Primary effusion lymphoma: cytopathologic diagnosis using in situ molecular genetic analysis for human herpesvirus 8

Primary effusion lymphoma is a form of diffuse large B-cell lymphoma with neoplastic cells largely limited to proliferation within major body cavities. Human herpes virus-8 is both integral to and required for an unequivocal diagnosis of primary effusion lymphoma. Prior methods for virus identification include DNA extraction with Southern blot analysis or in situ hybridization from paraffin-embedded samples. Our aim is to examine the utility of human herpesvirus-8 identification performed directly on smears from effusion samples by reverse transcriptase in situ polymerase chain reaction in patients with primary effusion lymphoma. Smears and cell block of body cavity fluids from five patients with effusions (three pleural, one peritoneal, and one both pleural and peritoneal) were examined microscopically by conventional Papanicolaou and Romanowsky (Diff-Quik) staining, and by reverse transcriptase in situ polymerase chain reaction for human herpesvirus-8 detection. In situ hybridization was performed also for Epstein-Barr virus (EBER-1, -2), T-cell receptor-beta, and kappa (kappa) and lambda (lambda) mRNA in all cases. Five adults ranged from 40-81 years of age. Three adults were HIV positive, one was a renal transplant recipient, and the oldest patient (Case 3) had the unusual distinction of a normal immune status. Two of three HIV-seropositive patients had concurrent Kaposi sarcoma. All samples were cytologically similar with lymphocytes having large-cell, plasmablastic, and immunoblastic morphology. Malignant cells from effusions were as follows: human herpesvirus-8 positive (all five cases), exhibited kappa monoclonal light chain (five cases), Epstein-Barr virus positive (three cases), and T-cell beta-gene receptor positive (two cases). Diffuse large B-cell lymphoma was evident in one peritoneal nodule (< 10% human herpesvirus-8 positive cells in contrast to > 90% positive in effusions, all kappa positive). Six other tissue specimens (lung, bone marrow, spleen, lymph node) were human herpesvirus-8 negative, and showed no evidence of lymphoma. Reverse transcriptase in situ polymerase chain reaction demonstrated near-complete restriction of human herpesvirus-8-infected malignant lymphoid cells to those in body cavities. Definitive diagnosis of primary effusion lymphoma is possible directly from cytologic smears/cell block by combining cytologic morphology with reverse transcriptase in situ polymerase chain reaction detection of human herpesvirus-8.