Helicobacter pylori antibodies and gastric cancer: a gender-related difference

Authors


*Corresponding author. Tel.: +45 35455569/44440458 (home); fax: +45 35456869, E-mail address: lpa@biobase.dk

Abstract

Helicobacter pylori has been proposed as a causative agent of gastric cancer. The aim of this study was to define serum antibodies response against different H. pylori antigens in patients with gastric cancer. Serum samples were collected from 115 Lithuanian patients with non-cardia gastric cancer and 110 age- and sex-matched controls without cancer. Heat-stable, low-molecular-mass, and outer membrane proteins were used as antigens to analyze serum IgG antibody response against H. pylori by enzyme-linked immunosorbent assay. Seroprevalence of H. pylori using low-molecular-mass antigen was significantly higher in gastric cancer patients, compared to controls (77% versus 57%, p < 0.05). Significant differences in the prevalence of H. pylori infection between gastric cancer patients and controls were found in females using all three studied antigens: heat-stable (98% versus 84%, p < 0.05), low-molecular-mass (88% versus 48%, p < 0.05) and outer membrane proteins (78% versus 57%, p < 0.05). In males, no significant differences were revealed between gastric cancer patients and controls. There may be other cofactors in addition to H. pylori that are important for the development of gastric cancer. H. pylori seems, however, to be a more important for development of gastric cancer in females than in males or males may have more confounding risk factors for gastric cancer than females.

1Introduction

Helicobacter pylori is likely one of the most common chronic bacterial infection observed worldwide [1]. Different acquisition rates of H. pylori infection have been observed in various ethnic groups and populations. Such parameters as socio-economic status, environmental factors, genetic predisposition, or social and cultural background could probably play a role in the prevalence of H. pylori infection [2,3].

Helicobacter pylori is the main cause of chronic gastritis and peptic ulcer disease [4,5]. A number of studies have consistently shown an association between infection with H. pylori and gastric cancer [6–8]. In Lithuania, 35.4 males and 25.6 females per 100,000 individuals developed gastric cancer in 2002. According to the Lithuanian Cancer Register, gastric cancer remains the second leading cause of cancer death (25.4 deaths in males and 18.4 in females per 100,000 population) in this country as well as worldwide. In the literature, the male/female ratio ranges from 1.4 to 4 for gastric cancer, whereas the seroprevalence of H. pylori is equal in both genders. This discrepancy remains unexplained.

The aim of this study was to define serum antibodies response against different H. pylori antigens in patients with and without gastric cancer from Lithuania. IgG antibodies against the semicrude heat-stable (HS), low-molecular-weight (LMW) and outer membrane proteins (OMP) were measured by an enzyme-linked immunosorbent assay (ELISA) technique.

2Materials and methods

2.1Patients

The studied subjects were 115 consecutive patients (66 males, and 49 females) with non-cardia gastric cancer (median age, 59.6; range, 30–89 years). History of dyspepsia, peptic ulcer or other gastrointestinal disease, smoking and alcohol intake habits was recorded. The control group consisted of 110 age- and sex-matched in-patients (56 females and 54 males; median age, 57.2; range, 30–89 years), admitted to Kaunas Medical University Hospital and Vilnius Oncology Centre for illnesses unrelated to gastrointestinal disorders: diabetes mellitus (21), miscellaneous gynaecological disorders (23), osteoporosis (3), pneumonia (10), hemiparesis (14), glaucoma (16), cataract (11), coronary heart disease (12). Tumour staging was based on the histopathological (pTNM) classification system [9]. Venous blood samples were collected from gastric cancer and control patients in the period of October 1996–May 1997. The study was approved by the local medical ethics committee of Kaunas Medical University Hospital.

2.2Antigen preparation for serology

Outer membrane proteins (OMP) was prepared by acid-glycin extraction of H. pylori (strain CCUG 17874) [10], heat-stable (HS) antigen was prepared by boiling H. pylori (strain CH20429) in buffered saline with 1% Triton X-100 followed by filtration through a 0.22 μm filter [11], and low-molecular-mass (LMW) antigen was prepared by ultrafiltration of H. pylori (strain CH20429) [12].

