
null
31.3
11
40.6
59.4
34.4
21
AG/GG GSTM1Ex4 1 10 1>-
65.6
25.0
8
AA
EPHX1 exon 4 A [ G H139R (rs2234922)
CC
TT/TC
39
75
62
52
41
74
12
104
n = 32
75.0
n = 117
*HP (-)
24
HP (?)
8
EPHX1 exon3 T [ C Y113H (rs1651740)
25.0
24
GA
75.0
84.4
GG
TNFA -308G [ A (rs1800629)
AA
GG/GA
34.4
65.6
21.9
78.1
25.0
75.0
9.4
27
11
SULT1A1 638G [ A (rs9282861)
21
(GC/AT)/(AT/AT)
7
25
8
24
3
%
34.2
65.8
54.4
45.6
35.7
64.4
10.3
89.7
0.503
0.146
0.894
0.032
p value
19
96
10
107
32
85
38
79
41
76
7
n = 117
n = 32
%
HP (?)
*HP (-)
p73 G4C14-to-A4T14 (rs1801173/rs2273953) (GC/GC)
GA/AA
GG
p53 IVS6 ?62A [ G (rs1625895)
16 bp/wt plus 16 bp/16 bp
wt/wt
53 intron3 (16 bp insertion)
CC
Table 3 continued
8
14
10
12
9
14
3
21
n = 25
36.4
63.6
45.5
54.6
39.1
60.9
12.5
87.5
%
vacA (m2)i
HP (?)
16.5
83.5
8.6
91.5
27.4
72.6
32.5
67.5
35.0
65.0
6.0
%
10
46
2
55
45
38
17
40
19
38
5
n = 57
23
42
37
28
19
45
8
58
n = 66
35.4
64.6
56.9
43.1
29.7
70.3
12.1
87.9
%
0.934
0.351
0.406
0.607
p value
cagA (-)i
HP (?)
vacA (m1, m1/m2)
0.273
0.194
0.437
0.247
0.284
p value
5
16
14
7
5
17
2
20
n = 22
23.8
76.2
66.7
33.3
22.7
77.3
9.1
90.9
%
9
47
8
49
12
19
20
37
21
36
2
n = 57
vacA (s2)i
17.9
82.1
3.5
96.5
54.2
45.8
29.8
70.2
33.3
66.7
8.8
%
16.1
83.9
14.0
86.0
38.7
61.3
35.1
64.9
36.8
63.2
3.5
%
33
57
45
45
33
57
10
81
n = 92
36.7
63.3
50.0
50.0
36.7
63.3
11.0
89.0
%
vacA (s1, s1/s2)
cagA (?)
0.264
0.168
0.161
0.575
p value
0.801
0.047
0.103
0.548
0.695
p value
Mol Biol Rep
123
123 8
22
7
GA/AA 21 11
(GC/GC)
(GC/AT)/(AT/AT)
p73 G4C14-to-A4T14 (rs1801173/rs2273953)
25
GG
p53 IVS6 ?62A [ G (rs1625895)
wt/wt 16 bp/wt plus 16 bp/16 bp
24 8
3
53 intron3 (16 bp insertion)
29
CC
34.4
65.6
21.9
78.1
75.0 25.0
9.4
90.6
54.8
17
CG/GG
p53 Ex4 ?119C [ G (rs1042522)
AC/CC
AA
45.2
8
MTHFR 1298A [ C (rs1801131) 14
25.0
14
TT
43.8
10
CT
31.2
25.0
75.0
71.9 28.1
3.1
96.9
CC
MTHFR 677C [ T (rs1801133)
24
2L/LL
IL1-RN VNTR
CC/TC TT
23 9
1
IL1B-511 T [ C (rs16944)
31
21.9
7
TT
78.1
25
32
85
38
79
76 41
7
110
53
56
17
56
44
7
109
101 15
10
106
13
104
n = 117
n = 32
%
HP (?)
*HP (-)
CC/CT
IL1B ? 3954C [T (rs1143634)
CC
TT/CT
IL1B -31C [ T (rs1143627)
Table 3 continued
27.4
72.6
32.5
67.5
65.0 35.0
6.0
94.0
48.6
51.4
14.5
47.9
37.6
6.8
93.2
87.1 12.9
8.6
91.4
11.1
88.9
%
0.437
0.247
0.284
0.497
0.541
0.366
0.002
0.039
0.294
0.113
p value
7
18
7
18
16 9
3
21
12
9
3
8
14
0
24
24 1
2
23
1
24
n = 25
28.0
72.0
28.0
72.0
9.0 36.0
12.5
87.5
57.1
42.9
12.0
32.0
56.0
0.0
100.0
96.0 4.0
8.0
92.0
4.0
96.0
%
vacA (m2)i
HP (?)
19
47
20
46
47 19
2
63
32
32
10
36
20
6
59
55 10
4
61
9
57
n = 66
28.8
71.2
30.3
69.7
71.2 28.8
3.1
96.9
50.0
50.0
15.1
54.5
30.3
9.2
90.8
84.6 15.4
6.2
93.9
13.6
86.4
%
vacA (m1, m1/m2)
0.941
0.830
0.506
0.087
0.570
0.072
0.142
0.140
0.536
0.177
p value
8
14
10
12
13 9
3
19
9
12
3
11
8
1
21
20 2
4
18
2
20
n = 22
36.4
63.6
45.5
54.5
59.1 40.9
13.6
86.4
42.9
57.2
13.6
50.0
36.4
4.5
95.5
90.9 9.1
18.2
81.8
9.1
90.9
%
vacA (s2)i
24
68
27
65
62 30
3
89
44
41
12
44
36
6
86
78 13
6
85
11
81
n = 92
26.1
73.9
29.4
70.7
64.4 32.6
3.3
96.7
51.8
48.2
13.0
47.8
39.1
6.5
93.5
85.7 14.3
6.6
93.4
12.0
88.0
%
vacA (s1, s1/s2)
0.335
0.147
0.461
0.050
0.465
0.972
0.594
0.519
0.097
0.522
p value
Mol Biol Rep
85.7
Adj ORa
CI 95 %
Any cancer, father
8.10
1.04–62.76
8.18
1.04–64.55
EPHX1 exon3 T [ C Y113H (rs1651740)
0.35
0.13–0.94
0.42
0.13–1.42
14.3 13
78 76.2
23.8
10.9 10 0.0
82 100.0
0.223
0.106 89.1
% n = 92 %
CI 95 %
IL1B-511 T [ C (rs16944)
0.38
0.15–0.97
0.36
0.12–1.46
IL1-RN VNTR
0.19
0.06–0.58
0.45
0.12–1.69
n = 22
5
16
0 8
56
9
4.0
73.9
26.1
86.2
12.1
0.180
511 (OR = 0.38, CI 95 % 0.15–0.97) and IL1-RN VNTR (OR = 0.19, CI 95 % 0.06–0.58) polymorphisms as protective in univariate analyses but the significance was not confirmed after adjustment for confounding factors in the multivariate analysis. A positive father‘s any cancer history was confirmed associated with an increased risk of H. pylori positivity, both on univariate (OR = 8.10, CI 95 % 1.04–62.76), and multivariate (OR = 8.18, CI 95 % 1.04–64.55) analyses.
