Blood, 15 September 2000, Vol. 96, No. 6, pp. 2304-2306
BRIEF REPORT
Lack of serologic association of human herpesvirus-8
(KSHV) in patients with monoclonal gammopathy of
undetermined significance with and without progression to
multiple myeloma
Dharam V. Ablashi,
Louise Chatlynne,
David Thomas,
Dimitra Bourboulia,
Matthew B. Rettig,
Robert A. Vescio,
Dimitri Viza,
Parkash Gill,
Robert A. Kyle,
James R. Berenson, and
James E. Whitman Jr
From Advanced Biotechnologies Inc, Columbia, MD;
Georgetown University School of Medicine, Washington, DC; Departments
of Oncology and Molecular Pathology, Windeyer Institute of Medical
Science, University College London, United Kingdom;
Division of Hematology and Oncology, Veterans Affairs, West Los Angeles
Medical Center and University of California/Los Angeles, Los Angeles,
CA; Laboratoire d'immunologie, Faculté de Medicine,
Saints Peres, Paris, France; Division of Hematology,
Department of Medicine, University of Southern California, Los Angeles,
CA; and Division of Hematology, Mayo Clinic, Rochester, MN.
 |
Abstract |
Because human herpesvirus-8 (HHV-8) DNA has been found in multiple
myeloma (MM) patients by polymerase chain reaction, it was suggested
that HHV-8 may play a role in the transformation of monoclonal
gammopathy of undetermined significance (MGUS) to MM. Therefore, 362 MGUS sera with and without progression to MM were tested for IgG
antibody to HHV-8. Only 7.8% of the MGUS sera contained HHV-8 antibody
to lytic proteins, and IgG antibody to HHV-8 latent antigen was even
lower than lytic antibody (2.9%). No differences were observed in the
distribution of antibody to HHV-8 in sera from MGUS patients who
progressed to MM. The seroprevalences of HHV-8 in MGUS (7.8%),
MM (5.4%), and healthy donors (5.9%) were similar, thus arguing for
the lack of epidemiologic evidence of HHV-8 participation in the
pathogenesis of MM. MGUS patients were immune competent in response to
Epstein-Barr virus (EBV) infection because 97% contained antibody to
EBV virus capsid antigen.
(Blood. 2000;96:2304-2306)
© 2000 by The American Society of Hematology.
| |
Introduction |
After Rettig et al1 presented
evidence that human herpesvirus (HHV)-8 DNA is present in the
bone marrow dendritic cells of patients with multiple myeloma (MM),
other reports quickly followed confirming2-10 or refuting
the association of HHV-8 with MM, using polymerase chain reaction (PCR)
techniques with various HHV-8 primers.11-16 In addition, a
few reports also showed that MM patients lack antibody to
HHV-8.11-14,16-20 Gao et al,21
however, did find HHV-8 IgG antibody in MM patients by latent nuclear
antigen (LNA) immunoblot in 81% of the cases. The HHV-8
antibody data in this report were not convincing enough to establish a
serologic relation of MM with HHV-8.14 MM patients often
have antibodies to either 
-herpesvirus (Epstein-Barr virus [EBV])
or 
-herpesviruses (cytomegalovirus, HHV-6, HHV-7), which supports
the hypothesis that MM patients have no defect in their immunologic
syste ms.11-13,16,17,19
One fourth of patients with monoclonal gammopathy of
undetermined significance (MGUS) develop MM or related serious B-cell disorders. Because Rettig et al1 suggested that HHV-8
(also called KSHV [Kaposi sarcoma-associated herpesvirus])
may be required for the transformation from MGUS to MM and for
perpetuation of the growth of malignant plasma cells, we tested sera
from 362 MGUS patients and 110 MM patients. To make sure our serologic assays were able to detect accurately IgG antibody to HHV-8, we used a
whole-virus enzyme-linked immunosorbent assay (ELISA), latent
antinuclear antigen immunofluorescence assay (LANA IFA), and an IFA to
lytic antigens. To see whether MGUS patients were immunocompetent, we
also tested study sera for the presence of EBV virus capsid antigen
(VCA) antibody.
| |
Study design |
MGUS sera were collected and frozen at 
20°C by Drs M. B. Rettig, J. R. Berenson, and R. A. Kyle. Samples of MM,
Kaposi sarcoma/human immunodeficiency virus-1 (KS/HIV), and
classic KS were obtained frozen from Drs M. Kaplan, A. Friedman-Kien,
D. Viza, and P. Gill. Donor sera were from a well-characterized
panel used at Advanced Biotechnologies Inc (Columbia, MD). All
sera were screened for antibodies to lytic proteins by the
whole-virus lysate ELISA kit (Advanced Biotechnologies
Inc).22 Positive sera were confirmed by IFA for lytic
HHV-8 antigens.22 D. Bourboulia did the testing for IgG
antibody to LANA by the IFA method of Simpson et al23 in
the laboratory of Dr C. Boshoff in London. The EBV-VCA ELISA was done
using native gp125 as a source of antigen.22 All samples were coded before testing.
| |
Results and discussion |
Table 1 shows the
seroprevalence of antibodies to HHV-8 lytic proteins for MGUS, MM, KS,
and healthy controls. Twenty-six of 362 MGUS sera (7.1%), 6 of 110 MM
sera (5.4%), 20 of 20 KS sera (100%), and 6 of 102 sera of healthy
blood donors (5.9%) were positive by ELISA for IgG antibodies to HHV-8
antigen. For confirmation, the positive samples were retested by IFA,
and all tested positive except for 2 of the MGUS sera and 2 of the
donor sera (Table 2). These results show
that the prevalence of antibody to lytic HHV-8 proteins in MGUS and MM
patients is similar to that observed in the healthy donor population
(Table 1). To ensure that we were not missing patients who had antibody
only to latent proteins, we also used the LANA IFA to test randomly
picked MGUS sera (95), MM sera (65), KS sera (20), and donor sera (50).