2.3OMP-ELISA

Sera were examined for IgG antibodies against H. pylori by ELISA, as described earlier by Lelwala-Guruge et al. [10]. Plastic microtiter plates (Maxi Sorp, NUNC, Roskilde, Denmark) were coated with 100 μl of the antigen (5 μg per well). One hundred microliters of a test serum was diluted to 1:800 and added to each well. The sera were tested in duplicate. Fifty microliters alkaline phosphatase-labelled anti-human IgG (DAKO, Glostrup, Denmark) diluted to 1:500 was added to each well. The reaction was developed by adding 100 μl of 1 mg ml−1p-nitrophenyl phosphate (Sigma, St. Louis, USA) to each well. The reaction was stopped by adding 50 μl of 2 N NaOH. Absorbance values at 405 nm were recorded and expressed as relative antibody activity. A pool of human IgG (KABI/Pharmacia, Stockholm, Sweden) was used as a reference standard. The cut-off levels for positive, negative or borderline results have been estimated in Swedish populations by comparing with other diagnostic tests for H. pylori[10].

2.4HS and LMW ELISAs

IgG antibodies against HS and LMW H. pylori antigens were measured by ELISA technique as previously described [10,11]. Briefly, antigen concentrations were optimized by checkerboard titration and adjusted to follow the same procedure. Microtiter plates (Life Technologies, Roskilde, Denmark) were coated overnight with 100-μl HS antigen preparation diluted 1:100 in phosphate buffer saline or 100-μl LMW preparation (10 μg ml−1) diluted 1:10 in phosphate buffer saline. Plates were washed and 100-μl serum samples diluted 1:500 were added to the plates. Horseradish peroxidase-conjugated rabbit antibodies to human IgG (DAKO, Copenhagen, Denmark, Nr P214) diluted 1:2000 was added to each well. To determine the enzyme activity, 4 mg of ortho-phenyl-diamine were diluted in 10-ml citric acid buffer; 15 μl of 30% H2O2 was added just before of the use, and then 100 μl of the solution placed to each well. The chromogenic reaction was stopped with 250 μl of H2SO4. The absorbance was read in a spectrophotometer at 492 nm. The test values corrected for day-to-day and plate-to-plate variations by dilutions of control sera. The results are expressed as ELISA units (Eu). The cut-off levels for positive, negative or borderline results with both antigens have been estimated in Danish populations by comparing with other diagnostic tests for H. pylori[11,12].

2.5Statistical analysis

Statistical evaluations were carried out by corrected χ2 test. P values ≤0.05 were considered statistically significant.

3Results

The overall H. pylori seropositivity in 115 gastric cancer patients and 110 controls assessed using HS, LMW and OMP antigens is shown in Table 1. A significant difference (p < 0.05) between gastric cancer and control patients was revealed using LMW antigen. The prevalence of H. pylori infection increased with age, from approximately 75% at the age 30–39 to 100% at the age 80–89 in gastric cancer patients, and from 43% to 90% in controls, respectively.

Table 1.  IgG antibody response to HS, LMW and OMP antigens in 115 gastric cancer patients and 110 controls detected by ELISA
IgG antibody responseGastric cancerControls
HS, n (%)LMW, n (%)OMP, n (%)HS, n (%)LMW, n (%)OMP, n (%)
  1. HS, heat-stable antigen; LMW, low-molecular-mass antigen; OMP, outer membrane proteins.
    HS: >800 Eu was considered positive; 400–799, borderline; <400, negative.
    LMW: >400 Eu was considered positive, 100–399 borderline and <100 negative.
    OMP: >35 of relative antibody activity values were considered positive, 25–34 borderline, <25 negative.

  2. *Significant difference between gastric cancer patients and controls (p < 0.05).

