Immunohistochemical screening for Epstein Bar virus (EBV) among Sudanese Patients with Esophageal Cancer
Authors: Hussain Gadelkarim Ahmed1,2, Mohmad Ahmad Babikir Ibraheem Beely3, Mohammed Siddig Abd El Aziz4, Fawaz D. Alshammari5, Ahmed Abdalla Agab Eldour3.
Affiliations: 1- Department of Pathology, College of Medicine, University of Hail, KSA,
2- Department of Histopathology and Cytology, FMLS, University of Khartoum, Sudan.
3- Department of Medical Laboratory, Faculty of Medicine, University of Kordofan, Sudan 4-Department of Histopathology and Cytology, Faculty of Medical Laboratory Science, Sudan University for Science and Technology, Sudan.
5- Department of Medical Lab. Science, University of Hail, KSA.
Correspondence to: Prof. Hussain Gadelkarim Ahmed, Department of Pathology, College of Medicine, 2440, University of Hail, Kingdom of Saudi Arabia (KSA)
Abstract:
Objective: The aim of this study was to screen for the existence of Epstein Barr Virus (EBV) in esophageal cancer (EC) tissue samples obtained from Sudanese patients. Methodology: In this retrospective study 102 formalin fixed paraffin wax embedded tissue’s blocks were retrieved from histopathology laboratories in Khartoum City, Sudan and subsequently tested by Immunohistochemical methods for the presence of EBV. All specimens were previously diagnosed as having esophageal carcinoma. Results: As regards Immunohistochemistry (IHC) staining for EBV, positive findings were revealed in 22 (21.5%) and 79/ (78.5%) of the study subjects were negative. Of the 55 males with esophageal cancer, 15/55 (27%) were identified with EBV infection and the remaining 40/55(73%) found without EBV infection. Of the 46 females with esophageal cancer, 7/46(13%) were found positive for EBV immunostaining and the remaining 39/46(87 %) were found negative for EBV. Conclusion: EBV seemed to have reasonable etiological effect in the occurrence of esophageal cancer in Sudan. Crucial preventive strategies are extremely needed to reduce the burden of esophageal cancer in Sudan.
Key words: Esophageal cancer, Sudan, EBV
Introduction:
Esophageal cancer (EC) occurs commonly, particularly in Asia, and it is the sixth leading cause of cancer deaths worldwide [1]. Esophageal cancer classically happens in one of two forms, squamous cell carcinomas (SCCs) arising from the stratified squamous epithelial lining of the organ, and adenocarcinomas (AC) arising from columnar glandular cells that replace the squamous epithelium [2]. Sarcomas and small cell carcinomas usually constitute less than 1%-2% of all esophageal cancers [3].
The major risk factors for esophageal cancer include; gastro-esophageal reflux [4], tobacco smoking, alcohol drinking [5], viruses [6], susceptibility genes [7], low levels of intake of fruits and vegetables [8]. The relationship between viral infection and esophageal cancer was previously reported for several viruses including; human papillomavirus (HPV) particularly subtype 16, herpes simplex virus 1 (HSV-1), Epstein-Barr virus (EBV) and cytomegalovirus (CMV). However, the mechanisms of the viral action in addition to geographic variation in infection rates of these viruses still need more efforts [9]. EBV is an oncogenic virus that has been described to be linked to Burkitt’s lymphoma, Hodgkin’s disease, breast cancer, gastric carcinoma, nasopharyngeal carcinoma and leiomyosarcoma [10]. However, whether EBV is associated to esophageal SCC still controversial: there are limited studies and their conclusions vary [11].
Therefore, the aim of this study was to screen for the presence of EBV in esophageal cancer tissue samples obtained from Sudanese patients.
