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ESCC is characterized by a striking variation in geographical distribution and a high mortality rate. The high-risk countries include Eastern Turkey, the Former Soviet Union, Iraq, Iran, Western and Northern China, Hong Kong, and Japan, named as the Asian esophageal cancer belt. In addition South Africa, Brazil and France have been reported as high-risk areas . In most countries the incidence of ESCC among the population is 1.5-2.5 and 2.5-5/100.000 for women and men, respectively, whereas high-risk countries such as northern China or South Africa have an incidence up to 246/100.000 .
In Sudan, esophageal cancer is the most common gastrointestinal tumor. It is the fourth most common cause of cancer in Sudanese males, and the fifth most common in females. According to a recent report by the World Health Organization (WHO), it was estimated that the 5- year prevalence of esophageal cancer in Sudan is 2.4%, with a mortality rate of 6.6%. Thus it is obvious that esophageal carcinoma has a major health burden in Sudan.
A number of environmental factors could be important in the carcinogenesis. Recent evidence has implicated an etiological role of certain micro-organisms, such as bacteria, fungi and viruses, in the development of esophageal carcinoma either by producing carcinogens or by Interacting directly with the host cell. [6,7] The development of esophageal cancer is a multifactorial process associated with a variety of risk factors. Cumulative evidence suggests that tobacco smoking, heavy alcohol drinking, micronutrient deficiency, and dietary carcinogen exposure may cause the disease. However, even in the at-risk population, only a portion of exposed individuals develop the cancer in their life span, indicating that there may be important genetic basis rendering such individuals susceptible to the disease.
1.1. Esophageal cancer and its association with infection:
1.1.1. Human Papilloma Virus:
The relationship between HPV and ESCC has been suspected since the initial reports by Syrjanen et al in 1982[8,9]. Since then many studies have reported inconsistent results. Meta-analyses by Syrjänen showed that HPV DNA was detected in 15.2% of ESCCs. Similarly, a study in Finland showed that ESCC patients have elevated serum antibody against HPV-16 when compared to blood bank donors . While a study in Norway has confirmed the Finnish study , another study in Sweden could not confirm it . On the other hand, a study conducted in Shaanxi Province, China, where esophageal cancer risk is known to be high; ESCC patients have elevated serum antibodies against HPV-16. However, none of these studies has tried to confirm the presence of HPV in esophageal carcinoma tissues.
High incidences of HPV positivity have been reported among esophageal squamous cell carcinoma tissues in high-risk areas such as China, Korea, South Africa, and Alaska. But HPV is rarely identified in esophageal squamous cell carcinoma in Japan, the United States, the United Kingdom, the Netherlands, and low-risk areas of China. There is obvious geographic variation, which may help explain the differences in rates of HPV seen around the world in esophageal cancers. Variation also exists by HPV type, with the following types associated with esophageal squamous cell carcinoma: 5, 6, 9, 11, 16, 18, 20, 20, 21, 24, 25, 27, 31, 32, 33, 39, 45, 52, 53, 58, 67, and 87. major HPV type linked with esophageal cancer is HPV 16. There has been a substantial amount of research into HPV 16, including potential mechanisms. The strength of an association for HPV 16 and esophageal squamous cell carcinoma is relatively high, with one study reported an odds ratio as high as 6.3 (95% CI: 1.6-23.7). 
1.1.2. Herpes Simplex Virus (HSV):
HSV is a member of the herpes virus family. There are at least two identifiable antigenic types of HSV: HSV-1 frequently causes herpetic lesions in the head and neck region, whereas HSV-2 is most frequently associated with genital infections.  Among the visceral organs, the esophagus is the most frequent site of involvement by HSV particularly in immunocompromised subjects. [16-18] HSV esophagitis has been recognized as a distinct clinical entity for a long time. Laboratory studies indicate that HSV is capable of transforming the morphological phenotype of rodent cell. In a number of epidemiological studies, increased levels of HSV antibodies have been detected in patients with head and neck cancers, as compared to carefully matched controls. . in two separate studies in China, the infection rates of HSV in esophageal cancer tissue were reported to be 30% , 31.7%  However, another study conducted in China, showed no evidence of infection with HSV in esophageal cancer tissues. .
