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Keywords:

  • multiple myeloma;
  • Waldenström macroglobulinemia;
  • non-Hodgkin's lymphoma;
  • lymphotropic herpesviruses;
  • HHV-8

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

It has been suggested that human herpesvirus 8 (HHV-8), also known as KSHV (Kaposi's sarcoma-associated human herpesvirus), might possess a promoting effect in the development and progression of monoclonal gammopathies. In this study, the presence of Epstein-Barr virus (EBV), human cytomegalovirus (CMV), human herpesvirus 6 (HHV-6) and human herpesvirus 8 (HHV-8) were tested in patients with multiple myeloma (MM) using both serologic and nucleic acid amplification techniques. The transient reactivation or continuous presence of EBV, CMV, HHV-6 and HHV-8 could be detected in, respectively, 36, eight, 13 and 29 of 69 MM patients; nine, one, four and six of 16 monoclonal gammopathy of unknown significance patients; and seven, four, zero and five of 10 Waldenström's macroglobulinemia patients. The total number of MM patients was 95. HHV-8 PCR-positivity was significantly more frequent in the MM group than in the control group of patients with non-Hodgkin's lymphoma (NHL). However, serologic testing did not reveal significant differences between the two patient groups. The number of MM patients with concomitant herpesvirus infections as detected by PCR was as follows: 15 double, seven triple and two quadruple virus nucleic acid positive. In 13/95 MM patients, the simultaneous presence of acute EBV infection and HHV-8 PCR-positivity was detected compared with none of the control group (P=0.009). These results indicate that in addition to HHV-8, the transitional reactivation of EBV may also play a role in the pathogenesis of MM.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Human herpesvirus-8 (HHV-8) has been discovered in lesions of Kaposi's sarcoma (KS) patients and has already been proved to be the causative agent of both sporadic and HIV-associated forms of the disease (Chang et al., 1994; Corbellino et al., 1996). Latent infection of bone marrow stromal cells by HHV-8 has been implicated in the development of multiple myeloma (MM) as well as in the conversion of MGUS (monoclonal gammopathy of unknown significance) into full-blown myeloma (Parravicini et al., 1997; Ma et al., 2000; Hermouet et al., 2003; Fonseca et al., 2004). However, considerable controversy exists in the literature: data from different investigators either attempt to prove or refute this hypothesis. There is a noticeable dichotomy in the results published, certain groups consistently producing data to affirm and others to refute the hypothesis of viral involvement (Kikuta et al., 1997; Parravicini et al., 1997; Mikala et al., 1999; Leāo et al., 2002; Barillé-Nion et al., 2003; Fonseca et al., 2004).

One important question has not been raised in connection with the pathogenesis of gamma herpesviruses. Many of these viruses possess mechanisms to counteract the antiviral response exerted by the infected host at the level of cell–cell interaction (Chang et al., 1994; Foster-Cuevas et al., 2004), despite the ongoing replication of HHV-8 in endothelial and spindle (Boshoff et al., 1995), lymphoma (Renne et al., 1996) and dendritic cells (Said et al., 1997). The molecular nature of these regulatory processes are so heterogeneous, such as expression of interleukins and receptors (Chang et al., 1994; Burger et al., 1998) induced by other herpesviruses, that changes caused by regulatory or epigenetic modification of latently infected cells by other herpesviruses (Tsai et al., 2002; Kiss et al., 2003; Li & Minarovits, 2003; Salamon et al., 2003) or even by papovaviruses (Shivapurkar et al., 2004) might influence the latency or replication stage of these viruses, and therefore may possibly contribute to the pathogenic process of MM.

The aim of the current study was to test the simultaneous presence of four different lymphotropic herpesviruses in Hungarian MM patients, Waldenström's macroglobulinemia (WM) and MGUS. We are aware that molecular differences have been found in the HHV-8 viruses of MM and KS patients (Ma et al., 2000). The possible role of different herpesviruses, such as HHV-8, in the etiology and pathogenesis of monoclonal gammopathies has been addressed by several groups, yet the systematic complex search for involvement of multiple lymphotropic herpesviruses has not yet been discussed.

In this study serology and nested PCR techniques were used for the detection of the viral genomes and specific immunologic responses of Epstein-Barr virus (EBV also known as HHV-4), human cytomegalovirus (CMV or HHV-5), human herpesvirus-6 (HHV-6) and HHV-8 in bone-marrow aspirates and peripheral blood samples of all MM patients. Similar samples of patients diagnosed with different types of non-Hodgkin's lymphoma (NHL) were used as control (Corbellino et al., 1996). Several patients were sampled and tested multiple times during the course of their disease and repetitive viral serology was also carried out.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Samples

Patients' samples were taken after obtaining their informed consent. The study was approved by the Institutional Review Board of the National Medical Center and was done according to the principles of the Helsinki Convention. In total, 95 patients with monoclonal gammopathy were examined, of whom 16 had MGUS and 10 had WM. The number of gammopathy-negative controls with NHL was 44.