Positive110 (96)89 (77)*85 (74)97 (88)63 (57)72 (66)
Borderline3 (2.6)17 (15)10 (8.7)11 (10)36 (33)9 (8.2)
Negative2 (1.7)9 (7.8)20 (17)2 (1.8)11 (10)29 (26)

The IgG antibody response to H. pylori varied depending of antigen preparation in gastric cancer patients and control individuals and significant difference in the antibody levels regarded positive between gastric cancer patients and controls was found with the LMW antigen (Table 1).

The prevalence of H. pylori infection in gastric cancer patients assessed using HS, LMW, OMP antigens was, respectively, 62 (94%), 46 (70%), 47 (71%) in males and 48 (98%), 43 (88%), 38 (78%) in females (Table 2). Significant gender-related difference (p < 0.05) in gastric cancer patients was found using LMW antigen. Control patients showed no significant differences between males and females (Table 2). Significant differences in the prevalence of H. pylori infection between females with gastric cancer and females without were found using all three studied antigens: HS (98% versus 84%, p < 0.05), LMW (88% versus 48%, p < 0.05) and OMP (78% versus 57%, p < 0.05). In males, no significant differences were revealed between gastric cancer patients and controls (Table 2).

Table 2.  IgG antibody response to HS, LMW and OMP antigens regarded positive for H. pylori in patients with gastric cancer and control individuals detected by ELISA
IgG antibody responseGastric cancerControls
HS, n (%)LMW, n (%)OMP, n (%)HS, n (%)LMW, n (%)OMP, n (%)
  1. HS: heat-stable antigen >800 Eu was considered positive;
    LMW: low-molecular-mass antigen >400 Eu was considered positive;
    OMP: outer membrane proteins >35 of relative antibody activity values were considered positive.
    Significant difference between gastric cancer patients and controls (p < 0.05).

Males62 (94)46 (70)47 (71)50 (91)36 (67)40 (74)
Females48 (98)43 (88)38 (78)47 (84)27 (48)32 (57)
Total110 (96)89 (77)85 (74)97 (88)63 (57)72 (66)

Seroprevalence of IgG antibodies against H. pylori in the I–IV gastric cancer stage patients is shown in Table 3. The number of H. pylori seropositive individuals with early and later stages of gastric cancer did not differ significantly.

Table 3.  Stage-related H. pylori seropositivity in 115 gastric cancer patients
Tumour stageNo. of patientsH. pylori seropositivity
HS, n (%)LMW, n (%)OMP, n (%)
  1. HS, heat-stable antigen; LMW, low-molecular-mass antigen; OMP, outer membrane proteins.

I + II4645 (97.8)36 (78.3)36 (78.3)
III + IV6965 (94.2)53 (76.8)49 (71.0)

In our study, 79 (69%) gastric cancer patients and 52 (47%) controls were seropositive with all three antigens; 84 (73%) and 59 (54%), with two antigens, respectively. Five (4.3%) gastric cancer patients and three (2.7%) controls were negative by all three tests.

Gastric cancer males significantly (p < 0.05) more frequently smoked (70% and 41%, respectively) and consumed alcohol (89% and 69%, respectively), compared to male patients of the control group. Comparison of females with gastric cancer and females without did not reveal significant differences in the smoking habit (20% and 13%, respectively) and alcohol consumption (67% and 57%, respectively).

4Discussion

Development of gastric cancer is a complex and poorly understood process. It is clear that besides chronic gastritis caused by H. pylori, dietary factors, high salt and nitrate intake, smoking and, possibly, alcohol consumption are additional risk factors for development of gastric cancer [13–17].