13.9
22 0.230 87.9 58 96.0
% n = 66 %
8 GA
HP Helicobacter pylori, icagA cytotoxin associated gene A, vacA vacuolating cytotoxin A
16.5
83.5 96 24 GG
75.0
19
10 15.6 5 AA
TNFA -308G [ A (rs1800629)
25.0
8.6
0.273
6
17
1
24 0.194 91.5 84.4 GG/GA
SULT1A1 638G [ A (rs9282861)
27
107
% n = 117 n = 32
%
HP (?) *HP (-)
Adjusted by age, any cancer (father), IL1B-511T [ C
Discussion
n = 25
vacA (m1, m1/m2) vacA (m2)i
HP (?)
OR
OR odds ratio, CI confidence interval
p value Table 3 continued
Table 4 Risk factors for Helicobacter pylori positivity from univariate and multivariate analyses
a
p value
vacA (s2)i
vacA (s1, s1/s2)
p value
Mol Biol Rep
Our study identified the presence of father history for any cancer as a significant risk factor for H. pylori infection in gastric cancer patients. From the univariate analysis, CVDs are reported to be less common among cagA? subjects. Wine consumption was associated with higher occurrence of H. pylori vacA m1 virulent subtype. As for the genetic factors, polymorphisms in EPHX1 exon 3, IL1B-511 and IL1-RN genes resulted as protective factors towards H. pylori infection in the univariate analysis. Additionally, our results show that homozygous genotype of SULT1A1 and the variant genotype of IL1-RN were more common among gastric cancer subjects infected with cagA positive strains. Lastly, MTHFR 677 variant allele was more common among patients infected with strains carrying the vacA m1 virulent subtype, while p53 exon 4 homozygote variant was less common among patients infected by vacA s1 virulent subtype. Reports in the past few years suggested that low socioeconomic status [37], low educational level [38] and large family size [39] are associated with H. pylori infection. Alcohol intake is reported to be inversely associated [40], while data on smoking are contradictory [37, 41]. Several genetic polymorphisms of the host have also been suggested to influence susceptibility toward H. pylori infection [42–47], among them polymorphisms of IL1B-511, IL1A889, TNFA-308, fucosyltransferase and myeloperoxidase genes. These data are derived both from studies conducted
123
Mol Biol Rep
among healthy individuals and patients with various gastric diseases, including gastric cancer. A family history of gastric cancer has been previously reported as risk factor for H. pylori infection in children [48]. North American consensus statement suggested testing for H. pylori in children with positive family history of gastric cancer [49]. However, the same observation has not been reported in the adult population [38]. Our study shows that any history of cancer of the father is a risk factor for H. pylori infection. However sub-analysis did not show that the history of gastric cancer is associated with H. pylori infection. The way in which any cancer history of the father influences the susceptibility to H. pylori infection remains currently unexplained. Our study shows that CVDs are less common among patients infected with cagA? strains. Several studies in the literature assessed the relationship between CVD and H. pylori infection, and cagA strains [50–53], with discordant results. Scho¨ttker et al. [53] recently reported an inverse association between infection with H. pylori cagA? positive strains and fatal cardiovascular events. Although they suppose that constant stimulation of the immune system of the host by cagA? H. pylori could be protective towards fatal cardiovascular events, this theory does not explain how cagA? strains could act protectively against chronic cardiovascular conditions. Alcohol consumption is reported to be inversely associated with H. pylori infection [40], and this association is stronger for wine than for beer drinkers [40], that might be explained by additional direct bactericide nature of wine. In our study we did not observe a difference in alcohol consumption among H. pylori positive and negative cases, though the virulent vacA m1 subtype was more frequent among wine drinkers respect to never drinkers. We can argue that that the high virulence properties of these strains allow them to survive in hostile environment, though this should be demonstrated by in vivo studies. Our study shows that the variant alleles IL1B-511 and IL1-RN genes might be protective factors against H. pylori infection in univariate analysis, as the variant genotypes are more common among H. pylori negative gastric cancer cases. IL1B-511 and IL1-RN polymorphisms affect the expression levels of IL-1b, a potent proinflamatory citokyne [29]. Increased IL-1b production induces stronger inflammatory response of the host to H. pylori but also inhibits gastric secretion thus influencing the susceptibility towards infection. Hosts with H. pylori infection who carry the IL-1B-511 T/T genotype or IL1-RN 2 might have a stronger IL-1b production [54]. Proinflammatory polymorphism of IL1B-511 has been reported to favour H. pylori infection [42, 43], however this association has not been reported for IL1-RN [42, 43]. Nevertheless, one study reported that IL1-511 T allele is associated with reduced
123
risk of H. pylori infection [55]. Larger studies to come should clarify if proinflammatory component of these genes acts protectively towards H. pylori infection or the inhibition of gastric secretion prevails in its favoring. EPHX1 codes for enzyme which is playing a role in detoxification and activation of many carcinogens. EPHX1 polymorphism is reported to be changing enzyme activity influencing susceptibility towards colorectal and lung cancers [56, 57]. Our univariate analysis showed for the first time, that the homozygote genotype of EPHX1 exon 3 is protective against H. pylori infection in gastric cancer patients. We reported that polymorphisms in MTHFR 677, SULT1A1 and p53 exon 4 interact with H. pylori virulent cagA positive and vacA s1 as well vacA m1 subtypes. Previous report did not show difference in H. pylori presence among patients with different MTHFR 677 genotypes [58]. Although it has been reported that SULT1A1 [22] and p53 exon 4 [59] polymorphisms have an influence on gastric cancer carcinogenesis, no investigation so far addressed an impact of these two polymorphisms to H. pylori susceptibility. Further studies on these issues including larger number of cases are required, in view of few subjects carrying the variant alleles of these gene. It has been reported that cagA positive H. pylori strains identified from patients with gastric lesions were more likely to carry the vacA s1 and vacA m1 subtypes [60]. Infection with strains which are simultaneously cagA and vacAs1/m1 positive is associated with progression of gastric precancerous lesions towards malignancies [61]. Nevertheless, a recent study from Brazil [62], although including small number of cases, reported high prevalence of vacA s1 genotype among cagA negative gastric cancer patients infected with H. pylori. Virulent vacA m and vacA s, H. pylori strains isolated from gastric cancer patients in our study were also less likely to be cagA positive. We can argue that, in absence of cagA positivity, carrying at least one factor of vacA virulence might play a crucial role in gastric cancer development. The shortcoming of our study is the small number of cases included, and 26.9 % missing data on patients education and 22 % on H. pylori vacA m subtypes. Another important issue regards the time of H. pylori infection acquirement, and because this cannot be assessed we cannot be sure that exposure to the investigated risk factors preceded the infection, except the genetic ones. On the other hand, this study addresses the effect of a large list of putative risk factors, that include several genetic host risk factors. In conclusion, our study shows that father history for any cancer is a risk factor for H. pylori infection. Polymorphism in IL1B-511, IL1-RN and EPHX1 exon 3 genes could be protective towards H. pylori infection. Further studies including a larger number of cases are required.
Mol Biol Rep Acknowledgments This work has been supported by the AIRC (Associazione Italiana Ricerca sul Cancro) IG 2013 N.14220, and from the Italian Ministry of Health (GR-2011-02347943). The work of Dr Nikola Panic was supported by the ERAWEB project, funded with support of the European Community. The work of Dr Emanuele Leoncini was supported by Fondazione Veronesi.