Bone-marrow aspirates and blood samples taken with anticoagulant [ethylenediaminetetraacetic acid (EDTA)] were processed for virological examinations. Samples were centrifuged at low speed at room temperature and the plasma and cells separated. Buffy coat cells were separated and stored at −20°C for further investigation or directly mixed with the lysis buffer and processed for DNA purification.

Cells and viruses used as positive controls

EBV DNA positive controls were prepared from B95-8 marmoset cells (ATCC: CRL 1612), and the AD-169 (ATCC: VR-538) strain of CMV was grown on primary human fibroblast cells (Varga et al., 1993). HHV-6-positive control DNA was prepared from commercial immunofluorescence slides purchased from Biotrin (Cat. no. V3HHV6). The freeze-dried spots of infected cells were dissolved with lysis buffer for 10 min [0.2 M Tris-HCl, pH 7.5, 25 mM EDTA, 0.3 M NaCl, 2% sodium dodecyl sulfate (SDS) and 20 μL proteinase (Proteinase K from Sigma)] at 37°C (25 μL per spot). The spots were scraped in order to improve deproteinization and the drops were collected with disposable tips for DNA isolation as described below. BCBL-1 cells were grown in RPMI-1640 completed with 2-mercaptoethanol (5 μM) and used for DNA isolation for HHV-8 control purposes or processed for histological control (Renne et al., 1996).

DNA isolation

Plasma, buffy coat and bone-marrow samples were mixed with lysis buffer [0.2 M Tris-HCl, pH 7.5, 25 mM EDTA, 0.3 M NaCl, 2% SDS and proteinase K (Sigma)] and incubated for 1.5 h at 37°C in a water bath. Deproteinization was done using Tris-buffered phenol and chloroform at ambient temperature. The aqueous phase was separated and precipitated by ethanol and incubated at −20°C overnight.

Following sedimentation of the DNA precipitate, samples were washed twice in 70% precooled ethanol, then dried for 1 h at room temperature. The DNA was redissolved in 20 μL nuclease-free distilled water and stored at −20°C until used further in PCR assays.

PCR

Four conventional and four nested (HHV-4, HHV-5, HHV-6, HHV-8) PCR assays were selected from the literature for our studies (Aubin et al., 1993; Chang et al., 1994; Mitchell et al., 1994; Lakeman & Whitley, 1995; Pozo & Tenorio, 1999; Markoulatos et al., 2000). PCR procedures were carried out following the instructions of the relevant authors. Table 1 details the characteristics of the PCR systems and target genes for the different herpesviruses studied.

Table 1.   Characteristics of the PCR systems as well as target genes for the different herpesviruses studied
VirusPCR typeSize of the product (bp)Target geneReferences
  1. EBV, Epstein-Barr virus; CMV, cytomegalovirus; HHV, human herpesvirus

HHV-4 (EBV)Simple171Large internal repeatMitchell et al. (1994)
Nested (first)194  
(second)54DNA polymerasePozo & Tenorio (1999)
HHV-5 (CMV)Simple150Glycoprotein BMitchell et al. (1994)
Nested (first)194  
(second)78DNA polymerasePozo & Tenorio (1999)
HHV-6Simple380Major capsid proteinAubin et al. (1993)
Nested (first)194  
(second)68DNA polymerasePozo & Tenorio (1999)
HHV-8Simple233Minor capsid proteinChang et al. (1994)
Nested (first)194  
(second)97DNA polymerasePozo & Tenorio (1999)

Detection of herpesvirus-specific antibodies

Measurement of the antibodies was carried out by using a Tetra-methil-benzidine (TMB)-based capture enzyme-linked immunosorbent assay technique (MEDAC Ltd), according to the manufacturer's protocol. Detection of HHV-6 antibodies was done using a HHV-6 IgG Enzyme immunoassay (EIA) kit (Biotrin International Ltd). Detection of HHV-8 antibodies was carried out as described previously (Juhász et al., 2001; Juhász et al., 2001).

