Absence Of Human Herpesvirus 8 Dna Sequences In Leucapheresis Products And Bone Marrow Samples Of Patients With Advanced Multiple Myeloma
2000-06-01

We read with interest the paper by 3 demonstrating that human herpesvirus 8 (HHV-8) DNA was found to be absent in 23 different leucapheresis products from 22 multiple myeloma patients. These findings are in agreement with the results of some other recently published studies demonstrating leucapheresis products to be negative for HHV-8 DNA ( 1; 52). Thus, the presence of HHV-8 in leucapheresis products of myeloma patients as reported by 6 has not been confirmed by several other investigators. We examined the leucapheresis products of 30 multiple myeloma patients for the presence of HHV-8 DNA sequences by nested polymerase chain reaction (PCR) in our hospital. There were 19 men and 11 women with a median age of 53 years (range 40–73 years). All patients exhibited active disease and were classified as Durie and Salmon stage II or III (five patients stage IIA, one IIB, 19 IIIA and five IIIB). The isotype distribution was IgG in 17 patients, IgA in 10, light chain in two and one patient suffered from a non-secretory multiple myeloma. Primers for nested DNA PCR were derived from the highly conserved open reading frame (ORF) 26 of HHV-8 (outer primers: KS-802 5′-cggactacatccaaattatgcag-3′ and KS-1263 5′-ggtacatggacagatcgtcaag-3′; inner primers: KS-983 5′-ttttagccgaaaggattccacc-3′ and KS-1221 5′-gatccgtgttgtctacgtccag-3′). The detection limit of our assay was 0·8 fg DNA or 15 copies of a 15-kbp HHV-8 genomic fragment cloned into a phage lambda-derived vector in the presence of 250 ng genomic cellular DNA. No cross-reactivity was observed with clinical isolates of other herpesviruses (HSV-1, HSV-2, VZV, HCMV, EBV, HHV-6). Thus, our assay was highly specific and sensitive for the detection of HHV-8 DNA sequences in clinical samples. Using 250 ng of DNA isolated from leucapheresis products (equivalent to about 37 500 cells), we failed to detect HHV-8-specific DNA in any of our myeloma patients. As the virus has also been identified in the bone marrow from myeloma patients ( 4), bone marrow samples from 16 multiple myeloma patients were also examined by PCR (12 men and four women with a median age of 51 years, range 36–63 years). All patients presented with active disease according to Durie and Salmon (two patients with stage IIA, 11 with IIIA and three with IIIB). The isotype distribution was IgG in eight patients, IgA in five and light chain in three patients. Again, none of them tested positive for HHV-8 DNA in our assay. As negative PCR results might be due to unspecific inhibitors of Taq polymerase, samples were evaluated for inhibitory effects. β-Globin sequences were successfully amplified from all of the samples, indicating that the lack of HHV-8 detection was not due to the presence of inhibitors. Although our PCR assay seemed to be slightly less sensitive than the assay used by 3, our results further support the observation that HHV-8 is not present in leucapheresis products from multiple myeloma patients undergoing stem cell collection for autologous transplantation. The issue of whether or not apheresis cells of myeloma patients harbour HHV-8 DNA is of great importance as dendritic cells derived from leucapheresis products and pulsed with monoclonal immunoglobulin are used for immunotherapy in myeloma patients. Furthermore, if leucapheresis products used for autologous transplantation were infected with HHV-8, current therapeutic concepts for the treatment of multiple myeloma would have to be re-evaluated because viral interleukin 6 has been shown to be functionally active on human myeloma cells by stimulating their growth in vitro. For these reasons, it seems to be necessary to clarify further the discrepant results observed in this area. Local differences in the HHV-8 seroprevalence of myeloma patients as well as technical differences have been mentioned as possible explanations. In summary, we were able to confirm the results described by 3 by screening leucapheresis products from multiple myeloma patients for HHV-8 DNA sequences by nested PCR. In addition, HHV-8 DNA was also not detectable in 16 bone marrow samples. Thus, at the moment, the evidence of HHV-8 infection in multiple myeloma is not sufficient to warrant a critical re-evaluation of the use of apheresis cells for the treatment of multiple myeloma patients. We gratefully acknowledge the excellent technical assistance of Ina Westdörp, Department of Medical Virology, Tübingen, Germany.