Materials and Methods:
In this study we investigated 102 Sudanese patients with esophageal carcinomas, 55 were males and 46 were females (males’/females’ ratio, 1.20:1.00), aged between 12 and 98 years with mean age of 60 years old, were diagnosed as having esophageal carcinomas, were investigated for the presence of HR-HPV subtypes 16&18 using LMP1 (Latent membrane protein 1 (LMP1) of EBV) Antibody immunohistochemistry. The diagnosis was established depending on clinical examination and histological features of the biopsy. EC diagnosis was confirmed base on Royal College of Pathologists criteria (Royal College of Pathologists, 2005). The esophageal cancer including 91/102 (89.2%) squamous cell carcinomas (SCCs) and 11/102 (10.8%) adenocarcinoma.
The sample included full coverage of patients with esophageal lesions referred to Radiation and isotope Centre Khartoum with available sample quantity and data. Ethical consent was obtained from ethical committee of the Faculty Research Board and Hospital.
LMP1 immunohistochemistry (IHC) was performed on formalin-fixed paraffin embedded (FFPE) tissue sections using kits from (Dako Agilent Technologies Company). Following antigen retrieval using Citrate buffer (pH 6) for 30 minutes, sections were incubated with mouse monoclonal LMP1, and then EnVision_ System HRP anti-mouse was employed, followed by diaminobenzidine (DAB) chromogen then counterstained with Harris’ Hematoxylin. Known LMP1-positive sections were used as positive controls, and sections without addition of primary antibody were used as negative controls. All LMP1 IHC slides were semi quantitatively evaluated by two investigators for intensity of staining in the cell nucleus and cytoplasm. Intensity was scored as 0 (none), 1(weak), 2 (moderate), or 3 (strong), with 0 or 1 scores regarded as negative and 2 or 3 categorized as positive. LMP1 scoring was performed without knowledge of EBV infection status.
Results
The present study retrieved 102 tissue blocks prepared from esophageal carcinoma’s specimens (91patients were with squamous cell carcinoma and 11 were with adenocarcinoma). Of the 102 study subjects 55 were males and 46 were females their age ranging from 12 to 98 years old with a mean age of 60 years. The majority of the study populations were at the age range 66-75 years constituting 26 patients followed by age ranges, both 56-65 and < 45 years representing 24 patients. Moreover, 16 patients were identified among age group 46-55 years, hence only 12 patients were found among age group 76+ years, as indicated in Fig1.
Figure 1. Description of the study population by age
Regarding IHC staining for EBV, positive findings were revealed in 22 (21.5%) and 79/ (78.5%) of the study subjects were negative, as shown in Microphotographs 1 & 2. Of the 55 males with esophageal cancer, 15/55 (27%) were identified with EBV infection and the remaining 40/55(73%) found without EBV infection. Of the 46 females with esophageal cancer, 7/46(13%) were found positive for EBV immunostaining and the remaining 39/46(87 %) were found negative for EBV. The association of EBV risk with male sex was OR (CI) = 2.08(0.77-5.7), P =0.145.
According to cancer type, of the 91 cases of the Squamous cell carcinoma, 19/91 (21%) were found with positive EBV and the remaining 72/91(79%) were negative. Of the 11 cases of the Adenocarcinoma, 3(27.3%) were found with positive EBV and the remaining 8 (72.7%) were negative, as indicated in Table 1. The association of EBV risk with Squamous cell carcinoma was OR (CI) = 0.70(0.17-2.9), P =0.63, as indicated in Table 1, Fig2.
Table 1. Distribution of IHC results of EBV by sex and cancer type.
Sex Positive Negative Total
Males 15 40 55
Females 7 39 46
Total 22 79 101
Cancer type
Squamous 19 72 91
Adenocarcinoma 3 8 11
Total 22 80 102
Figure 2. Description of IHC results of EBV by sex and cancer type
Concerning IHC staining results of EBV with age distribution, the highest positive results were found in age group 56-65 years, representing 7/23(32%), followed by 46-55, <45, 66-75 and 76+ constituting 6/22(27.3%), 4/22(18.2%),3/22(13.5%) and 2/22(9%) respectively, whereas; the groups with the highest negative results were 66-75 years, representing 23/80(28.8%) followed by <45,56-65 and (46-55& 76+) constituting 20/80 (25%), 17/80 (21.2%), and 10/80(12.5%) for each, respectively, as shown in Table 2, Fig 3.