Statistical analyses

The statistical data analyses were carried out with 2 × 2 contingency tables and Fischer's exact test. Differences were considered to be statistically significant at P≤0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The results of nested PCR and immunoglobulin G (IgG) serology for the different herpesviruses in the monoclonal gammopathies and control groups are shown in Tables 2 and 3, respectively. Patients included in our study were sampled at diagnosis and in 38/95 resampling was performed at subsequent relapses. At diagnosis 21/69 MM patients were at stage II and 48/69 patients at stage III, according to the staging system of Durie and Salmon. Twenty-three of the 69 MM patients were sampled multiple times. In our data analysis, a patient was considered positive for any given virus if at any time point during the disease course the virus could be detected. No patients were found in whom exclusively the first sample tested positive for HHV-8 DNA, but in 19 patients positivity appeared only at relapse. The number of PCR-positive MM patients was found to be 36 (EBV), eight (CMV), 13 (HHV-6) and 29 (HHV-8). The PCR results for the control (NHL) group were as following: the number of EBV-, CMV-, HHV-6- and HHV-8-positive patients was, 23 four, eight and one, respectively. The difference between the MM and the control groups was only significant for HHV-8 (P<0.0001).

Table 2.   Frequencies of EBV, CMV, HHV-6 and HHV-8 PCR-positivity in patients with multiple myeloma (MM), monoclonal gammopathy of unknown significance (MGUS) and Waldenström's macroglobulinemia (WM) compared with the control group
 nMMP vs. controlnMGUSP vs. controlnWMP vs. controlnControl (NHL)
Positive (n)Positive (%)Positive (n)Positive (%)Positive (n)Positive (%)Positive (n)Positive (%)
  1. Positive, no. of patients with nucleic acid of the respective virus.

  2. n, number of patients; NS, not significant; EBV, Epstein-Barr virus; CMV, cytomegalovirus; HHV, human herpesvirus.

EBV
PCR693652NS16956NS10770NS442352
CMV
PCR69812NS1616NS10440NS4449
HHV-6
PCR691319NS16425NS1000NS44818
HHV-8
PCR692942<0.000116638<0.000910550<0.00044412
Table 3.   Frequencies of EBV, CMV, HHV-6 and HHV-8 IgG antibodies in patients with multiple myeloma (MM), monoclonal gammopathy of unknown significance (MGUS) and Waldenström's macroglobulinaemia (WM) compared with the control group
 nMMP vs. controlnMGUSP vs. controlnWMP vs. controlnControl (NHL)
Positive (n)Positive (%)Positive (n)Positive (%)Positive (n)Positive (%)Positive (n)Positive (%)
  1. Positive, number of patients with IgG antibodies against the respective virus.

  2. n, number of patients; NS, not significant; EBV, Epstein-Barr virus; CMV, cytomegalovirus; HHV, human herpesvirus; IgG, immunoglobulin G.

EBV
IgG524790NS1212100NS8788NS272593
CMV
IgG524688NS121192NS88100NS271867
HHV-6
IgG521835NS12650NS8450NS271348
HHV-8
IgG5248NS1218NS800NS2600

Two small cohorts of patients with monoclonal gammopathies were also analysed. The number of PCR-positive MGUS patients were nine (EBV), one (CMV), four (HHV-6) and six (HHV-8). Four of the patients exhibited EBV IgM antibodies, indicating primary infection, and these patients were simultaneously positive for EBV and HHV-8 DNA. A small subgroup of 10 patients with WM was also examined. Nested PCR findings were seven (EBV), four (CMV), zero (HHV-6) and five (HHV-8) positive of 10 patients. One of the patients exhibited EBV IgM antibodies, indicating primary infection, and this patient was simultaneously positive for EBV and HHV-8 DNA. In the MGUS group, six of 16 patients were positive for HHV-8; five of 10 WM patients were found to be positive for this virus. These results were also significantly different from that of the control group.

Serological (IgG) results in the MM, MGUS and WM groups versus the control group did not reveal significant differences. In contrast to the 47 (of 52) EBV-seropositive MM patients, only four patients were found to carry antibodies specific to HHV-8. In addition, 46 of 52 and 18 of 52 patients with MM were seropositive to CMV and HHV-6, respectively.

Upon examining two small cohorts of MGUS and WM patients we detected seropositivity in MGUS patients (n=12) in the following numbers: 12 (EBV), 11 (CMV), six (HHV-6) and one (HHV-8). Result for the WM patients (n=8) were seven (EBV), eight (CMV), four (HHV-6) and zero (HHV-8).

The serological (IgG) findings in the control group for EBV, CMV, HHV-6 and HHV-8 were as follows: of 27 patients, 25, 18, 13 and zero were positive, respectively. The difference in seropositivity between EBV, CMV, HHV-6 and HHV-8 may indicate that the patients are immunotolerant to the antigens of HHV-8.