For the samples tested, 3 MGUS (3.2%), 3 MM (4.6%), 18 KS (90%), and 2 healthy donors (4.0%) were LANA positive. The prevalence of antibody
to the LANA in MGUS and MM is even lower than that of antibody
to lytic proteins (Table 1). Analysis of HHV-8 antibody status in MGUS
patients with and without progression to MM indicated no change in
seroprevalence (Table 3). This argues
against any role for HHV-8 in the transformation or pathogenicity of
MGUS to MM.
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Table 1.
Distribution of HHV-8 (KSHV) IgG antibody in sera from
MGUS, MM, KS, and healthy donors detected by whole-virus lysate ELISA
and by IFA to latent antigens
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Table 2.
MGUS, MM, KS, and healthy donors testing positive by HHV-8
whole-virus lysate ELISA and then tested by IFA to lytic antigens
for confirmation
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These data are consistent with other antibody studies done
largely on MM populations showing a lack of epidemiologic evidence for
HHV-8 involvement in MM.11,12,16,17,19,20 Chauhan et al5 reported the analysis of 53 MM sera and found no
antibody to latent or lytic proteins of HHV-8; they did, however, find HHV-8 DNA by PCR in the MM tissues. Whitby et al17 also
failed to detect HHV-8 antibody in 4 MGUS patients who subsequently
developed overt MM. Two other MGUS sera in their study were antibody
positive but the patients had no evidence of MM after 36 and 40 months, respectively. The only study reporting the presence of HHV-8 antibodies in MM patients is by Gao et al.21 Using an immunoblot
method instead of IFA,16,19 they reported the
detection of antibodies to ORF-64 and LNA antigens.21 With
the use of IFA, one can observe the difference in staining patterns
between nonspecific and specific reactivity, but with the immunoblot
method, it is possible to score only positive and negative results. If
any nonspecific reactivity survived the blocking step, it would appear
on the immunoblot as a positive result. This difference in technique
may account for the discrepancy in results with other researchers.
Therefore, on the strength of our data and that of the other
researchers mentioned earlier, we conclude that it is
extremely doubtful that HHV-8 has a role in the progression to MM.
To establish that MGUS patients are competent to make antibody to
herpesviruses, we also tested the same 95 MGUS sera for IgG antibody to
EBV-VCA using a native EBV (gp125 protein) ELISA. Ninety-two of 95 MGUS
sera tested (96.8%) were positive for EBV-VCA antibody, demonstrating
that MGUS patients were immunocompetent and responded to EBV infection
and other human herpesviruses, as shown previously in MM
patients.12,17,19 Both EBV and HHV-8 are classified as
human -herpesviruses.
The work done by Rettig et al1 was based largely on
PCR HHV-8 DNA data, as opposed to the present report, which is a study of serologic antibodies. It is interesting to note that in studies done
to compare the frequency of PCR data with that of
serology,24,25 the percentage of
HIV-1+/KS+ patients positive for HHV-8 DNA in
peripheral blood mononuclear cells by PCR is much lower than the
percentage found positive for HHV-8 IgG antibodies by IFA for lytic
proteins. It is therefore curious that Rettig et al1 could
find cells positive for HHV-8 DNA by PCR, but could not detect HHV-8
antibodies in these patients. Berenson and Vescio2
attributed the lack of serologic responses to HHV-8 in MM to
interpatient differences as well as to consistent changes in ORF-65
sequencing derived from MM as compared to primary effusion B-cell
lymphoma and KS patients. They argued that because ORF-65 is
responsible for a major part of the serologic response to HHV-8,
deletion of a base pair likely results in a change in the protein
product, and this may explain the lack of or low level of serologic
response to HHV-8 in MM patients.2 Although such deletions
may cause a change in the function of the protein, it is unlikely that
they would cause much change in antigenic response.
In light of this mounting molecular evidence based on HHV-8 data and
the fact that these same patients lack HHV-8 antibody, MM patients may
carry another herpesvirus yet to be identified. This theoretical
herpesvirus could share common sequences with HHV-8 but be
immunologically distinct from HHV-8.22,26 In much the same
way, HHV-8 and EBV share homologous DNA sequences,26 but
lack serologic cross reactivity.22
| |
Acknowledgments |
We thank Ms Ruth Weinroth, Ms Alice Long, and Ms Kelly Lehr for
their assistance in the preparation of this report.
| |
Footnotes |
Submitted January 5, 2000; accepted May 12, 2000.
Supported in part by grant CA62242 from the National
Institutes of Health.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Dharam V. Ablashi, Advanced Biotechnologies Inc,
9108 Guilford Road, Columbia, MD 21046-2701; e-mail:
dablashi{at}abionline.com.
| |
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Abstract
Introduction
