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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3643-3653
Human Herpesvirus 8 Infection in Patients With POEMS
Syndrome-Associated Multicentric Castleman's Disease
By
Laurent Bélec,
Ali Si Mohamed,
François-Jerôme Authier,
Marie-Charlotte Hallouin,
Aye Myat Soe,
Sylvie Cotigny,
Philippe Gaulard, and
Romain K. Gherardi
From Groupe d'Etude et de Recherche sur le Nerf Et le Muscle
(GERMEN, EA 2347); Faculté de Médecine de Créteil,
Université Paris XII-Val de Marne; Département de
Pathologie, Hôpital Henri Mondor, Créteil; and INSERM U430
and Laboratoire de Virologie, Hôpital Broussais, Paris, France.
 |
ABSTRACT |
The polyneuropathy, organomegaly, endocrinopathy, M protein, skin
changes (POEMS) syndrome is a rare multisystemic disorder associated
with osteosclerotic myeloma and multicentric Castleman's disease
(MCD). Human herpesvirus type 8 (HHV-8) DNA sequences have been
detected in lymph nodes of about 40% of human immunodeficiency virus
(HIV)-negative patients with MCD, and in bone marrow stromal cells of
patients with multiple myeloma. Considering these data, we investigated
the presence of HHV-8 in 18 patients with POEMS syndrome (9 with MCD),
by nested polymerase chain reaction (N-PCR) to detect DNA sequenses in
various cells and tissues obtained by biopsy or at autopsy (13 patients, of whom 7 had MCD), and by an immunofluorescence assay to
detect anti-HHV-8 IgG antibodies in blood (18 patients, of whom 9 had
MCD). Detection of HHV-8 DNA was performed using three different N-PCR,
targeting nonoverlapping regions in open reading frame (ORF) 25 and
ORF26. Seven of 13 (54%) POEMS patients had HHV-8 DNA sequences in
their tissues, as assessed by all three N-PCR, and 9 of 18 (50%) had
circulating anti-HHV-8 antibodies. HHV-8 was mainly detected in the
subset of POEMS patients with MCD (6 of 7 [85%] for DNA sequences; 7 of 9 [78%] for antibodies). The percentage of positive N-PCR was higher in lymph nodes than in bone marrow samples (P < .02).
Sequencing of amplicons showed a homogeneous restricted variability in
the ORF26 region, characteristic of the minority subgroup B defined by
Zong, and responsible for isoleucine and glycine substitutions at amino
acid positions 134 and 167. These findings strongly suggest an
association of HHV-8 infection with POEMS syndrome-associated MCD.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN herpesvirus type 8 (HHV-8), a
 2-Herpetovirinae (genus Rhadinovirus),1
may be implicated in the pathogenesis of distinct diseases depending on
the immune status of the host and viral tissue tropism.2
Sequences of HHV-8 have been initially identified in acquired
immunodeficiency syndrome (AIDS) and non-AIDS-associated Kaposi's
sarcoma.3-5 Subsequently, HHV-8 was also detected in primary effusion lymphomas, also known as body-cavity-based
lymphomas,6,7 in lymph nodes and peripheral blood
mononuclear cells (PBMC) of patients with multicentric Castleman's
disease (MCD),8-12 and in bone marrow dendritic cells from
patients with multiple myeloma.13
A certain degree of polymorphism exists in the open reading frame (ORF)
26 of HHV-8 genome.14 Its analysis has been proposed for
genetic subgrouping of viral strains.14 A homolog to the human interleukin-6 (IL-6) is present in HHV-8 genome,15,16 and viral IL-6 is suspected to play a role in the pathogenesis of
diseases in which IL-6 acts as a growth factor, including Kaposi's sarcoma, primary effusion lymphoma, multiple myeloma, and
MCD.13
MCD is a nonneoplastic lymphoproliferative disorder of unknown
significance, characterized by angiofollicular lymph node hyperplasia in multiple lymphoid organs that may appear as an idiopathic
disorder,17,18 or may be associated with a variety of
