Epstein-Barr virus (EBV)  is a ubiquitous human γ-herpes virus infecting more than 90% of the population worldwide  and plays a significant role as a cofactor in the process of tumorogenesis. It has consistently been associated with a variety of malignancies including endemic Burkitt's lymphoma (BL) [1, 2], nasopharyngeal carcinoma [3, 4], T-cell lymphoma, Pyothorax-associated or methotrexate-associated B-cell lymphoma, Primary effusion lymphoma, gastric carcinoma , EBV associated hemophagocytic syndrome and approximately 50% of Hodgkin's diseases. Moreover it has been associated with different types of lymphoproliferative diseases especially in immunocompromised patients . In immunocompromised patients with impaired cell mediated immunity, acute EBV infection is associated with the development of lymphoproliferative disease, with mortality rates between 50-80%. In addition to that, recipients of solid organ transplants, the incidence of post transplant lymphoproliferative disease (PTLD) ranges from 1% to 15% . Prevalence of EBV in Pakistani Burkitt's lymphoma patients is 80% which is significantly higher than in BL in North America .
EBV replicates in the epithelial cells of the mouth, tongue, salivary glands, and oral cavity and then it spread into the B-cells which are the main host cell type for its latent infection . Upon entry into B cell, the EBV proteins are expressed in a cascade manner. Every EBV-transformed cell carries multiple extrachromosomal copies of the viral episome and constitutively expresses a limited set of viral gene products referred to as latent proteins, which comprise of: Six (6) EBV nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C and -LP) [9–11], three latent membrane proteins (LMPs 1, 2A and 2B) and transcripts from the BamHIA region of the viral genome namely BART transcripts [11, 12]. In addition to the latent proteins, EBV-transformed cell also show abundant expression of the small, non-polyadenylated non-coding RNAs, EBER1 and EBER2. This pattern of latent EBV gene expression, which appears to be activated only in Burkitt's lymphoma, is referred to as 'latency I' [9–13]. Interleukin-10 (IL-10) an anti-inflammatory cytokine (also known as human Cytokine Synthesis Inhibitory Factor (CSIF)), is known to be an important regulator in cell transformation . EBV+ (positive) Burkitt's lymphoma (BL) cells express high level of IL-10 as compared to the EBV- (negative) BL cell lines . On the other hand, the expressional levels of IL-10 are extremely low and/or negligible in normal tumour cell lines [15, 16]. Approximately 10 fold increased expression of the IL-10 in Burkitt's lymphoma cell lines is reported . These elevated levels of EBER and IL-10 have also been known to increase cell's tumorigenicity [17, 18]. This expression profile could be used as power full tool in the expressional profiling studies as well as relative quantification of EBERs in the tissue samples.
For tissue preservation, formalin fixation followed by paraffin wax embedding is mostly used to maintain the morphological features of the original tissue . The Formalin Fixed and Paraffin Embedded (FFPE) tissues are used in various immunohistochemistry (IHC) techniques for localizing Epstein-Barr viral nucleic acid and/or protein to the tumour cells. Previously EBER in-situ hybridization (ISH) was considered as the most excellent test for detection and localization of latent EBV in tissue samples [20, 21]. Other techniques including automated EBER ISH and automated LMP-1 IHC with increased sensitivity and specificity have recently became useful diagnosis tool for EBV related diseases [22–24].
ISH reagents associated limitations like limited target site accessibility and capacity to penetrate into whole mount tissue specimens confines its reliability . Although, both techniques (fluorescent in-situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR)) have inevitable advantages in the field of diagnostics but comparative evidence of more target specificity and sensitivity of RT-PCR for the detection of minimal residual disease in long-term monitoring of patients has increased its importance in some applications . In addition to that quantitative real time PCR which has ability to detect as low as two fold changes in the gene expression, is rapidly replacing immunological assays [27, 28]. Numerous therapeutic agents aiming EBV associated tumour regression are beginning to emerge [29, 30]. These targeted drug delivery systems induce viral lytic gene expression [30, 31] and therefore, require more sensitive quantitative analysis of viral as well as cellular gene expression to monitor the effect of these therapeutic treatments.
This study was aimed to develop a precise Real time PCR based laboratory methodology to detect and quantify EBV encoded RNAs in FFPE Burkit's lymphoma samples of Pakistani origin. Furthermore, anti-inflammatory cytokine (IL-10) expression in EBV+ (positive) samples were characterized to indicate its relevance with viral persistence in tumour cells. These studies will introduce novel potential biomarkers as well as new insight in the current diagnostic trends of Pakistan.