Expression profiling of Epstein-Barr virus-encoded microRNAs from paraffin-embedded formalin-fixed primary Epstein-Barr virus-positive B-cell lymphoma samples

Epstein-Barr virus (EBV) is implicated in a range of B-cell malignancies and expresses unique microRNAs (EBV-miRNAs). Due to the requirements for high-quality RNA, studies profiling EBV-miRNA in EBV-positive lymphomas have been restricted to cell-lines or frozen samples. However, the most commonly available archived patient material is paraffin-embedded formalin-fixed (FFPE) tissue. This has impeded the widespread profiling of EBV-miRNA expression in clinical samples. The requirements for accurate EBV-miRNA real-time RT-PCR quantitation in FFPE tissues representing a broad-spectrum of EBV-positive lymphomas were determined systematically, including where the neoplastic cells are sparse relative to the non-malignant infiltrate. The level of cellular EBV-load correlated strongly with the sum of EBV-miRNA expression and the number of EBV-miRNAs detectable. As calibrators for cellular EBV-load, the sum EBV-miRNA was optimal to EBV-genome copy number and EBER2 expression level, with the added advantage of not requiring additional assays. EBV-miRNA was profiled reliably within archival FFPE tissue in 14/23 patients, but not in tissues with low abundance EBV. This method enabled specific and simultaneous detection of numerous EBV-miRNAs in FFPE lymphoma samples that contain EBV at high to medium levels, making it as a useful tool for studies of EBV-miRNA in the majority of diagnostic biopsies.

► A novel method for EBV-microRNA real-time RT-PCR quantitation in formalin-fixed paraffin-embedded tissues is described.
► Requirements for samples with sparse neoplastic cells were determined.
► A broad-spectrum of Epstein-Barr virus-positive formalin-fixed paraffin-embedded lymphoma samples was used.