19.
20. Conflict of interests
The authors have no conflict of interests.
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Susceptibility to Helicobacter pylori infection: results of an epidemiological investigation among gastric cancer patients Nikola Panic • Elena Mastrostefano • Emanuele Leoncini • Roberto Persiani • Dario Arzani Rosarita Amore • Riccardo Ricci • Federico Sicoli • Stefano Sioletic • Milutin Bulajic • Domenico D’ Ugo • Walter Ricciardi • Stefania Boccia
•
Received: 14 May 2013 / Accepted: 3 February 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The aim of this study was to identify the clinical, demographic, lifestyle factors and selected genetic polymorphisms that affect the susceptibility towards Helicobacter pylori (H. pylori) infection in gastric cancer patients. Histological confirmed gastric adenocarcinoma cases that underwent curative gastrectomy between 2002 and 2012 were included. Gastric biopsy samples were obtained to determine the H. pylori status, and further cagA status and vacA m and s genotypes by polymerase chain reaction. Patients were interviewed with structured questionnaires, and blood samples were collected for EPHX1, GSTM1, GSTT1, IL1B, IL1RN, MTHFR and p53 genotyping. Proportions were compared in univariate analysis, while the relation between putative risk factors and H. pylori status and genotype were measured using logistic regression analysis. One hundred forty-nine gastric cancer patients were included, of which 78.5 % were H. pylori positive. Among positive patients
50 % were cagA?, 72.5 % vacA m1 and 80.7 % vacA s1. The presence of cagA was less frequent among vacA m1 (p = 0.031) and vacA s1 (p = 0.052) subtypes. The presence of father history for any cancer was a significant risk factor for H. pylori infection [adjusted odds ratio (OR) = 8.18, 95 % confidence interval (CI) 1.04–64.55]. EPHX1 exon 3 T [ C (OR = 0.35, CI 95 % 0.13–0.94), IL1B-511 T [ C (OR = 0.38, CI 95 % 0.15–0.97) and IL1-RN VNTR (OR = 0.19, CI 95 % 0.06–0.58) polymorphisms were protective towards H. pylori infection in the univariate analysis. Wine consumption was associated with higher risk of carrying the H. pylori vacA m1 virulent subtype (p = 0.034). Lastly, cardiovascular diseases were less common among cagA positive subjects (p = 0.023). Father history of any cancer is a risk factor for H. pylori infection. Polymorphisms in IL1B-511, IL1-RN and EPHX1 exon 3 genes might be protective towards H. pylori infection.
N. Panic E. Mastrostefano E. Leoncini D. Arzani R. Amore W. Ricciardi S. Boccia (&) Institute of Public Health-Section of Hygiene, Universita` Cattolica del Sacro Cuore, Rome, Italy e-mail: [email protected]
S. Sioletic The Ludwig Center at Dana-Farber/Harvard Cancer Center, Boston, MA, USA
N. Panic M. Bulajic Faculty of Medicine, University of Belgrade, Belgrade, Serbia N. Panic M. Bulajic University Clinical-Hospital Center ‘‘Dr DragisaMisovicDedinje’’, Belgrade, Serbia
M. Bulajic University Clinical Hospital ‘‘Santa Maria della Misericordia’’, Udine, Italy S. Boccia IRCCS San Raffaele, Pisana, Rome, Italy
R. Persiani F. Sicoli D. D. Ugo Department of Surgery, Universita` Cattolica del Sacro Cuore, Rome, Italy R. Ricci Department of Pathology, Universita` Cattolica del Sacro Cuore, Rome, Italy
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Keywords Helicobacter pylori Gastric cancer Genetic polymorphism Family history
Introduction Gastric cancer accounts for about 10 % of all cancers worldwide, being the fourth most common cancer and the second cause of cancer death [1]. Incidence and mortality are high in Japan, China, Russia and most central and eastern Europe, where the prevalence of Helicobacter pylori (H. pylori) is relatively high compared to western Europe and the USA (60–90 % vs. 30–40 % of the population) [2]. Gastric carcinogenesis is the result of a complex interaction between environmental and genetic factors. Among the environmental, H. pylori infection, tobacco smoking, low fruit and vegetable intake, salt or salty food intake and the lack of food refrigeration plays a major role, while for the genetic factors accumulating evidences report that the individual genetic susceptibility to gastric cancer involves several genes with small effects [3–9]. H. pylori is proven to be group 1 carcinogen for gastric cancer [10], as it induces chronic inflammation of the gastric mucosa [11]. Some reports suggest that up to 85 % of noncardia gastric cancers are H. pylori related [12]. The virulence of H. pylori is determined by polymorphisms of the cagA and vacA genes, coding for certain bacteria virulence factors. CagA codes for a protein that alters numerous signaling pathways associated with malignant transformation of cells [13], while VacA is a secreted pore-forming toxin that causes cell vacuolation, apoptosis, and inhibition of T cell activation and proliferation [14]. CagA is present in 60–70 % of H. pylori strains in Western population and almost 100 % in East-Asia [15], while vacA is present in literally every H. pylori strain. However there is a variation in vacA vacuolation activity depending on differences in middle (m1/m2) and signal (s1/s2) regions [16]. The infection with virulent strains of H. pylori, carrying cagA [17] and the more active forms of vacA (s1/m1 forms) [16, 18], is reported to increase the risk of developing gastric cancer respect to the less virulent forms. Though not being a necessary component cause [8], H. pylori infection represents certainly a key step in gastric carcinogenesis, as testified by the observation that the eradication of H. pylori decreases the risk of precancerous lesions [19] and gastric cancer [20]. In this sense, the identification of the lifestyle and host genetic factors that influence the susceptibility to H. pylori infection have the potential to lead to more specific prevention strategies towards gastric cancer. The aim of our study was to identify the factors influencing the infection of H. pylori among a set of gastric
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cancer patients. The demographics, lifestyle, family history characteristics, and selected genetic polymorphisms were investigated in association with H. pylori infection status, and with the presence of cagA and vacA m1 and s1 virulent strains.