Table 2. Distribution of IHC results of EBV by age
Age group Positive Negative Total
<45 years 4 20 24
46-55 6 10 16
56-65 7 17 24
66-75 3 23 26
76+ 2 10 12
Total 22 80 102
Figure 2. Description of IHC results of EBV by age
Discussion:
The prevalence rates of esophageal cancer varies according to geographic regions and related risk factors. Many risk factors, including EBV, may be involved in the development of esophageal cancer. Although there is no a clear-cut association between EBV infection and EC, but there is evidence that EBV infection may contribute to esophageal cancer in certain high-risk populations [12].
However, in the present study we found a prevalence of 21.5% of infection of EBV among Sudanese patients with EC. Relatively higher prevalence rates were previously reported in some studies. In study from German investigated 37 patients with esophageal carcinoma (Esophageal squamous cell carcinomas (n=23) and adenocarcinomas (n=14)) for the presence of human EBV DNA, EBV was detected in 35% of squamous cell carcinomas and 36% of adenocarcinomas [13]. Nevertheless in the present study EBV positive was found in 27.3% of the cases of the adenocarcinoma and 21% of the cases of SCC. In another study an association between EBV infection and the development of esophageal carcinoma has been reported in 35.5% of the cases [14]. Although, It is strongly suspected that the EBV plays a role in the genesis of nasopharyngeal carcinoma, but some studies reported that, EBV is infrequently associated with esophageal SCC, and may appear through tumor-infiltrating lymphocytes in some advanced lesions [15,16]. However, the Prevalence rates of EBV in gastro-esophageal cancer varies between 5 and 20%, which may depend on the patient groups in different studies [17-19]. EBV-associated tumori-genesis appears to be rather restricted to gastric cancer while the role of EBV in other parts such as esophageal carcinomas appears to be insignificant in most parts of the globe [20].
To the best of our knowledge there no study investigated the role of the EBV in the etiology of EC from Sudan. Most of the studies in this context from Sudan, tested the association between EBV and nasopharyngeal carcinoma. Almost all of these studies found some degrees of association between EBV and nasopharyngeal carcinoma. In one study from Sudan, EBV genes were detected in 92/150 (61.3%) of patients with nasopharyngeal carcinoma [21]. Another study from Sudan has tested 43 Sudanese patients with nasopharyngeal carcinoma for the presence of EBV using EBER-ISH. All nasopharyngeal carcinoma biopsies (100%) were positive for EBER1 in almost all carcinoma cells [22]. These studies may give some degree of suspicion for EBV as possible etiological factor contributing to the etiology of EC in Sudan.
In regard to the age the prevalence of EBV infection in the current study was 27% in males and 13% in females. Since there is only small studies in regard to the relationship between EBV and EC, the relationship of sex and EBV can’t be strongly but, but studies on the association of EBV and nasopharyngeal carcinoma indicates similar findings to our series.
In regard to the age most of cases with positive EBV expression were found among age 56-65 years. Although cancer in general accumulate at elderly people, but this need further research in regard to the start of infection and initiation of carcinogenesis.
In conclusion EBV seemed to have reasonable etiological effect in the occurrence of esophageal cancer in Sudan. Crucial preventive strategies are extremely needed to reduce the burden of esophageal cancer in Sudan.
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Microphotograph 1. Esophageal carcinoma showing positive EBV expression (immunohistochemical staining using LMP1 antibody), EnVision_ System HRP. X100.
Microphotograph 2. Esophageal carcinoma showing positive EBV expression (Immunohistochemical staining using LMP1 antibody), EnVision_ System HRP. X400.
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