Prevalence of Helicobacter pylori among Sudanese patients diagnosed with colon polyps and colon cancer using immunohistochemistry technique

Objectives Infection with the bacteria Helicobacter pylori has been classified as class one carcinogen associated with increasing susceptibility of gastritis and gastric carcinoma. This study is aiming at investigating the prevalence of H. pylori among colon polyps and colon cancer patients. A descriptive cross-sectional hospital-based study was conducted between February and June 2017. Sixty-nine formalin-fixed paraffin blocks collected from colon polyps and colon cancer patients to detect H. pylori using immunohistochemistry technique. Results Of the 69 patients included in the study, 39 (56.5%) males and 30 (43.5%) were females, their age ranged from 21 to 80 years with a mean age of 47.1 ± 19.7. Of the 69 colon polyps and colon cancer patients, 44 (63.8%) were diagnosed as adenocarcinoma, 10 (14.5%) colitis, 15 (21.7%) juvenile polyposis syndrome. The results of immunohistochemistry technique showed the presence of 16 (23.2%) positive patients for H. pylori infection. Of these 16, 13 (81.3%) patients were diagnosed with adenocarcinoma and 3 (18.7%) patients were diagnosed with juvenile polyps. The results of H. pylori detection among the different colon polyps and colon cancer patients were showing a statistically significant association for H. pylori infection and adenocarcinoma, P value 0.028.


Introduction
Colorectal cancer (CRC) is accounted as the third most common malignancy and the third most common cause of death due to cancer in both men and women in the US [1]. CRC mostly arises from adenomatous polyps (adenomas) and from hyperplastic polyps [2,3]. Early diagnosis and surgical removal of these polyps have associated with the decreased in the incidence of mortality [4][5][6].
Therefore, clinicians started to focus in recent years on the prevention measures that decrease the incidence of developing CRC; although, researchers started to explore the role of several infectious organism and their ability to increase or induce CRC [7][8][9][10]. For instance, many epidemiological studies have linked the infection of Helicobacter pylori to colorectal neoplasm either through high prevalence of H. pylori seropositivity among CRC or colorectal polyp patients [11][12][13], or through the presence of bacterial products and their trophic effects on colon mucosa [14][15][16][17]. Moreover, few studies have linked the presence of H. pylori in the stomach or colon with colon cancer and/or polyps [18][19][20][21][22][23].
It is well known that H. pylori predisposes to develop gastric cancer precursor lesions, thus it has been classified as class 1 carcinogen [24]. A recent meta-analysis correlating between H. pylori and extra-gastric malignancies revealed a statistically significant relationship of H. pylori infection with both colon cancer and polyps [25]. Also, H. pylori infection linked with colorectal lesions appeared to be more common in African Americans compared to the Caucasian population in the US [26,27].
Epidemiological studies have confirmed a causal relationship between H. pylori and gastric cancer, and the colonic phenotype of H. pylori-related intestinal metaplasia (IM) [28]. Thus, association of H. pylori in various gastrointestinal cancers has been investigated, moreover, Helicobacter DNAs were prevalent in more than 50% of hepatobiliary cancer cases [29]. Helicobacter species, which may colonize the biliary tract, have been implicated as a possible cause of hepatobiliary diseases ranging from chronic cholecystitis and primary sclerosing cholangitis to gall-bladder carcinoma and primary hepatic carcinomas [30]. Therefore, the hypothesis that H. pylori would also be associated with colon lesions needs to be investigated. In Sudan, no reports addressing this manner were existed. Previous studies investigating the seroprevalence of H. pylori among Sudanese using ELISA and rapid immunochromatographic tests for the detection of H. pylori IgM and IgG antibodies, reported a seroprevalence ranging from 20% up to 70% [31][32][33][34]. Therefore, the aim of this study was to investigate the presence of H. pylori infections among Sudanese patients diagnosed with colon polyps and colon cancer and to correlate between its presence and the type of the lesions.

Sample and data collection
This is a preliminary, descriptive study aimed to investigate the frequency of H. pylori infections among Sudanese patients diagnosed with colon cancer. Data were collected from 69 patients attended the National Laboratory and Alrahma Laboratory between February and June 2017.
During colonoscopy, presence of abnormal tissue, such as clumps of cells; polyps, formed on the inside of the colon, the pathologists obtained colon biopsies. Colon biopsies were fixed with formalin and processed into paraffin embedded blocks. Formalin-fixed paraffin blocks were used for the immunohistochemical detection of H. pylori. Ethical approval was previously obtained by the pathologists of each hospital before colon polyps' biopsies were taken.

Preparation of the formalin fixed paraffin blocks
Four sections from each formalin-fixed paraffin block to increase detection sensitivity were obtained with a thickness of 4 µm using Rotary microtome (LEICA RM2125RT). All sections were de-waxed with two changes of Xylene for 3 min and then dehydrated in descending concentrations of Methanol starting from absolute Methanol through 90% and lastly, a concentration of 70% for 2 min in each concentration, and then washed using distilled water.

