Presence of the Helicobacter pylori in Esophageal Squamous Cell Carcinoma Samples

1MSc Student in Medical Microbiology, Students Research Committee, Babol University of Medical Sciences, Babol, Iran 2Infectious Diseases and Tropical Medicine Research Center, Babol University of Medical Sciences, Babol, Iran 3Department of Advanced Technology in Medical Biotechnology, Tehran University of Medical Sciences, Tehran, Iran 4Department of Biostatistics and Epidemiology, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran 5Department of Internal Medicine, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran 6Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran Int J Enteric Pathog. 2016 August;4(3):e37902

tries.][7] As one of the most common bacteria in the world, H. pylori was considered carcinogenic in 1994. 8Different factors are involved in the pathogenesis of gram-negative bacteria, which include urease, vacuolating cytotoxin (VacA), and cytotoxin-associated gene A (CagA) protein, as well as the host immune system response. 9Based on the reports, H. pylori infection in stomach, which causes atrophic gastroenteritis and reduction of gastric acid and serum pepsinogen, reduces the risk of esophageal ADC. 10 Recent studies showed an inverse relationship between H. pylori and ADC of the esophagus. 11Preliminary studies showed that H. pylori infection was higher in ESCC tissue than esophageal normal tissue. 12Several studies have been conducted in different regions mainly based on serological findings; however, they show contradictory results on the relationship between H. pylori and ESCC.Therefore, the relationship between H. pylori infection and ESCC is still unknown and requires further studies. 13ran is located in a geographic area with high prevalence and incidence of ESCC, especially in northern and northwestern areas.Therefore, it is important to identify the environmental factors of esophageal cancer. 14

Objectives
The high prevalence of esophageal cancer in Iran, especially in northern areas, carcinogenicity of H. pylori, and reports of some studies on the presence of the bacteria in the tissue of esophageal cancer led us to examine the relationship between H. pylori and esophageal cancer in Mazandaran province.

Patients and Samples
One hundred seventy cancerous samples and 107 non-cancerous esophagus samples were collected from the archive of pathology centers (pathology laboratory of Shahid Beheshti hospital in Babol and central laboratory of pathobiology in Amol).The data were collected by questionnaire and analyzed from the patient's medical records.All the cancerous samples were diagnosed by the pathologist and they were reviewed before including in the study to approve their diagnosis.Five-micron cuts were prepared and collected from each paraffin-embedded tissue block.

DNA Extraction
After transferring the samples to the laboratory, paraffin was removed and DNA was extracted using High Pure PCR Template Preparation kit (Roche Diagnostics,  Mannheim, Germany) from esophageal cancer tissue.The quality and integrity of extraction was evaluated by real-time polymerase chain reaction (PCR) analysis for ribonuclease P gene, as described previously. 15lymerase Chain Reaction After setting up the experiment, PCR was used for detecting H. pylori DNA.A reaction mixture of 25 μL, including 14.85 μL of deionized water, 2.5 μL of buffer, 1 μL of MgCl 2 , 0.5 μL of each primer, 0.5 μL of dNTP, and 0.15 μL of Taq enzyme, was prepared in a micro tube.Next, 5 μL of DNA was added to the sample.Thermo cycler's program was as follows: (a) Heat start for 2 minutes at 95°C, (b) 30 cycles at 60, 90, and 72°C each for 30 seconds, and (c) Final extension at 72°C for 5 minutes.PCR products were then analyzed by a 1.5% agarose gel with electrophoresis.The PCR was carried out using the specific primers for glmM gene of H. pylori strain.Table 1 shows the sequence of these primers.The PCR product of this gene with glmM primer is a piece with a length of 294 bp. 16

Results
The samples were examined in terms of age, gender, anatomical location of the esophagus, and histopathologic diagnosis and residence status.Of 170 cancerous and 107 control samples, 84 (49.4%) and 67 (62.6%) were male, respectively.The mean age of patients and control group was 66.50 ± 11.05 and 62.50 ± 14.50, respectively.Similarly, among patients, 77.1% and 22.9% lived in the cities and rural area, respectively (Table 3).The analysis of lesions for different anatomical sites is shown in tables 2 and 3.In cancer patients, 35 (21.3%), 65 (39%) and 64 (39.6%) cases belong to upper third, middle third and lower third of the anatomical sites.
Of the 104 control samples, 24 (23.1%),32 (30.8%) and 48 (46.2%) samples were in the upper, middle and lower third of the esophagus, respectively.124 (72.9%) of esophageal cancer samples had been collected by biopsy and 46 patients (27.1%) by surgery.Tables 2 and 3 shows the frequency distribution of the samples in both groups in different parts of the esophagus in terms of gender, type of lesion, age, and place of residence.
Ribonuclease P gene was examined using the real-time PCR technique through examining and controlling the presence of DNA in the tissue extracted from samples while all the samples had sufficient DNA.In this study, no band indicating the presence of H. pylori DNA was seen in the agarose gel of the samples (Figure 1).

Discussion
Many studies examined ADC and most of them concluded the protective role of H. pylori and reported a reverse relationship between ADC and H. pylori.The cause of  this relationship has not been realized yet; however, one reason may be the presence of H. pylori, which neutralize the acidity of gastro-esophageal reflux, which is one of the major risk factors for ADC. 11Some studies reported an inverse relationship between H. pylori and ESCC, including a study in Taiwan in 2005 which showed a protective role for H. pylori in ESCC development. 17Similarly another study in Taiwan in 2009 used the same method and reported a protective role for H. pylori in ESCC occurrence. 18Findings of the separate studies in Urmia and Tabriz using serological methods showed an inverse relationship between H. pylori and ESCC development. 19,20A positive relationship was reported between H. pylori and ESCC in some countries, such as Sweden 12 and China 13 studies.
A study, which was conducted using Western blot analysis in Sweden in 2007, reported no relationship between infection by this bacterium and ESCC.A study using a serological method in China in the same year also showed no relationship. 21,22nother study in Australia in 2010 examined the prevalence of H. pylori in esophageal cancer using serological methods and polymorphism.The study showed an inverse relationship between H. pylori infection in ADC and EGJAC (esophageal-gastric junction), but showed no relationship between H. pylori and ESCC. 10 Some studies were conducted in this concern in Finland and Germany in 2011, which reported no relationship between H. pylori and ESCC. 23,24The present study did not show the presence of H. pylori in any esophageal cancerous and non-cancerous sample of the patients of Mazandaran province and no relationship was found between H. pylori and esophageal squamous cell carcinoma (ESCC) (P > .05).
With respect to the results and importance of the issue, further extensive epidemiological studies are recommended in different parts of Iran, preferably on fresh samples, to clarify whether H. pylori is really linked to esophageal cancer.

Table 1 .
Sequences of Primers Used for the glmM Gene Abbreviation: PCR, polymerase chain reaction.

Table 2 .
Demographic Features in Non-cancerous Esophageal Lesions

Table 3 .
Demographic Features in Cancerous Esophageal Lesions Abbreviation: SCC, squamous cell carcinoma.