Methods Study population Consecutive primary gastric adenocarcinoma patients with histological confirmation, who underwent a curative gastrectomy in the ‘‘A. Gemelli’’ teaching hospital during the period 2002–2012, were asked to participate in the study [21–24]. Gastric cancer cases were classified as intestinal and diffuse according to Lauren classification [25]. Clinical data (tumor site, lymphnode involvement, grade and stage) were retrieved from medical charts. Written informed consent was obtained from all study subjects. This study was performed according to the Declaration of Helsinki and was approved by the ethics committee of the Universita` Cattolica del Sacro Cuore. Data collection Patients were interviewed by trained medical doctors using a structured questionnaire to collect information on demographic data, lifestyle habits (smoking history, alcohol consumption, history of previous illness, dietary habits), comorbidities (cardiovascular diseases, diabetes, respiratory diseases) and family history of cancer (1st and 2nd degree). Questionnaires on lifestyle habits referred to 1 year prior the diagnosis of gastric cancer for cases. Cigarette smoking status was categorized as never and ever-smokers (including both current and former smokers). The amount of smoking was evaluated as pack-years. A pack-year is defined as the product of packs smoked per day and the total years of smoking. The study categorized drinkers in three categories-heavy drinkers (who consumed 24 g per day or more), moderate drinkers (12–23 g per day) and light drinkers (\12 g a day). A standard drink is equal to 12.0 g of pure alcohol. Gastric cancer cases and H. pylori genotyping A venous blood sample was drawn from each participant, collected into EDTA-coated tubes from which DNA was isolated from peripheral blood lymphocytes by standard methods. Identification of the microsomal epoxide hydrolase (EPHX1) exon3 and exon4 polymorphisms, EPHX1 exon3 T [ C (rs1651740) and EPHX1 exon4 A [ G (rs2234922), was performed using a polymerase chain
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reaction (PCR) followed by restriction-fragment length polymorphism analysis (RFLP) [26]. Gluthatione S-transferase M1 (GSTM1) and T1 (GSTT1) alleles were identified using a known multiplex PCR based method [27]. Corresponding primers and PCR–RFLP conditions to determine interleukin 1B (IL1B) polymorphisms, IL1B-31 C [ T (rs1143627) and IL1B-511 T [ C (rs16944), and the variable number of tandem repeats in interleukin 1 receptor antagonist (IL1-RN VNTR) polymorphism have been previously described by Ruzzo et al. [28]. Analysis of interleukin 1B exon 5 polymorphism (IL1B ? 3954 C [ T, rs1143634) was described by El Omar et al. [29]. Genotyping for methylenetetrahydrofolate reductase (MTHFR) polymorphisms, MTHFR 677C [ T (rs1801133) and MTHFR1298 A [ C (rs1801131) was performed by PCR– RFLP [30]. Genotyping for p53 exon 4 [rs1042522], p53 intron 3 [rs17883323] and p53 intron 6 [rs1625895] polymorphisms was performed as described by Wu et al. [31]. Genotyping of p73 exon 2 G4C14-to-A4T14 [rs227353/ rs1801173] was performed using PCR [32]. The polymorphic site at nucleotide 638 in exon 7 (Arg213His) (rs9282861) of the sulfotransferase gene (SULT 1A1) were genotyped by PCR–RFLP analysis [33]. Tumor necrosis factor alpha (TNFA) polymorphism TNF-308G [ A (rs1800629) was determined as described by Lee et al. [34]. Fresh gastric biopsies were obtained by each patient after gastrectomy, and DNA was extracted using standard methods. H. pylori positivity was determined as described by Mapstone et al. [35], while cagA status and vacA genotypes (m- and s-region) were identified by multiplex PCR as previously reported by Chattopadhyay et al. [36]. Ten percent of the total samples were randomly selected and reanalyzed with 100 % of concordance. Statistical analysis Descriptive analysis using proportion and mean ± SD was computed for categorical and quantitative variables, respectively. Differences between groups were calculated using a Chi squared, two-sample t-tests or Fisher’s exact tests, as appropriate. The relationship between H. pylori positivity, cagA status, vacA regions and putative risk factors were measured using the adjusted odds ratios (ORs) and their 95 % confidence interval (CI) from logistic regression analysis. Variables with a p value of \0.10 from the univariate analysis were considered candidates for the multivariate logistic regression model using a backward elimination procedure. The Stata software, version 11.0 was used (StataCorp 2007; Stata Statistical Software, College Station, TX, US).
Table 1 Clinical features and Helicobacter pylori status of gastric cancer patients (n = 149) n
%
Gender Male
78
52.3
Female
71
47.7
Age (years) \50
13
8.8
50–59
22
15.0
60–69 70–79
39 56
26.5 38.1
[80
17
11.6
Elementary school
52
47.7
High-school
29
26.6
Secondary education
28
25.7
Educational level
Tumor gradinga 1
2
2.3
2
25
28.4
3
61
69.3
No
38
31.4
Yes
83
68.6
1 2
28 33
23.7 28.0
3
34
28.8
4
23
19.5
Intestinal
76
58.0
Diffuse
55
42.0
Positive
117
78.5
Negative
32
21.5
cagA (-)i
57
50.0
cagA (?)
57
50.0
vacA (m2)i
25
27.5
vacA (m1, m1/m2)
66
72.5
Lymph nodes involvement
Tumor stage
Tumor histotype
Helicobacter pylori
Helicobacter pylori positive cases*
a
vacA (s2)i
22
19.3
vacA (s1, s1/s2) cagA (?), vacA (m1, m1/m2)
92 41
80.7 46.1
cagA (?), vacA (s1, s1/s2)
48
43.2
vacA (m1, m1/m2), vacA (s1, s1/s2)
55
61.1
cagA (?), vacA (m1, m1/m2), vacA (s1, s1/s2)
37
42.0
Missing value = 41 %
* Missing values: cagA = 3 %, vacA m = 22 %, vacA s = 3 % i
cagA: cytotoxin associated gene A
vacA: vacuolating cytotoxin A
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123
12–23
\12
Alcohol intake (g/day)
Ever drinkers
Never
Spirits
Ever drinkers
Never
Beer
Current drinker
Wine Never
12
57.1 %
19.1 %
12.5 %
4 4
87.5 %
9.4 %
90.6 %
68.8 %
31.2 %
28.1 %
28.1 %
15.6 %
56.3 %
31.8 %
18.2 %
50.0 %
±12.52
40.6 %
64.3 % 35.7 %
75.9 %
21.8 %
25.0 %
28
3
29
22
10
9 9
Current smokers
[20 pack years
Alcohol
5
Ex-smokers
Never
18
7
Secondary education
Cigarette smoking
4
Middle school
Elementary education
11
68.4
Educational level
13
Age (years)
18 10
Male sex
Demographic
1–2 3–4
Stage
22
12
Diffuse
Lymph nodes involvement
19
Intestinal
Histotype
Clinical characteristics
32
15
14
101
23
92
77
38
44
23
34
58
21
25
41
66.8
65
43 47
61
43
57
n = 117
n = 32
%
HP (?)
HP (-)
41.0 %
19.2 %
12.2 %
87.8 %
20.0 %
80.0 %
67.0 %
33.0 %
37.6 %
20.0 %
29.6 %
50.4 %
24.1 %
28.7 %
47.1 %
±11.13
56.5 %
47.8 % 52.2 %
66.3 %
78.2 %
75 %
%
0.346
1.000
0.164
0.848
0.321
0.252
0.557
0.490
0.111
0.127
0.334
0.672
p value
17
7
7
50
13
44
38
19
19
13
15
29
13
12
20
67.3
34
24 19
29
17
31
n = 57
cagAi (-)
HP (?)
42.5 %
17.5 %
12.3 %
87.7 %
22.8 %
77.2 %
66.7 %
33.3 %
33.3 %
22.8 %
26.3 %
50.9 %
28.9 %
26.7 %
44.4 %
±10.70
59.6 %
55.8 % 44.2 %
65.9 %
39.5 %
56.4 %
%
14
8
7
48
10
45
38
17
23
9
18
28
8
12
19
66.3
3
19 28
32
26
24
n = 57
cagA (?)
