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Next Article 
Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3157-3159
CONTROVERSIES IN HEMATOLOGY
HHV-8 Is Present in Multiple
Myeloma Patients
By
James R. Berenson and
Robert A. Vescio
From the Veterans Affairs West Los Angeles Medical Center and Jonsson
Comprehensive Cancer Center and Division of Hematology and Oncology,
University of California School of Medicine, Los Angeles, CA.
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ARTICLE |
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 marrow-derived 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
patients.18 Using PCR with multiple sets of HHV-8 primers,
we and others have failed to find HHV-8 in whole peripheral blood
mononuclear cells (PBMCs) from myeloma patients, except in a small
minority of cases.12,19 However, after enrichment of PBMCs
for cells bearing CD68 or CD83 surface markers that were present on the virally infected bone marrow stromal cells, we were able to detect virus in most of the 157 myeloma samples analyzed (70%) and one fourth
of the MGUS specimens, whereas only 1 of 63 normal subjects showed
viral presence.19 Other studies have failed to detect HHV-8
in peripheral blood samples enriched for dendritic cells by tissue
culture.20,21 However, these investigators have studied cells that lack the CD83 marker and bear other markers not present on
the virally infected cell type identified in our studies.
HHV-8 has previously been demonstrated to be present in sexual and
other body secretions,22 and antibodies to HHV-8 as well as
KS have been found with a higher prevalence among homosexual males with
a large number of sexual partners.23 Using the PCR assay
with the ORF26 primers, a similar study among family members of myeloma
patients was performed analyzing PBMCs enriched for CD68 or CD83.
Although approximately three fourths of the 36 myeloma patients studied
demonstrated HHV-8, only 2% (1/46) of the family members showed viral
presence in the enriched PBMCs.24 In these enriched PBMC
samples, PCR amplification of the KS330233 and vIRF primers
gave identical results. These results suggest either that HHV-8 is
absent in this group at risk for viral exposure or that the level is below the level of sensitivity of this PCR-based assay.
Finally, these findings make PCR artifact highly unlikely as an
explanation for our detection of HHV-8 in myeloma patients, because
studies of patients (mostly HHV-8 positive) and their family members
(consistently HHV-8 negative) were performed concurrently in a blinded fashion.
Some studies have shown interpatient differences in the sequence of
HHV-8 from ORF26.25 Sequencing bidirectionally using multiple primer pairs specific for several HHV-8-specific ORFs from
myeloma patients has recently been completed.26 First, PCR-amplified products from ORF26 using the KS330233
primers were sequenced from three different tissue sources (long-term
cultures of adherent bone marrow stromal cells, PBMCs enriched for
dendritic cells, and bone marrow core biopsies) from 4 patients.
Although interpatient differences existed, these three tissues showed
identical ORF26 sequences in the same patient except for a single
basepair substitution in only 1 sample. Further evaluation of single
tissue sources from an additional 12 patients' ORF26 segment showed
interpatient differences. In addition, compared with those sequences
derived from both HHV-8-infected lymphomas and KS tissues, ORF26
derived from myeloma patients showed specific consistent changes. A
similar analysis of ORF65 also showed interpatient differences as well as consistent changes compared with the sequence obtained from lymphoma
and KS tissues. These findings make PCR artifact unlikely as an
explanation for the amplified products produced using HHV-8 primers in
myeloma samples. Moreover, because this latter ORF is responsible for a
major part of the serological response to HHV-8, deletion of a basepair
likely resulting in changes in the resulting protein product may help
explain the lack27,28 or low level29
serological response to this virus observed in myeloma patients.
Thus, our studies confirm the presence of HHV-8 in the vast majority of
myeloma patients and suggest that specific viral products may
contribute to the pathogenesis of this disease. Attempts to detect this
virus must rely on highly sensitive PCR techniques using samples that
are known to contain higher levels of HHV-8 in these patients,
including bone marrow biopsies and peripheral blood enriched for
markers known to be present on the virally infected dendritic cells in
these patients' bone marrow. A major objective is to define the role
of this virus in the pathogenesis of this B-cell malignancy.
| |
FOOTNOTES |
Submitted August 19, 1998; accepted February 10, 1999.
Address reprint requests to James R. Berenson, MD, 111H, WLA VAMC,
11301 Wilshire Blvd, Los Angeles, CA 90073; e-mail:
BERENSON.JAMES{at}WEST-LA.VA.GOV.
| |
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