dysimmune conditions, including rheumatoid arthritis,19
Hodgkin's and B-cell non-Hodgkin's lymphomas,20-22 human
immunodeficiency virus (HIV) infection,23 and POEMS
syndrome.24
The POEMS (polyneuropathy, organomegaly, endocrinopathy, M protein,
skin changes) syndrome is a rare multisystemic disorder associated with
osteosclerotic myeloma.25-28 In addition to their osteosclerotic myeloma, about 50% of patients with POEMS syndrome have
MCD.24,26,29 Increased serum levels of IL-6 and several other proinflammatory and angiogenic cytokines are found in patients with POEMS syndrome, and are likely to play a role in expression of the
multiple manifestations of the syndrome.30-37 In the same way, a causal role is attributed to IL-6 in systemic manifestations of
MCD in patients without POEMS syndrome.17,38-42
The association of MCD with HHV-8 is very strong in HIV-infected
patients,8-12 in whom it is almost always associated with Kaposi's sarcoma.23 The association seems weaker in
non-HIV-infected patients.9,11,43-45 Interestingly, among
the 7 of 17 HIV individuals with MCD and HHV-8 DNA
sequences in lymph nodes reported by Soulier et al,9 1 had
a POEMS syndrome.
Considering these data, we investigated the presence of HHV-8 DNA
sequences in various cells and tissues of 13 patients with POEMS
syndrome. HHV-8 DNA sequences showing restricted variability in the
ORF26 region were found in 6 of 7 patients with MCD and 1 of 6 without MCD.
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PATIENTS AND METHODS |
Patients.
Thirteen patients with POEMS syndrome were evaluated for HHV-8 DNA
sequences (Table 1). The clinical and
pathologic findings of 10 of 13 patients have been previously
published.34 Twelve patients had an osteosclerotic myeloma.
The monoclonal protein consisted of IgA (6 patients), IgG (3 patients), both IgA and IgG (1 patient), and isolated
light chain (3 patients). Castleman's disease-like lesions were
detected in lymph nodes in 7 patients, and were classified as
hyaline-vascular type in 1 (POEMS-3), and mixed hyaline-vascular and
plasma-cell type in 6.
Eighteen POEMS patients, with (n = 9) and without (n = 9) MCD, were
evaluated for serum antibodies to HHV-8. They included all 13 patients
evaluated for HHV-8 DNA sequences, and 5 previously described
patients.34 All patients were seronegative for HIV.
Tissues.
The studied material consisted of PBMC (POEMS-7 and POEMS-13) and
tissues (11 of 13 patients) obtained by biopsy at various times of
disease evolution (11 patients) or by autopsy (POEMS-1, POEMS-2, and POEMS-3). Tissue samples had been fixed in buffered formalin, embedded in paraffin for histology, and kept at room temperature until analysis. They included 16 lymph node samples from 8 patients (including 13 samples with Castleman's disease from 6 patients), 21 bone marrow samples from 9 patients (including 10 samples
showing reactive plasmocytosis and 11 showing infiltration by myeloma),
6 spleen samples from 3 patients (all showing Castleman's disease),
and 2 PBMC samples.
DNA extraction.
Preparation of the paraffin wax-embedded tissue sections for polymerase
chain reaction (PCR) was performed as previously
described,46 with slight modifications. Ten sections of 15 µm were cut for each tissue and placed in a 1.5-mL Eppendorf tube.
Paraffin wax was removed by three successive washings with 1 mL xylene,
during 24 hours, 10 minutes and 5 minutes, respectively, at room
temperature. Between washings, the mixture was centrifuged at
1,000g for 10 minutes, and the supernatant
containing paraffin was removed. The pellet was then washed with 1 mL
of ethanol (70% vol/vol) for 10 minutes, at room temperature, and the
mixture was centrifuged (at 1,000g for 10 minutes). The pellet
was washed again with 1 mL of ethanol (50% vol/vol) for 10 minutes.
