Linking Helicobacter pylori with gallbladder and biliary tract cancer in Moroccan population using clinical and pathological profiles

It is of interest to assess the clinical and pathological aspects of Gallbladder and biliary tract carcinomas confirmed by histological data. It is also of further interest to evaluate the link between Helicobacter pylori and biliary tract cancers. Eighty-nine (89) cases (mean age 60±12 years) of Gallbladder and biliary tract cancer confirmed by histological data were enrolled for the study at the Department of Pathology in Mohammed VI University Hospital, in Morocco. The data such as age, sex, clinical and histo pathological features were collected. Bile duct specimens were investigated for H. pylori using Giemsa and immuno histo chemistry staining. Results show that bile duct stones were found in 53.9% of cases. It is known using histological data that adeno carcinoma is common accounting for 70 % of all bile duct tumors. Moreover, Helicobacter pylori was detected in 54% of cases linking with the presence of bile duct stones characterized by the histological subtype, the macroscopic classification and lymph node's presence (p<0.001). Thus, data collected suggest the potential association of Helicobacter pylori with gallbladder cancer possibly through the formation of bile duct stones.

The incidence and pathological characteristics of carcinomas of the gallbladder and biliary tract in Morocco are largely unknown, although several investigations were performed. According

Data collection
Data collection was done retrospectively from patient records, using a standardized form. The collected variables included age, sex, clinical and histopathological data such as the presence of gallstones, preoperative diagnosis, macroscopic aspect, and anatomical location, the degree of invasion, histological type, and grade of differentiation. The collected data used to support the findings of this study are available from the corresponding author upon request.

Detection of Helicobacter pylori
Detection of Helicobacter pylori was done by modified Giemsa staining. The confirmation was done subsequently by immunohistochemistry (IHC). A 4 µm section was cut from formalin fixed and paraffin embedded block, two different slides were prepared, which were stained by Modified Giemsa and anti-H.pylori antibody immuno stain.

Modified Giemsa:
Paraffin-embedded sections were dewaxed and rehydrated. Then it is incubated for 20 min in 1:10 Giemsa's solution in distilled water. After rinsing in distilled water, the section was quickly dehydrated through alcohol solutions before being cleared with toluene and mounted.

Statistical analysis
The ҳ2 or Fisher's exact test was used for statistical analysis to determine the relationships between the presence of H. pylori and categorical data, p < 0.05 was considered as statistically significant. The data was analysed by the SPSS V23 statistical program.  After the examination by immunohistochemistry and Giemsa staining for H. pylori (Figure 2), 48 (54%) of the patients were positive and 41 (46%) of the patients were negative. Thirty-five were positive by H. pylori immunostain and Giemsa with the presence of calculis. There was a strong association between the presence of H. pylori and the presence of calculis (P<0.001).
Moderately differentiated gallbladder and biliary tract cancer showing H. pylori positivity was present in forty-four. The p-value was <0.05 indicating a significant association between the presence of H. pylori and the differentiation of these malignancies, the histological subtype, the macroscopic variety and lymph node's presence. However, none of the demographic factors (gender and age), or the location of the tumor had a significant correlation with H. pylori (Table 2).

Discussion:
Gallbladder and biliary tract cancer is rare but carries a poor prognosis. Clinical and pathological evidence has suggested that inflammatory conditions involving the bile ducts predispose these ducts to carcinogenesis, however, the relationship between chronic inflammation and malignant transformation is still under debate [17,18]. These malignancies are difficult to diagnose in an early stage, the majority of patients develop symptoms only at an advanced stage of the disease. In term of tumor location, Gallbladder cancer is the most common malignancy in our investigation, representing 84.3% of all cases.  GBC is seen commonly in women. In contrast, CCA is marked with a male predominance with a ratio of 1:1. provide strong integrity to the hypothesis that Helicobacter is associated with biliary cell inflammation and proliferation.
Although, several pathways by which H. pylori might induce a cancerous change in bile duct has been proposed. One mechanism called "the peri genetic pathway" involves the enhanced production of free radicals close to H. pylori and an increased rate of host cell mutation. It has been proposed that Helicobacter induces inflammation and locally high levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6. According to the anticipated peri genetic mechanism, signaling molecules associated with inflammation, such as TNF-α, alter epithelial cell adhesion and lead to the dispersion and movement of mutated epithelial cells without the need for additional mutation in tumor suppressor genes [47].

Conclusion:
It is of interest to confirm the presence of H. pylori in Gallbladder and biliary tract cancers using clinical and pathological data. The results link H. pylori with the development of CCA and GBC. Thus, Helicobacter infection in CCA and GBC patients confirmed with histopathological data implies its association with the bile duct stone formation. Moreover, the association between H. pylori infection and the degree of differentiation of Gallbladder and biliary tract cancer is also ascertained. However, additional data is needed to evaluate the link between the carcinogenesis of H. pylori in the biliary tract using in vitro and in vivo models. Data on the risk factors of these malignancies including Helicobacter infections, gallstones, environmental factors, genetic susceptibility and possible regional differences is also relevant.