37.8 %
21.6 %
12.7 %
87.3 %
18.2 %
81.8 %
69.1 %
30.9 %
40.4 %
16.4 %
32.7 %
50.9 %
20.5 %
30.8 %
48.7 %
±11.88
54.6 %
40.4 % 59.6 %
66.7 %
60.5 %
43.6 %
%
Table 2 Demographics and lifestyle characteristics of 149 gastric cancer patients according to Helicobacter pylori status, and cagA status and vacA m and s-regions
0.873
0.943
0.545
0.784
0.437
0.612
0.673
0.639
0.585
0.144
0.939
0.098
p value
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[9 times/week Add salt to meals
%
25.0 %
Male sex
Demographic
3–4
1–2
13
10
18
22
12
Diffuse
Lymph nodes involvement Stage
19
Intestinal
Histotype
40.6 %
35.7 %
64.3 %
75.9 %
21.8 %
65
47
43
61
43
57
n = 117
n = 32
Clinical characteristics
HP (?)
HP (-)
3.6 % 3.3 %
1 1
Gastric cancer (first degree)
33.3 %
6.3 %
18.8 %
59.4 %
28.1 % 21.9 %
56.3 %
Any cancer (father)
Any cancer (first degree)
10
2
Respiratory diseases
Cancer family history
6
Diabetes
Cardiovascular diseases
19
9 7
5/9 times/week
Comorbidities
5 18
\5 times/week 15.6 %
40.6 %
13
Fresh vegetables
[9 times/week
53.1 %
2 17
6.3 %
23.8 %
5/9 times/week
5
%
56.5 %
52.2 %
47.8 %
66.3 %
78.2 %
75 %
10
24
41
14
18
54
39 23
52
20
61
44
7
31
0.111
0.127
0.334
0.672
p value
n = 117
n = 32
%
HP (?)
HP (-)
\ 5 times/week
Fruit
Dietary Habits
C24
Table 2 continued
13
11
6
14
6
14
n = 25
%
vacA (m2)i
HP (?)
9.0 %
23.1 %
36.9 %
12.1 %
15.5 %
46.9 %
35.1 % 20.0 %
46.8 %
18.0 %
54.5 %
39.3 %
6.3 %
39.8 %
%
32.6 %
54.2 %
64.7 %
35.3 %
77.8 %
17.6 %
3
10
19
8
9
33
19 10
23
13
30
22
4
16
n = 57
cagAi (-)
HP (?)
36
28
25
35
28
29
n = 66
%
57.1 %
52.8 %
47.2 %
64.8 %
82.5 %
67.4 %
vacA (m1, m1/m2)
0.457
0.019
0.716
0.280
0.660
0.214
0.816
0.640
0.376
p value
0.802
0.391
0.307
0.138
p value
11
13
8
14
7
13
n = 22
50.0 %
61.9 %
38.1 %
66.7 %
16.7
22.6 %
7
14
21
6
9
21
18 13
28
7
30
21
2
15
n = 57
%
vacA (s2)i
5.6 %
20.0 %
35.2 %
14.0 %
15.8 %
53.9 %
34.5 % 17.5 %
41.8 %
23.6 %
53.6 %
39.3 %
7.1 %
40.0 %
%
cagA (?)
53
33
33
46
35
42
n = 92
%
58.9 %
50.0 %
50.0 %
67.6 %
83.3 %
76.4 %
vacA (s1, s1/s2)
13.0 %
27.4 %
38.9 %
10.7 %
16.1 %
37.5 %
34.0 % 23.6 %
52.8 %
13.2 %
56.6 %
39.6 %
3.8 %
40.5 %
%
0.450
0.341
0.933
0.401
p value
0.320
0.379
0.690
0.592
0.967
0.023
0.425
0.320
0.856
p value
Mol Biol Rep
123
123
7
Secondary education
5
9
9
Ex-smokers
Current smokers
[20 pack years Alcohol
22
Current drinker
3
Ever drinkers
4
Ever drinkers
5
18
9
\5 times/week
5/9 times/week
[9 times/week
Fresh vegetables
28.1 %
56.3 %
15.6 %
40.6 %
13
5/9 times/week
[9 times/week
53.1 %
2
17
\ 5 times/week 6.3 %
23.8 %
5
Dietary Habits Fruit
C24
57.1 %
4
12
12–23
19.1 %
12.5 %
87.5 %
9.4 %
90.6 %
68.8 %
31.2 %
28.1 %
28.1 %
15.6 %
56.3 %
31.8 %
18.2 %
50.0 %
±12.52
\12
Alcohol intake (g/day)
28
Never
Spirits
29
Never
Beer
10
Never
Wine
18
Never
Cigarette smoking
4
11
68.4
39
52
20
61
44
7
31
32
15
14
101
23
92
77
38
44
23
34
58
21
25
41
66.8
n = 117
n = 32
%
HP (?)
HP (-)
Middle school
Elementary education
Educational level
Age (years)
Table 2 continued
35.1 %
46.8 %
18.0 %
54.5 %
39.3 %
6.3 %
39.8 %
41.0 %
19.2 %
12.2 %
87.8 %
20.0 %
80.0 %
67.0 %
33.0 %
37.6 %
20.0 %
29.6 %
50.4 %
24.1 %
28.7 %
47.1 %
±11.13
%
0.640
0.376
0.346
1.000
0.164
0.848
0.321
0.252
0.557
0.490
p value
8
12
3
12
12
0
5
5
3
4
21
4
21
12
13
6
3
9
12
5
5
8
68.8
n = 25
34.8 %
52.2 %
13.0 %
50.0 %
50.0 %
–
38.5 %
38.5 %
23.1 %
16.0 %
84.0 %
16.0 %
84.0 %
48.0 %
52.0 %
25.0 %
12.5 %
37.5 %
50.0 %
27.8 %
27.8 %
44.4 %
±12.56
%
vacA (m2)i
HP (?)
20
29
12
32
26
4
18
18
9
6
58
13
51
46
18
30
17
15
31
10
15
23
65.4
n = 66
32.8 %
47.5 %
19.7 %
51.6 %
41.9 %
6.5 %
40.0 %
40.0 %
20.0 %
9.4 %
90.6 %
20.3 %
79.7 %
71.9 %
28.1 %
46.2 %
27.0 %
23.8 %
49.2 %
20.8 %
31.3 %
47.9 %
±10.79
%
vacA (m1, m1/m2)
0.852
0.613
0.971
0.458
0.770
0.034
0.071
0.250
0.877
0.213
p value
11
8
3
12
10
0
6
6
3
4
18
7
15
13
9
7
5
6
11
5
3
9
67.6
n = 22
50.0 %
36.4 %
13.6 %
54.5 %
45.5 %
–
40.0 %
40.0 %
20.0 %
18.2 %
81.8 %
31.8 %
68.2 %
59.1 %
40.9 %
31.8 %
22.7 %
27.3 %
50.0 %
29.4 %
17.6 %
52.9 %
±12.76
%
vacA (s2)i
26
40
17
47
31
6
25
26
10
10
80
15
75
63
27
35
17
27
43
15
22
30
66.6
n = 92
31.3 %
48.2 %
20.5 %
56.0 %
36.9 %
7.1 %
41.0 %
42.6 %
16.4 %
11.1 %
88.9 %
16.7 %
83.3 %
70.0 %
30.0 %
39.3 %
19.5 %
31.0 %
49.4 %
22.4 %
32.8 %
44.8 %
±10.87
%
vacA (s1, s1/s2)
0.277
0.497
0.945
0.470
0.135
0.326
0.347
0.918
0.506
0.721
p value
Mol Biol Rep
HP Helicobacter pylori, icagA cytotoxin associated gene A, vacA vacuolating cytotoxin A
1.000
0.683 9.3 %
21.7 % 18
8 4.6 %
21.1 % 4
1 0.667
0.654 21.4 %
9.8 % 6
12
Results
4.2 %
26.1 % 6
1 0.457
0.019 23.1 %
9.0 % 10
24
1 Gastric cancer (first degree)
3.3 %
1 Any cancer (father)
3.6 %
0.897 34.9 % 30 36.4 % 8 0.812 36.1 % 22 33.3 % 8 0.716 36.9 % 41 33.3 % 10 Any cancer (first degree)
Cancer family history
0.303
0.459 13.2 % 12 9.1 % 2 0.292 15.4 % 10 8.0 % 2 0.280 12.1 % 14 2 Respiratory diseases
6.3 %
0.472 49.4 %
18.2 % 16
45 40.9 %
9.1 % 2
9 0.819
0.821 18.8 %
45.3 % 29
12 16.7 %
48.0 % 12
4 0.660
0.214 46.9 %
15.5 % 18
54
6 Diabetes
59.4 % 19 Cardiovascular diseases
18.8 %
0.739 19.5 % 17 22.7 % 0.798 19.1 % 12 16.7 % 4 0.816 20.0 % 23 21.9 % 7 Add salt to meals
Comorbidities
n = 117 n = 32
% HP (?) HP (-)
% Table 2 continued
5
% n = 92 % n = 22 % n = 66 % n = 25
vacA (m2)i
p value
HP (?)
