Guidelines for Interpreting EBER In Situ Hybridization and LMP1 Immunohistochemical Tests for Detecting Epstein-Barr Virus in Hodgkin Lymphoma

Histochemical stains demonstrate Epstein-Barr virus (EBV) in approximately 40% of all Hodgkin hymphomas, suggesting a role in tumorigenesis and the potentialfor EBV-targeted therapy. As research progresses, it is important to define criteria for interpreting histochemical stains. Four hematopathologists independently interpreted EBV-encoded RNA (EBER) and latent membrane protein 1 (LMP1) histochemical stains from 40 cases of Hodgkin lymphoma and then reviewed the stains as a group to resolve discrepancies and to develop interpretation guidelines. To call a Hodgkin case EBV-related, the EBER and/or LMP1 signal must be unequivocally present in Reed-Sternberg/Hodgkin (RS/H) cells. The cytologic features and distribution of stained cells should be matched with those on the corresponding HE-stained slide to help interpret whether the EBER or LMP1 signal is in malignant or reactive cells. The EBER signal is localized to the nucleus, whereas LMP1 is in the cytoplasm and surface membrane. In some cases, only a fraction of RS/H cells express these factors for technical or biologic reasons. Before calling a case EBER-negative, it is essential to show that tumor cell RNA is preserved and available for hybridization. LMP1 staining, although usually strong among all tumor cells in a given case, may alternatively be focal and weak, contributing to false-negative interpretation. EBER and LMP1 assays in combination are more effective than either assay alone for identifying EBV-related Hodgkin lymphoma.