Immunohistochemical Expression of Epstein Barr virus Antigen Latent Membrane

Different genetic and environmental factors appear to be involved in the pathogenesis of classical Hodgkin’s lymphoma. One of the important eatiological factors is the presence of Epstein Barr virus which is ssociated with Hodgkin's lymphoma in different proportions and this association is believed to be causal.
Overexpression of Bcl-2, which is an anti-apoptotic member of Bcl-2 family of proteins, was contributes to the development of some tumors like lymphomas. Several studies proved that EBV latent membrane protein-1, promote survival of Reed/Sternberg cells through many signaling pathways, it also up regulates the antiapoptotic Bcl-2 protein. Aim of the study : 1. To evaluate the immunohistochemical expression of the Latent Membrane Protein-1 of Epstein-Barr Virus and the anti-apoptotic protein Bcl-2 in Classical Hodgkin Lymphoma using a specified automated cellular image analysis system (Digimizer software analysis) and to correlate their expression with different clinicopathological parameters. 2. To find if there is any relation between the immunohistochemical expression of LMP-1 and Bcl-2 in classical Hodgkin’s lymphoma. Patients, Materials and Method : This retrospective study was done on fifty paraffin embedded blocks of lymph nodes biopsies from patients diagnosed as Classical Hodgkin’s Lymphoma. The control group consists of 20 age and gender matched subjects having reactive lymph nodes biopsies. The cases were selected from archived files of the Department of Pathology of different Teaching hospitals in Baghdad. For each case, three representative sections (4μm thickness for each) were prepared. One section stained with Hematoxylin and Eosin while other two sections were stained immunohistochemically for LMP-1 and Bcl-2.the work was conducted at Al-Khadhimiya teaching hospital laboratory. The positive staining pattern was brown diffuse cytoplasmic stain of Hodgkin/Reed-Sternberg cells for both markers. Slides were examined by light microscope at 40x magnification to identify the best representative area, three images were captured and the immunohistochemical staining was estimated by a specialized automated cellular image analysis system, Digimizer software, version 3.7.0. Data were analyzed using SPSS program (Statistical Package for Social Sciences) version 16 and Microsoft Office Excel 2007. P- Value of less than 0.05 was considered significant.
Results: In current study the mean age of patients was 30.24+2.31years old, the median was 27 years old and M:F ratio was 1027:1. Current study revealed that LMP-1 expression was detected in 90% of Hodgkin’s lymphoma cases versus 60% of control subjects. The majority of lymphoma cases showed strong LMP-1 expression (72%) whereas all positive control cases showed weak expression (60%). There was significant inverse correlation between LMP-1 expression and the age of the patients. So that all childhood HL cases were positive for LMP-1 antigen. Moreover the intensity of LMP-1 expression was significantly high in Mixed cellularity HL than other histological subtypes (p=0.028). On the other hand there was no statistical significant relations between the LMP-1 expression in HRS cells and the gender of the patients, site of lymph node biopsies and stage of the disease. In the current study the Bcl-2 expression was detected in 66% in lymphoma cases versus 80% of control subjects. Majority of lymphoma cases showed strong Bcl-2 expression (48%) and the rest showed moderate (16%) and weak expression of Bcl-2 (2%). Whereas 50% of control cases showed weak expression of Bcl-2 and 30% showed moderate expression. There was significant positive correlation between Bcl-2 expression and the age of the patients. Furthermore there was significant association between fractional area of Bcl-2 expression and the stage of the disease. Whereas there was no significant association between the Bcl-2 expression and the gender of the patients, site of lymph node biopsies, and histological subtypes of the disease. Finally, in current study there was no statistical significant relation between LMP-1 and Bcl-2 expression in HRS cells of Hodgkin’s lymphoma cases. Conclusions : 1. We may suggest a role for Epstein Barr virus in the pathogenesis of Hodgkin’s lymphoma. 2. The intensity of Bcl-2 immunostain was significantly stronger in lymphoma compared to control subjects (p=0.001). 3. The EBV antigen LMP-1 and Bcl-2 were independent markers although the two biological markers act on the same pathway. 4. The use of Automated Cellular Image Analysis System to quantitate immunohistochemical staining (Digmizer software ) is easy to use and flexible image analysis software and is simple to perform