vacA (m1, m1/m2)
p value
vacA (s2)i
vacA (s1, s1/s2)
p value
Mol Biol Rep
The clinical and demographic features of 149 gastric cancer patients included in study are reported in Table 1. Fifty-two percent of the cases were male, more than 70 % were aged C60 years old and 47.4 % had only elementary school education. Most cases were high grades of gastric cancer (grade 3: 69.3 %) and had lymph nodes involvement (68.6 %). Fifty-eight percent were intestinal histotype, and overall 78.5 % of the cases were H. pylori positive. Among the positive H. pylori cases, 50 % were cagA?, 72.5 % vacA m1 subtype and 80.7 % were vacA s1 subtype. Forty-six percent and 43.2 % were simultaneously cagA and, respectively, vacA m1 or vacA s1 positive, while 61.1 % were at the same time vacA m1 and s1 subtype. CagA?, vacA m1 and vacA s1 genotype was simultaneously present in 42 % of strains. Overall, cagA? strains were not associated with the co-presence of vacA m1 (p = 0.031) or vacA s1 (p = 0.052) subtype (data not shown). Data on demographics, lifestyle habits, comorbidities and cancer family history according to H. pylori status and H. pylori cagA status and vacA genotypes are reported in Table 2. CagA positive strains were more common among the diffuse histotype of gastric cancer respect to the intestinal, even though not significantly different. The proportion of current wine drinkers was higher among vacA m1 or m1/m2 strains (71.9 %) respect to vacA m2 (48.0 %) (p = 0.034). The consumption of more than 20 pack-years of cigarettes was more common in patients infected by vacA m1 or m1/m2 than vacA m2, although not significantly different (p = 0.071). Cardiovascular diseases (CVDs) were more frequent in patients infected by cagA negative strains than in cagA positive strains (p = 0.023). H. pylori positive patients were more likely to have a father with a history of any cancer compared with the H. pylori negative patients (23.1 vs. 3.6 %, respectively; p = 0.019). Table 3 reports the distribution of the studied polymorphisms in our gastric cancer patients. The variant genotypes of EPHX1 exon 3 (p = 0.032), IL1B-511 (p = 0.039) and IL1-RN (p = 0.002) gene were more common among H. pylori negative than positive patients. Concerning the H. pylori strains, IL1-RN VNTR (p = 0.051) and SULT1A1 (p = 0.047) variant genotypes were borderline more common among cagA positive cases. Lastly, MTHFR 677 variant allele was borderline more frequent among those infected with carriers of vacA m1 subtype (p = 0.072) than in m2, while p53 exon 4 homozygote variant genotype was less common in patients infected with vacA s1 subtype than in s2 (p = 0.050). The ORs for H. pylori positivity based on univariate and multivariate analyses are reported in Table 4. We identified EPHX1 exon 3 (OR = 0.35, CI 95 % 0.13–0.94), IL1B-
123
123 78.1
8
CG/GG
29
17
AC/CC
p53 Ex4 ?119C [ G (rs1042522)
14
AA
MTHFR 1298A [ C (rs1801131)
90.6
54.8
45.2
25.0
14
TT
43.8
10
31.2
25.0
75.0
28.1
71.9
3.1
96.9
21.9
CT
8
24
9
23
1
31
7
40.6
59.4
CC
MTHFR 677C [ T (rs1801133)
22
2L/LL
TT IL1-RN VNTR
CC/TC
IL1B-511 T [ C (rs16944)
TT
CC/CT
IL1B ? 3954C [T (rs1143634)
CC
TT/CT
25
13
IL1B -31C [ T (rs1143627)
19
68.8
22
null
31.3
10
65.6 34.4
25.0
8 21 11
75.0
24
wt
GSTT1 Ex5-49 1>-
null
wt
GSTM1Ex4 1 10 1>-
AA AG/GG
EPHX1 exon 4 A [ G H139R (rs2234922)
CC
TT/TC
EPHX1 exon3 T [ C Y113H (rs1651740)
110
53
56
17
56
44
7
109
15
101
10
106
13
104
39
75
62
52
74 41
12
104
n = 117
n = 32
%
HP (?)
*HP (-)
94.0
48.6
51.4
14.5
47.9
37.6
6.8
93.2
12.9
87.1
8.6
91.4
11.1
88.9
34.2
65.8
54.4
45.6
64.4 35.7
10.3
89.7
%
0.497
0.541
0.366
0.002
0.039
0.294
0.113
0.503
0.146
0.894
0.032
p value
52
28
25
8
31
18
1
56
6
51
7
50
5
52
19
35
31
23
38 19
5
52
n = 57
cagA (-)i
HP (?)
91.2
52.8
47.2
14.0
54.4
31.6
1.8
98.2
10.5
89.5
12.3
87.7
8.8
91.2
35.2
64.8
57.4
42.6
66.7 33.3
8.8
91.2
%
55
24
31
9
24
24
7
50
8
48
3
53
7
50
20
37
29
28
34 21
7
49
n = 57
cagA (?)
96.5
43.6
56.4
15.8
42.1
42.1
12.3
87.7
14.3
85.7
5.4
94.6
12.3
87.7
35.1
64.9
50.9
49.1
61.8 38.2
12.5
87.5
%
Table 3 Distribution of the selected gene polymorphisms of 149 gastric cancer patients according to Helicobacter pylori status, and cagA status and vacA m- and s-regions
0.219
0.339
0.405
0.051
0.544
0.168
0.381
0.991
0.490
0.592
0.368
p value
Mol Biol Rep
%
15.6
5
68.8
10 22
wt
19 13
wt
null
GSTT1 Ex5-49 1>-
null
31.3
11
40.6
59.4
34.4
21
AG/GG GSTM1Ex4 1 10 1>-
65.6
25.0
8
AA
EPHX1 exon 4 A [ G H139R (rs2234922)
CC
TT/TC
39
75
62
52
41
74
12
104
n = 32
75.0
n = 117
*HP (-)
24
HP (?)
