Gabal, Samia M.; Hosni, Hala N.; Elsheikh, Samar A. M.; Essa, Ahmed N.
The number of species belonging to the genus Helicobacter has rapidly increased over the past decade. The genus now includes at least 24 formally named species as well as numerous other Helicobacter spp. awaiting formal naming (Fox, 2002).
Helicobacter pylori (H. pylori) is the best known and the most important in terms of its global impact on human disease. It infects about 50% of the world population and its prevalence varies widely in different parts of the world, with average rates of 40–50% in western countries, rising to greater than 90% in the developing world (Peek and Blaser, 2002).
H. pylori is a gram-negative bacterium that has become well adapted to the human stomach through interaction with gastric epithelial cells (De Luca and Iaquinto, 2004). Chronic gastric infection with H. pylori causes inflammation and several gastric pathologies, including gastric ulcers and gastric cancer (Lochhead and El Omar, 2007).
Compelling evidence from epidemiological, histopathological, and animal studies has linked H. pylori infection to the subsequent development of gastric cancer (Uemura et al., 2001).
Despite the established relationship between H. pylori and gastric pathologies, the association between H. pylori and colorectal cancer is not very clear. Epidemiologic studies have used serology, PCR methods, C-urea breath tests, and circulating gastrin levels to examine colorectal neoplasia in relation to H. pylori infection and have produced conflicting results (Burnett-Hartman et al., 2008).
Infection of colorectal tissue with H. pylori may not be directly responsible for an increased risk for colorectal cancer but rather the byproducts of a gastric H. pylori infection (Hartwich et al., 2001a). One theory stems from the fact that gastric H. pylori infection leads to an increase in serum levels of gastrin, leading to hypergastrinemia (Mulholland et al., 1993), which is hypothesized to have proliferative effects on intestinal mucosa (Sobhani et al., 1993). A positive association has been found between hypergastrinemia and colorectal neoplasia (Georgopoulos et al., 2006).
Carcinogenesis through H. pylori infection involves inflammation, as well as deregulation of the cell cycle by the H. pylori protein, cytotoxin-associated gene A (CagA), which binds and activates SHP2 (a human phosphatase that can act as an oncoprotein), resulting in cell growth and motility (Lochhead and El Omar, 2007).
Associations between neoplastic colorectal lesions (adenomas and carcinomas) and H. pylori were either based on indirect evidence such as that from studies correlating these lesions with increased CagA+ levels (Hartwich et al., 2001b) or increased gastrin levels (Konturek et al., 2002), or on the direct correlation with H. pylori seropositivity (Fujimori et al., 2005). Other studies have failed to demonstrate this association based on seropositivity (Limburg et al., 2002).
Enterohepatic Helicobacter spp. are non-H. pylori Helicobacter spp. that naturally colonize the intestinal crypts; they are often associated with diarrhea and can cause bacteremia and systemic disease, including colonization of the biliary tract and induction of cholecystitis and hepatitis (and in some cases hepatic cancer). Immunocompromised hosts are particularly susceptible to these microorganisms (Solnick and Schauer, 2001).
It is possible that Helicobacter spp. are under-recognized causes of infective diarrhea in humans. Helicobacter spp. cultured from human diarrheal samples include H. cinaedi, H. canis, H. pullorum, H. fennelliae, H. canadensis, H. rappini, and other unclassified but related organisms (Fox, 2002).
Murine models have clearly shown that if the normal immune balances are altered then mucosa associated Helicobacter spp. induce a pathology similar to that of human inflammatory bowel disease. This is possibly because of their presence in mucous, the microbial niche closest to the susceptible mucosa (Shomer et al., 1997). The aim of our study was to determine the role of Helicobacter organisms in the development of colitis or colorectal neoplasia and to correlate Helicobacter immunohistochemical positivity with other clinicopathologic parameters.
Materials and methods
This work included a total of 50 patients, including 10 with a normal colon, 15 with colitis, 10 with colonic tubular adenoma, and 15 with colorectal carcinoma. Samples were obtained by collection of archived paraffin blocks from the Department of Pathology, Faculty of Medicine, Cairo University, during the period from January 2011 to September 2011.