After centrifugation, the last pellet was dried at room temperature for
about 45 minutes. Tissue samples and the snap-frozen PBMC were
processed for DNA extraction by the phenol-chloroform procedure, after
overnight digestion at 56°C with 100 µg/mL proteinase K, 0.5%
sodium dodecyl sulfate (SDS), 25 mmol/L EDTA, 100 mmol/L NaCl, and 10 mmol/L Tris-HCl (pH 8.3); DNA precipitation was performed with sodium acetate (0.25 mol/L), glycogen (100 µg/mL), and 2 vol of ethanol; the
resulting pellet was resuspended in 100 µL of 10 mmol/L Tris-HCl, and
the DNA was quantified by spectrophotometry.
Evaluation of DNA amplifiability.
The presence of DNA in each tissue extract and its amplifiability by
PCR were further assessed by amplifying a 110-bp fragment of the human
 -globin gene, as previously described.47 DNA was successfully extracted as assessed by PCR amplification of human -globin sequences, except in three samples that were not used in the
study. We further evaluated the relative intactness of DNA in one
sample of each evaluated tissue from POEMS patients, and the efficiency
of the PCR procedure in the different tissues used in the study. To
evaluate the level of amplifiable, ie, intact, target DNA, 1 µg of
the extracted DNA was serially fivefold diluted in distilled water (up
to 58), and each dilution was further subjected to
-globin PCR, as previously described.48 The resulting
amplicons were migrated on agarose gel and visualized under
UV. The level of amplifiable extracted DNA was expressed as
log5 of the last dilution giving a positive -globin PCR
(ie, an end-point dilution of 5x is expressed as x
log5). To evaluate efficiency of the PCR procedure, the
amplicons obtained with the -globin PCR amplification at the
55 dilution of extracted DNA were hybridized and
quantified by DNA enzyme immunoassay (DEIA).49 The
previously published 40-base oligonucleotide RS06 [5'-CTG ACT
CCT GAG GAG AAG TCT GCC GTT ACT GCC CTG TGG G-3'],47
which is complementary to the target sequence, was used as probe.
Briefly, amplified product (20 µL), denatured by heating, were added
to streptavidin-coated microtiter plates (Gen-Eti-K; Sorin Biomedica,
Saluggia, VC, Italy), preincubated overnight with 7 ng/well of
single-stranded 5'-biotinylated -globin RS06 probe, and
detected with an anti-double-stranded monoclonal antibody, after
hybridization for 1 hour at 50°C. Optical density (OD) of
hybridized products was read at 450 nm.
Nested PCR for HHV-8 DNA detection.
One microgram of DNA from each extract positive for -globin gene was
processed for HHV-8 DNA amplification, in parallel with three nested
PCR (N-PCR) conceived to amplify nonoverlapping regions within the
ORF25 and ORF26 of HHV-8 (Fig
1).1 Primers sets for amplification have been previously
described: for N-PCR1, the outer set was KS1/KS2 (KS1: 5'-AGC CGA
AAG GAT TCC ACC AT-3'; KS2: 5'-TCC GTG TTG TCT ACG TCC
AG-3'), originally described by Chang et al,3 and the
inner primer set was WH-1/WH-2 (WH-1: 5'-GTG CTC GAA TCC AAC GGA
TT-3'; WH-2: 5'-ATG ACA CAT TGG TGG TAT
AT-3')50; for N-PCR2, the outer set was 5'-AGG
CAA CGT CAG ATG TGA C-3' and 5'-GAA ATT ACC CAC GAG ATC
GC-3', and the inner set was 5'-CAT GGG AGT ACA TTG TCA GGA
CCT C-3' and 5'-GGA ATT ATC TCG CAG GTT
GCC-3'51; for N-PCR3, the outer set was 5'-GGC GAC ATT CAT CAA CCT CAG G-3', and 5'-ATA TCA TCC TGT GCG
TTC ACG AC-3', and the inner set was 5'-CGC ATG GAG GAC CTA
GTC AAT AAC-3', and 5'-GGT TGT AGT CAT TCT CGT CCA
GGG-3'.52 For each N-PCR, the outer PCR consisted of
an initial denaturation at 94°C for 5 minutes,
followed by 35 cycles of amplification (94°C, 45 seconds; 55°C,
45 seconds; 72°C, 60 seconds), and a final 15-minute elongation (72°C). Five microliters of the first PCR product was taken for the