Immunohistochemistry diagnosis
Immunohistochemistry diagnosis was performed on all the obtained sections. Known gastric sections containing H. pylori infection was used as positive and negative controls; for the negative control the primary antibody incubation step was omitted. All sections were pretreated to retrieve antigens at 97 °C for 10 min in citrate buffer solution, and then sections were blocked by 3% Hydrogen peroxide and absolute Methanol for 20 min at humidified chamber. Afterward, sections were blocked into Bovine serum Albumin (Thermo Fisher Scientific, Germany). A rabbit polyclonal antibody ULC3R (BioGenex, USA) (prepared from tissue culture supernatant diluted in PBS, pH 7.6 containing 5% BSA and 0.09% Sodium Azaide) against H. pylori was applied for 40 min, then washed in buffer solution for 5 min. Then, a polymer solution was applied for 15 min, and washed in buffer solution for 5 min. Chromogen solution was added for 10 min, and washed in distilled water. Finally, Mayer's Hematoxylin was added for 2 min, and then, sections were blued using running distilled water for 5 min. After bluing, sections were dehydrated, cleared, and mounted in DPX. Prepared sections were investigated microscopically by two experts' pathologists blindly without knowing the duplication of slides sections of each patient using X40 lens. Results were recorded into categories of positive and negative results; the dot like shape denoted the coccoid form of the organism as describe previously [35][36][37][38].

Statistical analysis
Descriptive data were analyzed using the Statistical Package for Social Science (SPSS-v20). Pearson Chi-square test was used to test the association of H. pylori infection with the different types of lesions. A P value < 0.05 was considered as a statistically significant.

Results
Of the 69 patients, there were 30 (43.5%) females and 39 (56.5%) males, their ages ranged from 21 to 80 years with a mean age 47.1 ± 19.8. 44 (63.8%) patients were diagnosed with adenocarcinoma, 10 (14.5%) patients were colitis, and 15 (21.7%) patients were juvenile polyposis syndrome. No statistically significant was observed for the association of gender and the pathological condition of each patient, P value = 0.649. Out of the 69 patients, 16 (23.2%) patients were positive for H. pylori infection. These 16 positive patients included; 13 (81.3%) patients diagnosed with adenocarcinoma and 3 (18.7%) patients diagnosed with juvenile polyps. The correlation between presence of H. pylori infection and the histopathological condition of patients were positively correlated (P value 0.028) ( Table 1).
In respect to Immunohistochemistry diagnosis, the bacteria were prominent and easier to detect in the immune-stained sections in several patterns including organisms attached to the epithelial cells or within the superficial mucus. And in some cases, the bacteria were masked by inspissated mucus or being positioned flat and closely opposed to the epithelial surface. Regarding the morphological appearance of the organism; H. pylori stained brown in color and take a dot and small curved shape in different sizes (Fig. 1).

Discussion
The exact role of H. pylori in the induction of colon cancer is still a debate between the scientific researcher communities; this is attributed to the controversial results obtained. In previous studies, H. pylori were linked to the development of gastric cancer [39], while others reported paradoxical results showing no association between H. pylori and gastric cancer susceptibility [22,40,41]. However, reports from Sudan regarding the possible link between H. pylori and colon cancer are scarce. Therefore, in the present study, we examined the presence of H. pylori using immunohistochemistry technique on colon polyps and colon cancer lesions of Sudanese patients underwent colonoscopy.
The results obtained from this study showed a positive correlation between the presence of H. pylori infection and the histopathological diagnosis, as H. pylori was prevalent in higher frequency in patients diagnosed with adenocarcinoma compared to those diagnosed as juvenile polyposis syndrome, and the result was statistically significant. This result also agrees with studies conducted by Jones et al. and Grahn et al. they investigated the presence of H. pylori among 59 patients diagnosed with colon adenocarcinomas using immunohistochemistry technique, and 77 colon and rectum cancer patients using molecular technique, correspondingly [20,39]. Jones et al., reported that H. pylori were detected in 10/59 adenocarcinoma cases which represent about 16.9% of the total cancer cases studied [39]. While, Grahn et al. showed that H. pylori were present in 27% of the patients; among the studied colon cancer, H. pylori were present in 11/42 (26%) patients [20].
Although, several studies failed to demonstrate any association between H. pylori and colon cancer, or even if this microorganism can colonize the colon [42][43][44][45][46]. This could only be attributed to the ability of demonstrating the H. pylori bacteria, which was achieved by the aid of the immunohistochemistry technique that allowed a better localization of H. pylori within the various colon lesions of colitis, polyps, and adenocarcinoma included in this study.
Interestingly, several theories were proposed regarding the exact role by which H. pylori induced colon cancer, one hypothesis is that colon cancer can be induced by toxins produced by H. pylori; however, this theory was based only on serological data [22,40,41,47]. Furthermore, some studies showed that colitis and colon cancer were also developed in experimental mice models infected with H. hepaticus [46]. Accordingly, the development of colon cancer seems most likely due to the interaction between toxins produced by the bacteria and the immune cells of the mice [46]. Therefore, the results we obtained from our study showing that H. pylori were present, nevertheless, it means that H. pylori infection is responsible for the induction and the development of colon cancer, since the presence of H.
pylori could be encountered as post-cancer incidence. This however, still requires more complicated experimental studies to investigate this hypothesis; yet, the role of H. pylori cannot be excluded due to this hypothesis. Therefore, this preliminary report needs further advanced experimental investigations to enable the

Conclusion
This study was able to demonstrate the presence of H. pylori in colon polyps and colon cancer using immunohistochemistry marker, besides the significant association in the presence of H. pylori with colon adenocarcinoma, indeed further studies are required to elaborate more in-depth about the exact role of H. pylori in the development of colon cancer.

Limitations
• In this study the sample size studied was relatively small, but still a significant association was observed.
A bigger sample size of colon cancer lesions and benign colon lesions; non cancer lesions, should be included in future studies to determine the significant association of H. pylori with adenocarcinoma among the Sudanese patients diagnosed with colon cancer.