Clinical data obtained from the pathology sheet are age and sex of patient; site of colorectal carcinoma; size of the tumor; growth appearance, whether fungating, ulcerating, or annular infiltrating; presence of distant metastasis; and surgical margins, whether positive or negative. Serial sections of 5 μm thickness were prepared from each block; one of them was mounted on a glass slide and stained using hematoxylin and eosin (H&E) for histological evaluation and another one was mounted on charged slides for immunohistochemical staining.
Histopathologic examination of H&E-stained slides was performed to detect the degree of inflammation in patients with colitis as well as the presence or absence of dysplasia in patients with tubular adenoma. Patients with colorectal carcinoma were classified according to the WHO classification of the tumors of the colon and rectum (Hamilton et al., 2010). Colorectal carcinoma was histologically graded as well, moderately, or poorly differentiated predominantly on the basis of the extent of their glandular appearance. Well-formed glands were present in greater than 75% of well-differentiated tumors, in 25–75% of moderately differentiated tumors, and in less than 25% of poorly differentiated carcinomas (Fenoglio Preiser et al., 2007).
Mucinous carcinoma was considered to be poorly differentiated or of grade III (Petras and Frankel, 2009).
Staging of colorectal carcinoma was performed according to the modified Duke’s (Astler and Coller, 1954, quoted from Cooper, 2004) and tumor, node, and metastasis staging systems (Green et al., 2002).
Immunohistochemical staining for anti-H. pylori antibody
The sections were deparaffinized in xylene; thereafter, they were hydrated with a series of graded alcohols (95–70%), distilled water, and PBS (at pH 7.5).
The slides were then immersed in 10 mmol/l citrate buffer (pH 6) and were pretreated twice in a microwave oven at 800 W for 4 and then 8 min.
Between each period of heating, evaporated fluid was replenished.
After a 25-min cooling period, the endogenous peroxidase activity was inhibited by incubation in 3% hydrogen peroxide (H2O2) for 5 min.
After washing with tris-buffered saline, the sections were incubated with the primary antibody for 1 h at room temperature. The primary antibody a polyclonal rabbit anti-H. pylori antibody, supplied in a liquid form that was ready to use (code: 760-2645; Lab Vision, California, USA).
The sections were washed in tris buffer and incubated with the avidin–biotin–peroxidase system for 30 min. The peroxidase reaction was detected by the addition of diaminobenzidine tetrahydrochloride.
All slides were rinsed well in tap water for 5 min, after which they were slightly counterstained with Mayer’s hematoxylin for 1–2 min and dehydrated in ascending alcohol concentrations.
The slides were cleared in xylene for three changes, after which Canada balsam and cover slips were applied.
Evaluation of the expression of anti-H. pylori antibody
Tissue sections were examined under high magnification (oil immersion, ×1000). Photographs were captured using a Nikon camera [D 60, China (Mainland)] attached to an Olympus microscope [China (Mainland)] and were considered positive when Helicobacter organisms were detected as thin coiled brown bacilli.
The data collected were analyzed using SPSS version 16.0 (SPSS Inc., Chicago, Illinois, USA) and the relationship between Helicobacter positivity and colitis and colorectal neoplasia was evaluated using the χ2-test. The P-value was used to assess the significance of the results. When the P-value was found to be less than 0.05, the results were considered to be statistically significant. The odds ratio (OR) was used to assess the relative risk, which indicates how much more likely anti-H. pylori antibody-positive patients are to develop colitis or colorectal neoplasia as compared with those who are negative for anti-H. pylori antibodies. If the OR is greater than one, colitis and colorectal neoplasia are more likely to develop. If the OR is less than one, colitis and colorectal neoplasia are less likely to develop.
This retrospective study was conducted on 50 patients, including 10 (20%) patients with a normal colon, 15 (30%) with colitis, 10 (20%) with colonic tubular adenoma, and 15 (30%) with colorectal carcinoma. Samples were obtained by collection of archived paraffin blocks from the Department of Pathology, Faculty of Medicine, Cairo University, during the period from January 2011 to September 2011.
The patients included 26 men (52%) and 24 (48%) women. Their ages ranged from 25 to 74 years, with a mean age of 49.5 years.