inner PCR, which consisted of an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of amplification (94°C, 60 seconds;
60°C, 60 seconds; 72°C, 60 seconds), and a final 15-minute elongation (72°C). The mix used for both outer and inner PCR
contained 25 pmol/L of each primer, 1.5 U of Taq DNA polymerase
(Phamarcia Biotech, Uppsala, Sweden), 200 µmol/L each dNTP, 10 mmol/L
Tris-HCl, 1.5 mmol/L MgCl2, and 50 mmol/L KCl. The 233 bp-PCR products (KS330233) obtained by the outer PCR of
N-PCR 1, and all final PCR products obtained by the three N-PCR (170 bp
for N-PCR1; 213 bp for N-PCR2; 115 bp for N-PCR3) were visualized under
UV transillumination by ethidium bromide staining after electrophoresis
on the same 2% agarose gel. These N-PCR are considered to be 100 to
1,000 times more sensitive for detecting HHV-8 sequences than the
previously published single PCR using the KS1/KS2 primers
set,3 and allow detection of less than 10 copies of HHV-8
genome under optimal conditions, as previously determined by
comparative serial dilutions of a DNA standard containing a known
amount of HHV-8 DNA.51 Presence of HHV-8 in a sample was
considered certain in case of positive results of all three N-PCR
evaluations, probable in case of two positive results, possible in case
of only one positive result.
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| Fig 1.
Relative map locations within the ORF25 and ORF26 of
HHV-8 genome of the three nested-PCR (N-PCR1, N-PCR2, and N-PCR3) used
as a diagnosis purpose for HHV-8 DNA sequence detection, of the N-PCR4,
used to obtain a 334-bp amplicon from the ORF26 DNA target, and of the
N-PCR5, used to obtain a 494-bp amplicon from the ORF75 DNA target,
sufficiently large to allow genetic variability analysis after direct
DNA sequencing. The 17.4-kb divergent locus-B of the HHV-8 genome,
supporting the vIL-6 and the bcl-2-like genes,61 and the
ORFs within the 20.7-kb KS5 fragment of HHV-8,1 are shown
in comparison with other equivalent loci and known genes in the
herpesvirus Saimiri (HSV). The upper portion of the figure illustrates
the arrangement of the ORFs in a contiguous 55-kb block at the
left-hand end of the HSV genome: solid bars refer to ORFs that are
common in all known -Herpetovirinae, shaded bars indicate
ORFs that are found in several 2-Herpetovirinae, and open
bars denote ORFs that are unique to HSV. The ORF25 of HHV-8 is thought
to be translated into a major capsid protein and the ORF26 into a
virion protein.1
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Estimation of HHV-8 PCR product amount.
A slightly modified N-PCR1, N-PCR1', was used to indirectly
estimate the levels of HHV-8 DNA target in tissue samples. We used a
procedure that insures a linear positive correlation between initial
target concentration and the final N-PCR product.52 To
avoid plateauing of the amplification curve, the round of amplification performed with the outer pair of primers consisted of 20 cycles only,
insuring that maximum concentration of PCR products did not exceed 10%
of the molarity of the outer primers, and the round of amplification
performed with the inner pair of primers consisted of 25 cycles only.
Five microliters of the first PCR product of N-PCR1' were
serially 10-fold diluted in distilled water (up to 109), and further subjected in parallel to the inner
amplification. The amount of final HHV-8 N-PCR1' product was
calculated from the last dilution of the first PCR product that gave a
signal after the second round of amplification. The results were
expressed as log10 of the last positive dilution obtained
from 1.0 µg of tissue DNA (ie, an end-point dilution of
10x is expressed as x log10). Interassay
variability determined by paired evaluations of 30 Kaposi's sarcoma
samples was 0.7 ± 0.6 log10 (mean ± standard error).