1.1.3. Epstein Barr Virus:
Epstein-Barr virus (EBV), a common herpesvirus that can infect both lymphoid cell and epithelial cell  has been associated with infectious mononucleosis  oral hairy leukoplakia  and nasopharyngeal carcinoma (NPC) . In addition, expression of EBV receptor (CD21) and EBV particles were identified in the oronasopharnyx. [26-27] Slaughter et al  hypothesized that epithelial surface of the upper aerodigestive tract might share a common carcinogen exposure and increase risk of cancer development, or a so-called "field cancerization." Because of the prevalence of EBV in NPC  and the anatomic vicinity of nasopharynx, buccal cavity, and tonsil to the esophagus, presence of EBV in esophagus could have implication in the pathogenesis of ESCC
A relationship between EBV and lymphoproliferative disorders (Burkett's lymphoma and Hodgkin's lymphoma) has been recognized. But up to now there have been few reports of a relationship with esophageal carcinomas. The EBV detection rates in these studies have varied from 1.8% to 35.5%.[30-34]
There were two early studies from 1999 and then a German group published two studies in 2002 and 2005.[35-38] Lam  conducted a study in Hong Kong on 40 sporadic mesenchymal tumors of the esophagus that underwent immunohistochemical study for EBV. None of the tumors were positive for either of the two EBV markers. Another early study, conducted in northern China on 51 esophageal squamous cell carcinoma specimens, found no link using in situ hybridization and PCR. .
A study of 72 squamous cell carcinoma and 40 adenocarcinoma specimens from Russia were assessed for EBV using PCR. . One-third of esophageal squamous cell carcinoma and adenocarcinomas had EBV DNA in the nuclei of tumor-infiltrating lymphocytes, suggesting that EBV plays no role in the development of esophageal cancer. In another earlier study from this group, just over one-third of esophageal squamous cell carcinomas (35%) and adenocarcinomas (36%) were positive for EBV DNA from 37 specimens. 
1.1.4. JC Virus:
JC virus (JCV) is a human polyomavirus that was isolated first from the brain of a patient suffering from progressive multifocal leukoencephalopathy (PML). . Polyomaviruses are a subfamily of nonenveloped DNA viruses with icosahedral capsids that contain small, circular, double-stranded DNA genomes.  It is known that JCV is the etiologic infectious agent of PML, a fatal demyelinating disease of the central nervous system. The virus is widespread throughout the population, with > 80% of adults exhibiting JCV-specific antibodies. It is believed that JCV infection takes place during early childhood and usually is subclinical. However, under conditions of immunosuppression, e.g., in patients with acquired immunodeficiency syndrome, JCV can emerge from latency to cause PML.  JCV can transform cells in culture and is oncogenic in laboratory animals.  JCV DNA sequences have been detected in several kinds of human malignancies, including brain tumors of glial origin, ,  medulloblastomas,  and colon carcinoma. 
Currently, there is only one study looking at human polyomavirus JC virus (JCV) and esophageal cancer development.  In this study, Molecular DNA of 70 esophageal biopsy specimens from individuals with various types of esophageal disorders (achalasia, reflux esophagitis, Barrett esophagus, adenocarcinoma, squamous call carcinoma) and healthy controls was analyzed. JCV DNA was isolated from 85% of normal esophageal tissues, and 100% of esophageal carcinomas. JCV DNA was found in normal, benign, and malignant esophageal tissue samples; however, it was active only in carcinoma cells where viral expression of proteins occurred. The investigators suggest that these data provide evidence that infection with JCV may play a role in the subsequent development of esophageal carcinoma. 
To the best of our knowledge, this is the first study that attempts to determine the role of different environmental and genetic factors in the predisposition to esophageal cancer in Sudan. Esophageal cancer is a common cancer in Sudan and constitutes a major health burden. Determining the magnitude that environmental and genetic factors play in the causality of oesophageal cancer in Sudan will help in sitting the appropriate plans that aim to reduce the prevalence of this disease.
The known environmental risk factors for esophageal cancer in other populations may contribute to the etiopathogenesis of the disease in Sudan.
The risk of the esophageal cancer may increase by consumption of certain local food.