Among patients with colorectal cancer, the sigmoid colon was the most commonly involved site [six (40%)], followed by the rectum [four (26.7%)], the transverse colon [three (20%)], and the right colon [two (13.3%)]. Nine (60%) patients had lesions less than 5 cm in diameter, whereas six (40%) had lesions greater than 5 cm in diameter.
Among patients with colorectal cancer, eight (53.3%) had the circumferential infiltrating type, six (40%) had the ulcerative type, and one (6.7%) had the fungating type. All the studied cases showed negative surgical margins.
As regards the histological class of patients with colorectal cancer, 11 (73.3%) patient were classified as having adenocarcinomas, two (13.3%) were classified as having mucoid adenocarcinoma, and two (13.3%) were classified as having as adenocarcinoma with neuroendocrine differentiation.
As regards the degree of differentiation, 14 (93.3%) patients with colorectal cancer showed moderate differentiation and only one (6.7%) patient showed poor differentiation.
As regards the depth of tumor invasion among patients with colorectal cancer, 13 (86.7%) patients had T3 tumors, one (6.7%) had a T2 tumor, and one (6.7%) had a T4b tumor.
As regards lymph node metastasis among patients with colorectal cancer, 11 (73.3%) showed lymph node metastasis, whereas four (26.7%) had no lymph node metastasis.
Among patients with carcinoma, four (26.7%) had Duke’s stage B2 carcinoma, one (6.7%) had Duke’s stage C1, nine (60%) had Duke’s stage C2, and one (6.7%) had class C3.
As regards immunohistochemical staining with anti-H. pylori antibodies, 14 (28%) patients showed positive staining, whereas 36 (72%) showed negative staining.
Among normal participants, anti-H. pylori antibody staining was positive in two (20%) and negative in eight (80%). Among patients with colitis, anti-H. pylori antibody staining was positive in five (33.3%) and negative in 10 (66.7%). Among patients with adenoma, anti-H. pylori antibody staining was positive in four (40%) and negative in six (60%). Among patients with carcinoma, anti-H. pylori antibody staining was positive in three (20%) and negative in 12 (80%).
Risk estimation showed that H. pylori positivity is less likely to be present in participants with a normal colonic biopsy (OR=0.583).
The correlation between colitis and H. pylori positivity among the study group was non-statistically significant (P=0.73).
Risk estimation showed that H. pylori positivity is more likely to be present in patients with colitis on colonic biopsy (OR=1.444).
The correlation between adenoma and H. pylori positivity among the study group was non-statistically significant (P=0.43).
Risk estimation showed that H. pylori positivity is more likely to be present in patients with adenoma on colonic biopsy (OR=2.000).
The correlation between adenoma and H. pylori positivity in the study group was non-statistically significant (P=0.43).
Risk estimation showed that H. pylori positivity is more likely to be present in patients with adenoma on colonic biopsy (OR=2.000; Tables 1–4).
Helicobacter is a genus of gram-negative bacteria, possessing a characteristic helical shape (Boyanova, 2011).
Some Helicobacter spp. have been found to be present in the lining of the upper gastrointestinal tract, as well as the liver, of mammals and some birds. The most widely known species of the genus Helicobacter is H. pylori, which infects up to 50% of the human population. Some strains of this bacterium are pathogenic to humans as they are strongly associated with peptic ulcers, chronic gastritis, duodenitis, and stomach cancer. H. pylori also serves as the type species of the genus (Ryan and Ray, 2003).
This study aims to determine the role of Helicobacter organisms in the development of colitis or colorectal neoplasia and to correlate Helicobacter immunohistochemical positivity with other clinicopathologic parameters.
This retrospective study was conducted on 50 patients, including 10 (20%) with a normal colon, 15 (30%) with colitis, 10 (20%) with colonic tubular adenoma, and 15 (30%) with colorectal carcinoma. Samples were obtained by collection of archived paraffin blocks from the Department of Pathology, Faculty of Medicine, Cairo University, during the period from January 2011 to September 2011.
