Epstein-Barr virus (EBV) detection and typing by PCR: a contribution to diagnostic screening of EBV-positive Burkitt's lymphoma

Abstract

Background: Epstein-Barr virus (EBV) is associated to the etio-pathogenesis of an increasing number of tumors. Detection of EBV in pathology samples is relevant since its high prevalence in some cancers makes the virus a promising target of specific therapies. RNA in situ hybridization (RISH) is the standard diagnostic procedure, while polymerase chain reaction (PCR)-based methods are used for strain (EBV type-1 or 2) distinction. We performed a systematic comparison between RISH and PCR for EBV detection, in a group of childhood B-cell Non-Hodgkin lymphomas (NHL), aiming to validate PCR as a first, rapid method for the diagnosis of EBV-associated B-cell NHL.

Methods: EBV infection was investigated in formalin fixed paraffin-embedded tumor samples of 41 children with B-cell NHL, including 35 Burkitt's lymphoma (BL), from Rio de Janeiro, Brazil, by in situ hybridization of EBV-encoded small RNA (EBER-RISH) and PCR assays based on EBNA2 amplification.

Results: EBV genomes were detected in 68% of all NHL. Type 1 and 2 accounted for 80% and 20% of EBV infection, respectively. PCR and RISH were highly concordant (95%), as well as single- and nested-PCR results, allowing the use of a single PCR round for diagnostic purposes. PCR assays showed a sensitivity and specificity of 96% and 100%, respectively, with a detection level of 1 EBV genome in 5,000-10,000 EBV-negative cells, excluding the possibility of detecting low-number EBV-bearing memory cells.

Conclusion: We describe adequate PCR conditions with similar sensitivity and reliability to RISH, to be used for EBV diagnostic screening in high grade B-NHL, in "at risk" geographic regions.

Figure 1

Molecular analysis of EBV-positive and negative Non-Hodgkin lymphomas. (A) Nested-PCR EBV genotyping. Expected sizes of nested PCR products were 250 bp (type-1) and 300 bp (type-2), amplified from an 801 bp fragment obtained in the first PCR reaction. Lane 1: Type-1 positive control (Raji cell line); lanes 2–3: EBV-positive type-1 patients; lane 4: Type-2 positive control (BC1 cell line); lane 5: EBV-positive type-2 patient; lane 6: EBV negative patient; lane 7: negative control (Ramos cell line); lane 8: PCR control (without DNA). (B) DNA amplification testing by multiplex PCR of constitutive β-globin, β-actin and Glyceraldehide-3 phosphate dehydrogenase genes (from bottom to top) corresponding to patients and controls in A. 2.5% agarose gel stained with ethidium bromide. M: molecular weight marker (100 bp ladder).

Figure 2

Sensitivity assays. Namalwa cells (2 EBV genome per cell) were serially diluted in the EBV-negative cells of Ramos cell line. PCR results showed that the method is able to detect 1 EBV genome in a background of 5 × 10³ (first reaction) (A) and 1 × 10⁴ negative cells (nested PCR) (B). 2.5% agarose gel stained with ethidium bromide.