8
EPHX1 exon3 T [ C Y113H (rs1651740)
25.0
24
GA
75.0
84.4
GG
TNFA -308G [ A (rs1800629)
AA
GG/GA
34.4
65.6
21.9
78.1
25.0
75.0
9.4
27
11
SULT1A1 638G [ A (rs9282861)
21
(GC/AT)/(AT/AT)
7
25
8
24
3
%
34.2
65.8
54.4
45.6
35.7
64.4
10.3
89.7
0.503
0.146
0.894
0.032
p value
19
96
10
107
32
85
38
79
41
76
7
n = 117
n = 32
%
HP (?)
*HP (-)
p73 G4C14-to-A4T14 (rs1801173/rs2273953) (GC/GC)
GA/AA
GG
p53 IVS6 ?62A [ G (rs1625895)
16 bp/wt plus 16 bp/16 bp
wt/wt
53 intron3 (16 bp insertion)
CC
Table 3 continued
8
14
10
12
9
14
3
21
n = 25
36.4
63.6
45.5
54.6
39.1
60.9
12.5
87.5
%
vacA (m2)i
HP (?)
16.5
83.5
8.6
91.5
27.4
72.6
32.5
67.5
35.0
65.0
6.0
%
10
46
2
55
45
38
17
40
19
38
5
n = 57
23
42
37
28
19
45
8
58
n = 66
35.4
64.6
56.9
43.1
29.7
70.3
12.1
87.9
%
0.934
0.351
0.406
0.607
p value
cagA (-)i
HP (?)
vacA (m1, m1/m2)
0.273
0.194
0.437
0.247
0.284
p value
5
16
14
7
5
17
2
20
n = 22
23.8
76.2
66.7
33.3
22.7
77.3
9.1
90.9
%
9
47
8
49
12
19
20
37
21
36
2
n = 57
vacA (s2)i
17.9
82.1
3.5
96.5
54.2
45.8
29.8
70.2
33.3
66.7
8.8
%
16.1
83.9
14.0
86.0
38.7
61.3
35.1
64.9
36.8
63.2
3.5
%
33
57
45
45
33
57
10
81
n = 92
36.7
63.3
50.0
50.0
36.7
63.3
11.0
89.0
%
vacA (s1, s1/s2)
cagA (?)
0.264
0.168
0.161
0.575
p value
0.801
0.047
0.103
0.548
0.695
p value
Mol Biol Rep
123
123 8
22
7
GA/AA 21 11
(GC/GC)
(GC/AT)/(AT/AT)
p73 G4C14-to-A4T14 (rs1801173/rs2273953)
25
GG
p53 IVS6 ?62A [ G (rs1625895)
wt/wt 16 bp/wt plus 16 bp/16 bp
24 8
3
53 intron3 (16 bp insertion)
29
CC
34.4
65.6
21.9
78.1
75.0 25.0
9.4
90.6
54.8
17
CG/GG
p53 Ex4 ?119C [ G (rs1042522)
AC/CC
AA
45.2
8
MTHFR 1298A [ C (rs1801131) 14
25.0
14
TT
43.8
10
CT
31.2
25.0
75.0
71.9 28.1
3.1
96.9
CC
MTHFR 677C [ T (rs1801133)
24
2L/LL
IL1-RN VNTR
CC/TC TT
23 9
1
IL1B-511 T [ C (rs16944)
31
21.9
7
TT
78.1
25
32
85
38
79
76 41
7
110
53
56
17
56
44
7
109
101 15
10
106
13
104
n = 117
n = 32
%
HP (?)
*HP (-)
CC/CT
IL1B ? 3954C [T (rs1143634)
CC
TT/CT
IL1B -31C [ T (rs1143627)
Table 3 continued
27.4
72.6
32.5
67.5
65.0 35.0
6.0
94.0
48.6
51.4
14.5
47.9
37.6
6.8
93.2
87.1 12.9
8.6
91.4
11.1
88.9
%
0.437
0.247
0.284
0.497
0.541
0.366
0.002
0.039
0.294
0.113
p value
7
18
7
18
16 9
3
21
12
9
3
8
14
0
24
24 1
2
23
1
24
n = 25
28.0
72.0
28.0
72.0
9.0 36.0
12.5
87.5
57.1
42.9
12.0
32.0
56.0
0.0
100.0
96.0 4.0
8.0
92.0
4.0
96.0
%
vacA (m2)i
HP (?)
19
47
20
46
47 19
2
63
32
32
10
36
20
6
59
55 10
4
61
9
57
n = 66
28.8
71.2
30.3
69.7
71.2 28.8
3.1
96.9
50.0
50.0
15.1
54.5
30.3
9.2
90.8
84.6 15.4
6.2
93.9
13.6
86.4
%
vacA (m1, m1/m2)
0.941
0.830
0.506
0.087
0.570
0.072
0.142
0.140
0.536
0.177
p value
8
14
10
12
13 9
3
19
9
12
3
11
8
1
21
20 2
4
18
2
20
n = 22
36.4
63.6
45.5
54.5
59.1 40.9
13.6
86.4
42.9
57.2
13.6
50.0
36.4
4.5
95.5
90.9 9.1
18.2
81.8
9.1
90.9
%
vacA (s2)i
24
68
27
65
62 30
3
89
44
41
12
44
36
6
86
78 13
6
85
11
81
n = 92
26.1
73.9
29.4
70.7
64.4 32.6
3.3
96.7
51.8
48.2
13.0
47.8
39.1
6.5
93.5
85.7 14.3
6.6
93.4
12.0
88.0
%
vacA (s1, s1/s2)
0.335
0.147
0.461
0.050
0.465
0.972
0.594
0.519
0.097
0.522
p value
Mol Biol Rep
85.7
Adj ORa
CI 95 %
Any cancer, father
8.10
1.04–62.76
8.18
1.04–64.55
EPHX1 exon3 T [ C Y113H (rs1651740)
0.35
0.13–0.94
0.42
0.13–1.42
14.3 13
78 76.2
23.8
10.9 10 0.0
82 100.0
0.223
0.106 89.1
% n = 92 %
CI 95 %
IL1B-511 T [ C (rs16944)
0.38
0.15–0.97
0.36
0.12–1.46
IL1-RN VNTR
0.19
0.06–0.58
0.45
0.12–1.69
n = 22
5
16
0 8
56
9
4.0
73.9
26.1
86.2
12.1
0.180
511 (OR = 0.38, CI 95 % 0.15–0.97) and IL1-RN VNTR (OR = 0.19, CI 95 % 0.06–0.58) polymorphisms as protective in univariate analyses but the significance was not confirmed after adjustment for confounding factors in the multivariate analysis. A positive father‘s any cancer history was confirmed associated with an increased risk of H. pylori positivity, both on univariate (OR = 8.10, CI 95 % 1.04–62.76), and multivariate (OR = 8.18, CI 95 % 1.04–64.55) analyses.