Positive and negative controls for N-PCR.
Tissues from 3 patients with AIDS and Kaposi's sarcoma were used as
positive controls for HHV-8 (2 lymph nodes with Kaposi's sarcoma
without MCD and 1 skeletal muscle with metastasis of
Kaposi's sarcoma). Different tissues from HIV
individuals were used as normal controls, and included 17 lymph nodes
with mild reactive changes and no MCD, 3 normal bone marrow biopsy samples, and 1 spleen tissue sample. All tissues were
paraffin-embedded, and DNA extraction was performed after paraffin
dissolution by xylene, as described above.
DNA sequence variability among HHV-8 samples.
Nucleotide sequence variability among HHV-8 DNA-positive samples giving
strong positivity by N-PCR1 was evaluated in the ORF26 region.14 For ORF26, 1 µg of DNA was subjected to N-PCR4,
using the outer set KS4 5'-AGC ACT CGC AGG GCA GTA CG-3'
and KS5 5'-GAC TCT TCG CTG ATG AAC TGG-3', previously
published,53 and the inner set LGH 1701 5'-GGA TGG
ATC CCT CTG ACA ACC-3' and LGH 1702 5'-ACG TGG ATC CGT GTT
GTC TAC G-3', previously used by Zong et al (Fig
1).14 Outer amplification was performed by
"touchdown" PCR54 for 14 cycles (94°C, 45 seconds; 60°C, 45 seconds, decreasing by 1°C per cycle;
72°C, 90 seconds), followed by 20 cycles (94°C, 45 seconds;
53°C, 45 seconds; 72°C, 90 seconds), with a final 15-minute
extension (72°C). The inner PCR consisted of 40 cycles (94°C,
45 seconds; 56°C, 45 seconds; 72°C, 90 seconds), and a final
15-minute elongation (72°C). Sequencing of the resulting 334 bp for
ORF26 PCR products was performed without prior cloning, using the
dideoxynucleotide chain termination method,55 according to
fluorescent-based cycle sequencing with dye dichlororhodamine-labeled terminators (ABI Prism dRhodamine Terminator Cycle Sequencing Ready
Reaction Kit; Perkin-Elmer, Applied Biosystems, Inc, Foster City, CA)
and an automated DNA sequencer (ABI Prism 310 Genetic Analyzer;
Perkin-Elmer, Applied Biosystems). Two opposing strands from each PCR
product were obtained by using the 5' and 3' inner primers,
and further aligned with the software Sequencer Navigator 1.0.1 (Applied Biosystems), and corrected manually. To avoid contamination, the sequencing procedure was carried out blind for the diagnosis, in a
laboratory remote from that in which detection N-PCR was performed, by
sets of five amplicons (belonging to both patients and controls)
analyzed discontinuously over a 4-month period. Distribution in three
subgroups of ORF26 sequences within single sets of amplicons insured
that contamination between tissue samples did not occur.
Phylogenetic analysis was based on comparison of 334-bp nucleotide
sequences of the ORF26 of HHV-8 detected in tissue samples from
HHV-8+ patients with POEMS syndrome, 1 HIV-infected patient
with Kaposi's sarcoma and MCD (patient KS/MCD), 8 HHV-8+
healthy African blood donors, living in the Central African Republic, and on 12 representative sequences previously used to define three subgroups of HHV-8 variability in ORF26 [from patients with
HIV-related Kaposi's sarcoma, living in the United States (KSHV AIDS,
C282 AIDS KS, ASM70 Lung KS, and AKS1 AIDS KS) or in Uganda (ST1 AIDS KS, ST2 AIDS KS, ST3 AIDS KS); from patients with non-HIV-related Kaposi's sarcoma, living in the United States (EKS1 non-AIDS KS) or in
Zaire (431 KAP Endemic KS); and from patients with HIV-associated body-cavity-based lymphoma, living in the United States: BCBL-1, BCBL2, and BCBLR AIDS Lym].14 KSHV AIDS is from the
original sequence published by Chang et al (GenBank accession no.