Parsonnet et al. [6], Kikuchi et al. [18] and Hansen et al. [19] reported that presence of H. pylori infection is a stronger predictor of gastric cancer in females compared to males. Our results also have revealed gender-related differences in prevalence of H. pylori among gastric cancer patients. Smoking and alcohol consumption were significantly more prevalent in males with gastric cancer than in males without, while these differences were not present in females. Hence, it may be considered that smoking and alcohol consumption as risk factors have stronger impact in males than for females. Increased IgG antibody levels against H. pylori were significantly found more frequent in females with gastric cancer than in males with gastric cancer and females without gastric cancer. However, it should be taken into account that retrospective case control studies, where H. pylori status is assessed only by serology after the diagnosis of cancer, underestimate the magnitude of the association between H. pylori and gastric cancer as a result of loss of infection in cases with the onset of disease [20]. H. pylori does not colonise areas with cancer, intestinal metaplasia, or atrophy and there is evidence that with the development of advanced gastric disease, the organism can be lost from the stomach [21]. A recent study from Iceland has shown that a decline in serum IgG antibody level is a risk factor of gastric cancer in of population-based cohort [22]. A study from Siponnen's group showed that H. pylori antibodies disappeared spontaneously within 10 years in almost one fourth of patients with advanced atrophic corpus gastritis and the disappearance of antibodies is accompanied by none or more than a mild improvement of the gastric mucosa [23]. Additionally, epidemiological and clinical studies [24,25] have shown that moderate alcohol consumption may facilitate the loss of chronic H. pylori infection in adults. Therefore, a higher prevalence of alcohol consumption in gastric cancer males in comparison to controls could be related with the loss of infection in some of our patients. However, differences in the smoking and alcohol consumption could not fully explain the gender-related difference in prevalence of H. pylori in our patients and other, yet unknown, host and environmental factors might be also important in the development of gastric cancer.

Some previous studies [26,27] have shown higher prevalence of H. pylori infection in patients with early stages of gastric cancer. However, our study did not reveal significant difference in H. pylori seroprevalence between early (I + II) and advanced (III + IV) gastric cancer stages patients, supporting the data of Kokkola et al. [28].

Many serodiagnostic tests have been developed to determine serum antibodies against H. pylori, such as agglutination and enzyme-linked immunosorbent assays used for clinical diagnosis and epidemiological studies. The explanation of great difference in the H. pylori seropositivity using three different antigens might be that HS and LMW-ELISA were standardized for the Danish population and OMP-ELISA for the Swedish population, and none of them were standardized for the Lithuanian population. High sensitivity and specificity test values cannot be extrapolated from one population to another [11,12]. Both, the heat-stable antigen and the outer membrane proteins probably have several common antigens and some different antigens that could explain the different results concerning the three antigens in this study. The optimal antigens for serodiagnosis of H. pylori infection should contain a high proportion of antigens common to all local strains and these should not cross-react with other bacteria or show non-specific binding with immunoglobulins.

Our findings indicate that an association of gastric cancer with LMW antigen might be more specific than with other H. pylori antigens. The difference in the number of borderline reactions between controls and patients with gastric cancer using this antigen was unexpected and cannot be explained from our previous experience. Even if a lower cut-off level had chosen, there would still have been a significant difference between females and males with gastric cancer. A relationship between H. pylori LMW antigen and gastric cancer has also been found in studies of Yamaoka et al. [29] and Vilaichone et al. [30], indicating that low-molecular-mass antigens may be either indirect markers for H. pylori-related diseases or even have specific active role in development of gastric cancer.

In conclusion, we have revealed gender-related difference in prevalence of IgG antibodies against H. pylori in Lithuanian gastric cancer patients. The pathogenesis of gastric cancer cannot be explained by infection with H. pylori alone. There might be other yet unknown cofactors. Nevertheless, H. pylori may be a more important factor for development of gastric cancer in females than in males or males may have more confounding risk factors than females.

Acknowledgements

This study was supported by a Grant from the Swedish Medical Research Council (16X-4723) and the European Helicobacter pylori Study Group (EHPSG).

We are grateful to Ms. Ingrid Nilsson and Ms. Bente Jensen for an excellent technical assistance, to Michael Weis Benson for a much helpful assistance on the biostatistics, to Professor Mecys Stukonis and Mads Bennedsen for sharing their knowledge and great ideas.

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