Esophageal cancer could be caused by infectious agents.
4.1. General objective:
The general objective of this study is to measure the infectious risk factors of esophageal cancer in Sudan.
4.2. Specific objectives:
To study the association between HPV, HSV, EBV, JCV and esophageal cancer in Sudanese patients.
To determine the biological effects of these viruses in patients with esophageal cancer.
To measure the sensitivity and the specificity of the immunohistochemsitry method in detection of viruses that may infect esophageal tissues.
5.1. Study design:
Considering our stated objectives a prospective hospital-based case-control study will be conducted.
5.2. Study Center:
Khartoum Teaching Hospital, Alshaab teaching Hospital, Radiation and Isotope Center Khartoum (RICK).
5.3. Study population:
Esophageal cancer patients (males and females) attending the respective hospitals will be included. The criteria for including patients in this study will be the following:
They must be diagnosed clinically and confirmed histologically as having squamous cell esophageal cancer.
Consent to participate in genetic testing.
Sudanese subjects (males and females) will be included. They will be matched to the patients with respect to their age, ethnicity and gender distribution.
The inclusion criteria for this group will be as follows:
They must not have any esophageal pathology at the time of conducting this study.
Consent to participate in genetic testing.
5.4. Data collection:
Sources of data will include: clinical examination (done by the physician in the respective hospitals), personal interview, a questionnaire and laboratory investigations.
A simple questionnaire is designed and used for each patient; it included complete demographic characteristics, present and family history of cancer, personal habits and medical history. Another questionnaire will be designed for control subjects including same questions.
5.5. Sample size:
The formula to calculate the sample size is:
n= tÂ² x p (1-p) Â
n = required sample size.
t = confidence level at 95% (standard value of 1.96).
p = estimated prevalence of the disease in the area.
m = margin of error at 5% (standard value of 0.05).
The prevalence of esophageal cancer in Sudan is 2.4%. By substituting this figure in the formula above, the calculated sample size is about 36 patients.
However, we will take a sample size to include 100 patients and 100 controls.
5.6. Data analysis:
The data will be cleaned and checked for consistency before entering it for analysis. Analysis will be conducted using PASW version 18. The alpha Î± level of significance will be set at 0.05. Descriptive analysis will be done. Chi-square and Fischer's exact test will be used to compare the difference in proportions between groups. Independent t-test will be used to test the differences in means between groups. Multiple logistic regressions for the effect of multiple risk factors on esophageal cancer will be conducted as well. Odd ratio will be calculated to measure the risk factors for esophageal cancer. Analysis will be displayed in appropriate table and figure format using Excel 2007.
5.7. Sampling Techniques:
Esophageal biopsies will be collected from patients during upper GI endoscopy. Biopsies will be kept frozen immediately after removal and then stored in liquid nitrogen until use.
For the control group, the esophagus must be cancer-free.
5.8. Laboratory Tests:
5.8.1. Histopatholgic examination:
The 4 Âµm thick paraffin sections will be cut from each specimen, placed on aminopropyltriethoxy-silane-treated slides and routinely stained with hematoxylin and eosin. All specimens will be examined under light microscopy to confirm the histopathological diagnosis of carcinoma and the tumors will be graded according to the WHO histological classification criteria.
Tumor tissue will be sectioned at 4 Î¼m in thickness and stained with hematoxylin and eosin for routine histological characterization.
Immunohistochemistry will be performed by using an avidin-biotin-peroxidase complex system.
Nonenzymatic antigen retrieval will be performed in 0.01 M sodium citrate buffer (pH 6.0) for 40 min at 95Â°C.
After the mixture is allowed to cool, peroxidase quenching in MeOH-% H2O2 for 20 min will be performed, followed by blocking in 5% normal horse serum for 2 h at room temperature.
Primary antibodies against viral and cellular proteins will be incubated overnight at room temperature in a humidified chamber. Primary antibodies to detect HPV, HSV, JCV, EBV proteins will include anti-HPV E6, rabbit anti-HSV I, II; rabbit anti-JCV Agnoprotein; anti-EBV LMP1, respectively.
In addition, to assess the biological effects of the above-mentioned viruses, expression of the following cellular and/or nuclear proteins will be assessed using specific monoclonal antibodies: TP53, p16, Ki67, cyclin D.