U40377),3 including the KS330233Bam fragment;
BCBL-1 and BCBL2 have been previously reported by Cesarman et
al.56 Phylogeny construction and evaluation were performed
using the Phylip software package,57 with the
matrix distance Fitch and Margoliash method.58 The tree
obtained by the Fitch and Margoliash method was statistically evaluated
using 100 bootstrap samples.59 The values of the branches represent the percentage of trees for which the sequences at one end of
the branch are a monophyletic group. Branches with bootstrap values
above 90% are usually considered to be robust, while values below 70%
are generally not confident enough to fully support a topology.
Amino acid sequences were inferred from ORF26 nucleotides sequences of
HHV-8 previously used for phylogenetic analysis, plus one
sequence from an HIV patient with monocentric
Castleman's disease (case 9)43 and three sequences of
HHV-8 detected in three cases of reactive lymphadenopathy (cases 6, 10, and 16),45 and further aligned, using the sofware GeneWorks
2.45 (IntelliGenetics, Inc, Mountain View, CA).
Because of striking restriction of genetic variability found in ORF26,
a control procedure was performed on another variable region, the
ORF75.14 For this purpose, we used 1 µg of the DNA extracts that had allowed successful ORF26 sequencing in POEMS patients
and in the patient KS/MCD used as control. The DNA was subjected to
domestic N-PCR5 (Fig 1), using the outer set LGH 1704 5'-GTA CGG
ATC CAC GGA GCA TAC-3' and LGH 1984 5'-CTA GAG ATC TGT TTA
GTC CGG AG-3', previously used by Zong et al14 and the inner set BM2 5'-GAG CAT ACA CCC ACG TCC AC-3'
(position 602 to 621) and BM1 5'-GGA GAA GAT AGG GCC CTT
GG-3' (position 128 to 147), determined within the sequence KS631
Bam DNA sequence of Chang et al,3 using the sofware Primer3
Test Pre-Release Output (internet address:
http://www.genome.wi.mit.edu//cgi-bin/primer/primer3_www.cgi); amplification of outer PCR was performed for 40 cycles (94°C, 60 seconds; 60°C, 60 seconds; 72°C, 90 seconds), with
a final 15-minute extension (72°C). The inner PCR consisted 40 cycles (94°C, 45 seconds; 60°C, 45 seconds; 72°C, 60 seconds), with a final 15-minute extension (72°C). The resulting
494-bp amplicon was subjected to direct sequencing, as described above.
HHV-8 serology.
Circulating IgG to HHV-8 late proteins were detected by
immunofluorescence assay done on a the KS-1 cell line from a
body-cavity-based lymphoma of an HIV patient,
infected by HHV-8 but not by Epstein-Barr virus (EBV) (HHV-8 IgG IFA
kit; Advanced Biotechnologies Inc, Columbia, MD). According to the kit recommendations, levels of anti-HHV-8 antibodies were estimated by immunofluorescence intensity assessed on a 0 to
4+ scale. Fifteen patients with multiple myeloma without
POEMS syndrome, 10 with HIV-1 infection and Kaposi's sarcoma, and 15 healthy individuals were used as controls for HHV-8 serology.
Statistical analysis.
Quantitative results were expressed as mean ± standard error.
Statistical analyses were performed using the Fisher's exact test and
the Mann and Witney U test. A P < .05 was considered significant.
| |
RESULTS |
The three tissues with evidence of Kaposi's sarcoma from three
patients with AIDS were positive by the three N-PCR. Among the 21 tissue samples from 21 HIV individuals, 19 were
negative by all three N-PCR (15 lymph nodes, 3 bone marrow and 1 spleen
tissue samples), and 2 showed possible presence of HHV-8 (2 lymph nodes
with 1 of 3 positive N-PCR evaluations).