The immunohistochemical detection method, rather than routine histochemical analysis using Giemsa, was used for the detection of H. pylori, as immunohistochemical staining is more specific and accurate than the Giemsa method (McNulty and Wyatt, 1999) and is more likely to detect nonspiral forms of the organism (Saito et al., 2000). In our study, tissue sections were examined under high magnification (oil immersion, ×1000) and were considered positive when Helicobacter organisms were detected as thin, coiled brown bacilli (Fig. 1).
As regards immunohistochemical staining with anti-H. pylori antibodies in this study, 14 (28%) patients showed positive staining for anti-H. pylori antibodies, whereas 36 (72%) showed negative staining. Among normal participants, anti-H. pylori antibody staining was positive in two (20%) and negative in eight (80%). Among patients with colitis, anti-H. pylori antibody staining was positive in five (33.3%) and negative in 10 (66.7%). Among patients with tubular adenoma, anti-H. pylori antibody staining was positive in four (40%) and negative in six (60%). Among patients with colorectal carcinoma, anti-H. pylori antibody staining was positive in three (20%) and negative in 12 (80%).
Our results show that the prevalence of H. pylori positivity is not statistically significant in participants with a normal colon or patients with colitis, adenoma, or carcinoma. However, risk estimation by determination of OR showed that H. pylori positivity is most likely present in patients with colitis (OR=1.444) and tubular adenoma (OR=2.000).
In a trial investigating the prevalence of H. pylori in patients with colonic adenomas and colorectal carcinoma using immunohistochemical methods, Jones et al. (2007) found significant prevalence of H. pylori in patients with tubular adenoma (OR=11.13), tubulovillous adenoma (OR=10.45), and colorectal carcinoma (OR=8.13) but insignificant prevalence in those with villous adenoma (OR=0.95). In contrast to this finding, Soylu et al. (2008) found a higher prevalence of H. pylori positivity in patients with villous polyps compared with those with other histologic types. The absence of H. pylori in patients with villous adenomas, as reported by several authors, does not necessarily indicate the lack of an association but might be supportive of the hypothesis of H. pylori migration after the development of the lesion (Jung et al., 2002).
Shmuely et al. (2001) reported that H. pylori CagA+ seropositivity is enhanced in gastric and colon cancers, whereas Fireman et al. (2000) demonstrated a correlation between H. pylori seropositivity and CA19-9 elevation in patients with colorectal carcinoma. In the evaluation of the colon pathologies of 332 patients with high-resolution colonoscopy by Mizuno et al. (2005), the increase in the incidence of adenomatous polyps and the decrease in the incidence of normal colonoscopy findings was found more significant in H. pylori IgG-seropositive patients than in seronegative patients. Although the underlying mechanism is not clear, the prevalence of gastric H. pylori infection was found to be increased, especially in colonic adenomas (71.4% in polyps, 55% in cancers; Meucci et al., 1997). In case reports of Cap polyposis that support the correlation between colorectal neoplasia and H. pylori, despite the inability to demonstrate the presence of H. pylori in polyps by immunohistochemical methods, eradication of the infection led to improvements in symptoms and polyps (Oiya et al., 2002; Akamatsu et al., 2004).
Although the direct and indirect relationships between H. pylori and colorectal neoplasia have widely been recognized, only the relationship between the presence of serologically positive H. pylori and colorectal neoplasia has been demonstrated (Maggio-Price et al., 2006; Rao et al., 2006). It has also been reported that the remote and local consequences of H. pylori infection might have a synergistic effect on the emergence or development of these neoplasms under certain conditions (Maggio-Price et al., 2006; Rao et al., 2006).
Bulajic et al. (2007) in their study on 83 patients with colorectal carcinoma investigated the presence of H. pylori DNA by PCR analysis of biopsy specimens from colorectal carcinoma patients and from those with normal mucosal tissues; 36 patients showed H. pylori IgG antibody positivity and the presence of H. pylori DNA was determined by PCR in the tissues of one patient with colorectal carcinoma and five participants with normal mucosa. However, no correlation between H. pylori positivity and colorectal carcinoma could be demonstrated. There exist serologic and colon tissue PCR studies that support (Moss et al., 1995; Siddheshwar et al., 2001) or reject (Luzza et al., 1996; Bell et al., 2003) a correlation between H. pylori and colorectal neoplasia.
Conflicts of interest
There are no conflicts of interest.
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