After incubation with primary antibodies, the sections will be incubated in biotinylated secondary anti-mouse or anti-rabbit antibodies for 1 h at room temperature, followed by incubation with avidin-biotin complex and will be developed with diaminobenzidine.
Slides will be counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted with Permount.
Negative control will be designed by using phosphate-buffered saline (PBS) instead of the monoclonal antibodies used in detection.
The immunoreactivity for all the proteins investigated in this study will be evaluated as strongly positive (++) when more than 50% of tumor cells stain, positive (+) when more than 25% of the cells stain, slightly positive (Â±) when more than 10% of the tumor cells stain, and negative (-) when less than 10% of the tumor cells stain or when the tumor cells completely lacked immunoreactivity. All slides will be observed by two independent pathologists, and the final concordant results will be adopted.
5.8.3. DNA extraction from tissue:
To detect viruses in esophageal tissues, genomic DNA will be extracted from tumor tissue (biopsy & resection specimens) using extraction kits. From 1g-mammalian cells, 2mg DNA can be expected.
To determine genetic polymorphism, DNA will be extracted from leukocytes using salting-out method.
The process involved:
1- Preparation of cells, Cells lyses and digestion, DNA extraction and DNA purification.
2- Electrophoreses of extracted DNA in agarose gel (It is the checking of the extracted DNA). DNA is detected on gels by staining with ethidium bromide, which has an intense fluorescence excited by ultra-violet radiation when it complexes with nucleic acids.
3- Determination of DNA concentration and purity will be assessed by the Nanodrop.
5.8.4. Polymerase Chain Reaction (PCR):
PCR technique will be used to detect the different viruses in the esophageal tissues.
PCR is an in vitro method for the enzymatic amplification of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands in the target DNA.
A repetitive series of cycles involving template denaturation, primer annealing, and the extension of the primers by DNA polymerase results in the exponential accumulation of the specific DNA segment.
The Standard Reaction:
It is impossible to describe a single set of conditions that will guarantee success in all situations.
The reaction components are (template, primers, Taq polymerase, dNTP's, and buffer).
Minor adjustments to these parameters will often transform a marginal PCR into one with excellent specificity and yield.
The tubes will then be placed in the PCR machine. The temperature of the reaction will be cycled (Denaturation, Annealing, and Extension of the reaction).
A pair of primers is usually designed using the published data for sequences to amplify different DNA fragments of the selective gene.
It is the primers more than anything else that determines the success or failure of amplification reaction, hence they:
Will be between 20-30 bases in length.
Should be with a random base distribution and with a GC content similar to that of fragment being amplified (50% content GC).
Should have sequences with no significant secondary structures (no palindrome).
Have either C or G on the end.
Should be checked against each other for complementarities, in particular avoiding primers with 3-overlaps which will reduce the incidence of (primer dimmers).
Primers that will be used to detect infection by viruses:
Table (1) lists the primers that will be used to detect the different viruses.
5'CAGTACGGCCCCGAGTTCGTGA 3'; 5'GTAGATGGTGCGGGTGATGTT 3'
Checking of the PCR Products:
The specificity of PCR is typically analyzed by evaluating the production of the target fragment by gel electrophoresis of 5Âµl DNA on 2% agarose gel stained with ethidium bromide.
6. Ethical considerations
Ethical clearance to conduct the study will be obtained from the Ethics Committee of the Faculty of Medicine, University of Khartoum. Initial consent will be obtained from authorities of the study areas. Individual permission for participation will be obtained through "witnessed informed verbal consent".
The purpose of the study and the nature of the procedure will be explained to the study subjects. The right of the participant to refuse to be involved in the study will be respected. Information given by the participant and the result of the test will be confidential.
7. Gantt chart
Weeks 2 to 4
Weeks 5 to 8
Weeks 12 to 13
Meeting health authorities and present the study proposal
Patients and controls attending the study areas
Writing of the thesis
Finalizing the study
Submission of the final project
1. DNA extraction Kit
2. PCR kit (Taq Polymerase, Primers, MgCl, Buffer)
4. Other Chemicals
8. Other costs (10%)