Blood 93/10

IN MYELOMA, THE ROLE of the bone marrow microenvironment has been shown to be of increasing importance in supporting the malignant plasma cell.1,2 Specifically, nonmalignant stromal cells in the bone marrow from myeloma patients have been shown to promote the growth and prevent the apoptosis of malignant plasma cells largely by the production of interleukin-6 (IL-6).1-3 Recently, a new member of the human herpesvirus family, HHV-8, was discovered in a case of Kaposi’s sarcoma (KS)4 and was also found to be present in patients with a rare form of B-cell lymphoma, primary effusion lymphoma, and multicentric Castleman’s disease.5,6 We have recently found this virus associated with multiple myeloma.7 In the acquired immunodeficiency syndrome (AIDS) population in whom KS and HHV-8 infection are relatively common, a higher risk of multiple myeloma has been also observed in a recently published study.8 In further support of the connection between this new virus and multiple myeloma, HHV-8 was found to encode an IL-6 homologue that was capable of stimulating growth and preventing apoptosis of murine9 and human myeloma cell lines.10 Previously, our laboratory had demonstrated the presence of HHV-8 in the adherent nonmalignant cell population from long-term cultures of bone marrow from myeloma patients.7 Moreover, approximately one fourth of patients with monoclonal gammopathy of undetermined significance (MGUS) also demonstrated virus in these bone marrowderived dendritic cells. Further characterization of the virally infected bone marrow stromal population showed dendritic cell features with a specific immunophenotype. Although some groups have been unable to detect virus in stromal cell populations derived from myeloma bone marrow, the phenotype of the cells generated in these cultures is unknown11 and could influence the results (see below). In our initial study, we could not detect HHV-8 in the fresh aspirate from myeloma patients, although this virus was readily detected after long-term culture. Others have also recently reported the absence of HHV-8 using polymerase chain reaction (PCR)-based techniques on fresh marrow aspirates from these patients.11,12 Some of these studies also did not detect this virus in myeloma cell lines, consistent with our identification of the nonmalignant dendritic cell as the HHV-8–containing population in these patients.10 Because the infected cells were initially found only in long-term cultures and not in the fresh bone marrow aspirates, further studies were performed on fresh bone marrow biopsies using both PCR and in situ hybridization techniques with HHV-8–specific primers and probes, respectively.13 In studies of nearly 50 myeloma patients, viral presence could be demonstrated using these techniques in myeloma bone marrow biopsy samples from 80% of these cases, whereas biopsies from patients with lymphomas, other cancers infiltrating the bone marrow, and normal subjects did not contain HHV-8. A French group confirmed the presence of HHV-8 in the bone marrow biopsies derived from the majority of the myeloma patients evaluated using the KS330233 primers, whereas it was not detected in any of the normal subjects’ bone marrow biopsies.14,15 Recent work has also shown the presence of HHV-8 in 80% of Turkish myeloma bone marrow specimens and none of the control samples.16 The presence of virus has now been confirmed using multiple primer pairs from different open reading frames (ORFs) of HHV-8 (ORF26, ORF65, ORF74, v-MIP-1, v-IRF, v-IL-6, T1.1, v-bcl-2, and v-cyclin D). In these studies, each of these HHV-8 primer pairs was optimized for temperature and buffer conditions. Importantly, without optimizing these conditions, the primers may be as much as three logs less sensitive to detect HHV-8. In addition, even after optimization of individual primer pairs, there are marked differences in sensitivity of primers from different HHV-8 ORFs. To further confirm the presence of HHV-8 in myeloma bone marrow, reverse transcription-PCR (RT-PCR) using RNA from fresh marrow biopsies has shown consistent expression of two HHV-8 transforming genes, vIRF and ORF74, in myeloma but not normal subjects’ specimens, which was confirmed by sequencing the amplified product.17 HHV-8 can be detected in the peripheral blood of most KS