13.9
22 0.230 87.9 58 96.0
% n = 66 %
8 GA
HP Helicobacter pylori, icagA cytotoxin associated gene A, vacA vacuolating cytotoxin A
16.5
83.5 96 24 GG
75.0
19
10 15.6 5 AA
TNFA -308G [ A (rs1800629)
25.0
8.6
0.273
6
17
1
24 0.194 91.5 84.4 GG/GA
SULT1A1 638G [ A (rs9282861)
27
107
% n = 117 n = 32
%
HP (?) *HP (-)
Adjusted by age, any cancer (father), IL1B-511T [ C
Discussion
n = 25
vacA (m1, m1/m2) vacA (m2)i
HP (?)
OR
OR odds ratio, CI confidence interval
p value Table 3 continued
Table 4 Risk factors for Helicobacter pylori positivity from univariate and multivariate analyses
a
p value
vacA (s2)i
vacA (s1, s1/s2)
p value
Mol Biol Rep
Our study identified the presence of father history for any cancer as a significant risk factor for H. pylori infection in gastric cancer patients. From the univariate analysis, CVDs are reported to be less common among cagA? subjects. Wine consumption was associated with higher occurrence of H. pylori vacA m1 virulent subtype. As for the genetic factors, polymorphisms in EPHX1 exon 3, IL1B-511 and IL1-RN genes resulted as protective factors towards H. pylori infection in the univariate analysis. Additionally, our results show that homozygous genotype of SULT1A1 and the variant genotype of IL1-RN were more common among gastric cancer subjects infected with cagA positive strains. Lastly, MTHFR 677 variant allele was more common among patients infected with strains carrying the vacA m1 virulent subtype, while p53 exon 4 homozygote variant was less common among patients infected by vacA s1 virulent subtype. Reports in the past few years suggested that low socioeconomic status [37], low educational level [38] and large family size [39] are associated with H. pylori infection. Alcohol intake is reported to be inversely associated [40], while data on smoking are contradictory [37, 41]. Several genetic polymorphisms of the host have also been suggested to influence susceptibility toward H. pylori infection [42–47], among them polymorphisms of IL1B-511, IL1A889, TNFA-308, fucosyltransferase and myeloperoxidase genes. These data are derived both from studies conducted
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among healthy individuals and patients with various gastric diseases, including gastric cancer. A family history of gastric cancer has been previously reported as risk factor for H. pylori infection in children [48]. North American consensus statement suggested testing for H. pylori in children with positive family history of gastric cancer [49]. However, the same observation has not been reported in the adult population [38]. Our study shows that any history of cancer of the father is a risk factor for H. pylori infection. However sub-analysis did not show that the history of gastric cancer is associated with H. pylori infection. The way in which any cancer history of the father influences the susceptibility to H. pylori infection remains currently unexplained. Our study shows that CVDs are less common among patients infected with cagA? strains. Several studies in the literature assessed the relationship between CVD and H. pylori infection, and cagA strains [50–53], with discordant results. Scho¨ttker et al. [53] recently reported an inverse association between infection with H. pylori cagA? positive strains and fatal cardiovascular events. Although they suppose that constant stimulation of the immune system of the host by cagA? H. pylori could be protective towards fatal cardiovascular events, this theory does not explain how cagA? strains could act protectively against chronic cardiovascular conditions. Alcohol consumption is reported to be inversely associated with H. pylori infection [40], and this association is stronger for wine than for beer drinkers [40], that might be explained by additional direct bactericide nature of wine. In our study we did not observe a difference in alcohol consumption among H. pylori positive and negative cases, though the virulent vacA m1 subtype was more frequent among wine drinkers respect to never drinkers. We can argue that that the high virulence properties of these strains allow them to survive in hostile environment, though this should be demonstrated by in vivo studies. Our study shows that the variant alleles IL1B-511 and IL1-RN genes might be protective factors against H. pylori infection in univariate analysis, as the variant genotypes are more common among H. pylori negative gastric cancer cases. IL1B-511 and IL1-RN polymorphisms affect the expression levels of IL-1b, a potent proinflamatory citokyne [29]. Increased IL-1b production induces stronger inflammatory response of the host to H. pylori but also inhibits gastric secretion thus influencing the susceptibility towards infection. Hosts with H. pylori infection who carry the IL-1B-511 T/T genotype or IL1-RN 2 might have a stronger IL-1b production [54]. Proinflammatory polymorphism of IL1B-511 has been reported to favour H. pylori infection [42, 43], however this association has not been reported for IL1-RN [42, 43]. Nevertheless, one study reported that IL1-511 T allele is associated with reduced
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risk of H. pylori infection [55]. Larger studies to come should clarify if proinflammatory component of these genes acts protectively towards H. pylori infection or the inhibition of gastric secretion prevails in its favoring. EPHX1 codes for enzyme which is playing a role in detoxification and activation of many carcinogens. EPHX1 polymorphism is reported to be changing enzyme activity influencing susceptibility towards colorectal and lung cancers [56, 57]. Our univariate analysis showed for the first time, that the homozygote genotype of EPHX1 exon 3 is protective against H. pylori infection in gastric cancer patients. We reported that polymorphisms in MTHFR 677, SULT1A1 and p53 exon 4 interact with H. pylori virulent cagA positive and vacA s1 as well vacA m1 subtypes. Previous report did not show difference in H. pylori presence among patients with different MTHFR 677 genotypes [58]. Although it has been reported that SULT1A1 [22] and p53 exon 4 [59] polymorphisms have an influence on gastric cancer carcinogenesis, no investigation so far addressed an impact of these two polymorphisms to H. pylori susceptibility. Further studies on these issues including larger number of cases are required, in view of few subjects carrying the variant alleles of these gene. It has been reported that cagA positive H. pylori strains identified from patients with gastric lesions were more likely to carry the vacA s1 and vacA m1 subtypes [60]. Infection with strains which are simultaneously cagA and vacAs1/m1 positive is associated with progression of gastric precancerous lesions towards malignancies [61]. Nevertheless, a recent study from Brazil [62], although including small number of cases, reported high prevalence of vacA s1 genotype among cagA negative gastric cancer patients infected with H. pylori. Virulent vacA m and vacA s, H. pylori strains isolated from gastric cancer patients in our study were also less likely to be cagA positive. We can argue that, in absence of cagA positivity, carrying at least one factor of vacA virulence might play a crucial role in gastric cancer development. The shortcoming of our study is the small number of cases included, and 26.9 % missing data on patients education and 22 % on H. pylori vacA m subtypes. Another important issue regards the time of H. pylori infection acquirement, and because this cannot be assessed we cannot be sure that exposure to the investigated risk factors preceded the infection, except the genetic ones. On the other hand, this study addresses the effect of a large list of putative risk factors, that include several genetic host risk factors. In conclusion, our study shows that father history for any cancer is a risk factor for H. pylori infection. Polymorphism in IL1B-511, IL1-RN and EPHX1 exon 3 genes could be protective towards H. pylori infection. Further studies including a larger number of cases are required.
Mol Biol Rep Acknowledgments This work has been supported by the AIRC (Associazione Italiana Ricerca sul Cancro) IG 2013 N.14220, and from the Italian Ministry of Health (GR-2011-02347943). The work of Dr Nikola Panic was supported by the ERAWEB project, funded with support of the European Community. The work of Dr Emanuele Leoncini was supported by Fondazione Veronesi.
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20. Conflict of interests
The authors have no conflict of interests.
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