Eight of the MM patients exhibited EBV IgM antibodies, indicating primary infection, and these patients were simultaneously positive for EBV and HHV-8 DNA. These eight patients were all carrying HHV-8 DNA either in the plasma or in the plasma and/or buffy coat. These markers might indicate recent reactivation of the Kaposi sarcoma herpesvirus upon acute EBV. The number of MM patients with concomitant herpesvirus infections as detected by PCR, was as follows: 15 double, seven triple and two quadruple virus nucleic acid positive. In 13/95 total MM patients, the simultaneous presence of acute EBV infection and HHV-8 PCR-positivity was detected compared with none of the control group (P=0.009)

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The data presented here seem to support the findings of Fleckenstein and others (Burger et al., 1998) who originally suggested a possible etiological role of HHV-8 in the pathogenesis of MM. Although only approximately half of our patients (42%) exhibited PCR-positivity suggesting HHV-8 infection, this frequency is significantly higher than in the control NHL group. The frequency of HHV-8 seropositivity in the groups is similar to that reported previously for Hungarian blood donors (2001). The lack of anti-HHV-8 antibodies in MM patients, i.e. the significantly higher frequency of HHV-8 PCR-positivity compared with seropositivity, requires further analyses. As the patients examined in our study possess humoral antibodies to EBV, CMV and HHV-6, and less frequently against HHV-8, a possible hypothesis is that HHV-8 was transmitted perinatally and a subsequent immune tolerance developed. The normal antibody responses to other members of the herpesvirus family do not support the hypothesis of generalized immune dysfunction in MM patients, although a secondary hypogammaglobulinemia cannot be ruled out.

There is no well-accepted explanation of the dichotomy observed in the literature with respect to the relationship between HHV-8 and MM. Geographic differences in HHV-8 detectability among myeloma patients are theoretically possible, and this hypothesis could be conveniently tested by switching samples between the different laboratories.

It is tempting to speculate on the high, yet not overwhelming (40–50%) HHV-8 positivity detected in our myeloma patient cohort. One may argue that – given the bone-marrow stromal cell as the host of the HHV-8 replication – our test system involving bone-marrow aspirates is an unreliable source of these cells. However, direct culturing of differentiable adherent stromal cells was attempted in a separate study of eight myeloma bone-marrow aspirates and was successful in six of the cases (bacterial and fungal infection occurred in the remaining two cases – data not shown). This effectively rules out the lack of stromal cells in the aspirates as a explanation for the lack of HHV-8 detectability. Nevertheless, the small number of adherent stromal cells present in the aspirates highlights the need for a sensitive nested PCR technique to detect HHV-8 nucleic acid.

MM is a genetically and clinically heterogeneous disease. Recently, (at least) five prognostically and biologically different subgroups have been described based on karyotype and type of cyclin expression. HHV-8 infection may play a role in the disease process of only one or more subgroups of myeloma patients but not in others. Therefore, patient selection in previous and also in this study may influence the results and helps to explain the obvious differences obtained. Further studies are needed to clarify the situation.

In the history of HHV-8 research, it has been exceedingly difficult to obtain lymphoid cell lines (e.g. effusion lymphoma) devoid of the EBV genome (Renne et al., 1996; Said et al., 1997). This raises the issue of a possible interaction between these two lymphotropic herpesviruses, at least in diseases of the lymphoid system. A similar interaction of HHV-6 and −8 has been described in vitro. EBV-seronegative patients may be prone to HHV-8 reactivation. The molecular basis of this observation may be the possible interaction of EBV early proteins with the activation of latently harbored HHV-8 genomes. This might occur also when the two viruses are not replicating in the same cell, but viral membrane proteins, i.e. ZEBRA, or interleukin-like molecules (IL10) shed by the EBV-producing B cells might interact with B cells or bone marrow cells carrying latent HHV-8 minichromosomes.

Our present observations may indicate a possible novel viral factor associated with MM. It may be possible that the continuously replicating myeloma cells release both EBV and HHV-8 as latent virus-infected cells are usually blocked in the G0–G1 phases of the cell cycle. When myeloma cells enter the S-phase, the availability of the DNA replication machinery may activate the DNA viruses persisting in a small proportion of cells. The absence of CMV reactivation can be understood given that the sites of latency are the CD34+ progenitor cells of monocytes. The main site of replication is the B lymphocytes and many other cells in the human body (Sindre et al., 1996).

In conclusion, the present study has further confirmed the potential association of MM and HHV-8. The data also indicate that in addition to HHV-8, the transitional reactivation of EBV may also play a role in the pathogenesis of MM.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The BCBL reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: BCBL-1 SP from Drs Michael McGrath and Don Ganem. This study was supported by a National Research Grant (OTKA T 033067 and ETT 186/2000). Helpful discussions with Ilona Mezey, Ildiko Visontai, Bernadett Pályi, and the outstanding technical assistance of Mrs Anna Marchut and Mrs Agota Pus-Weller are gratefully acknowledged.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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