The results of -globin amplification and HHV-8 detection in tissues
from patients with POEMS syndrome are reported in Table 1. The mean
end-point dilution of DNA amplifiable by -globin PCR was similar in
POEMS patients with and without MCD (log5 4.0 ± 0.36 v log5 4.60 ± 0.44), assessing similar DNA
intactness in the two groups. The levels of amplified -globin gene,
assessed by OD of DEIA hybridized product,49 were similar
in the two groups (0.67 ± 0.14 v 0.90 ± 0.21). DEIA
hybridization also indicated a similar efficiency of the -globin PCR
performed with DNA extracted from paraffin-embedded material, if one
except a somewhat lower PCR efficiency on spleen tissue of POEMS
patients with MCD. Taken together, these results allowed
reliable comparison between the two groups. HHV-8 DNA was detected in 7 of 13 patients with POEMS, including 6 of 7 with MCD and 1 of 6 without MCD.
Patients with POEMS syndrome and MCD.
Among the 33 tested samples evaluated in this group, presence of HHV-8
was established in 21 samples (3 positive N-PCR), probable in 1 sample
(2 positive N-PCR), possible in 6 samples (1 positive N-PCR), and not
found in 5 samples. As a whole, 6 patients with MCD were found to have
HHV-8 DNA sequences detected by all three N-PCR evaluations in their
tissues. In these patients, the proportion of overall positive N-PCR
detections in lymph node samples (32 of 36 positivities) was higher
than in bone marrow samples (25 of 39, P < .02); it appeared
also slightly higher than in spleen samples (12 of 18, P < .07). Results in bone marrow samples infiltrated by myeloma (13 of 24)
did not differ significantly from those of bone marrow samples showing
reactive plasmocytosis (12 of 15, not significant [NS]).
Among all tissues positive for HHV-8 by N-PCR1, only two lymph nodes
(POEMS-1 and POEMS-4) were slightly positive on agarose gel by the
nonnested outer KS330233 PCR.
Patients with POEMS syndrome without MCD.
Among the 13 tested samples evaluated in this group, presence of HHV-8
was fully assessed in 1 sample (3 positive N-PCR), possible in 1 (1 positive N-PCR), and not found in 11 (3 negative N-PCR). The
positivities were detected in the bone marrow samples of a Black
African patient (POEMS-11); they were undetectable by the nonnested
KS330233 PCR.
Estimation of HHV-8 PCR product amount.
The mean amount of final HHV-8 PCR product obtained from tissues of
POEMS patients (1.7 ± 0.2 log10) was significantly
lower than in HIV-associated Kaposi's sarcoma controls (8.7 ± 0.3 log10) (P < .0001)
(Figs 2 and 3).
The amount of HHV-8 PCR product estimated from lymph nodes of POEMS
patients (2.5 ± 0.3 log10) was higher than that from
the other tissues of POEMS patients (1.2 ± 0.1 log10)
(P < .02).
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| Fig 2.
Indirect estimation of HHV-8 viral load in tissues by
end-point dilution of final HHV-8 N-PCR1' product obtained with a
linear amplification procedure, in Kaposi's sarcoma (KS) samples from
three AIDS patients (AIDS-1 to AIDS-3), and in lymph nodes (LN), spleen
(S), bone marrow (BM), PBMC, and myeloma (Myel) samples from POEMS
patients with (POEMS-1 to POEMS-7) or without (POEMS-11) multicentric
Castleman's disease.
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| Fig 3.
Vizualization by electrophoresis on a 2%
agarose/ethidium bromide gel of serial 10-fold dilutions of final HHV-8
N-PCR1' amplified products in Kaposi's sarcoma (KS) sample from
AIDS-1, and in lymph node (LN), spleen (S), bone marrow (BM), and
myeloma (Myel) samples from POEMS-1. The molecular size (in base pairs,
bp) of the expected amplified product for HHV-8 ORF26 (170 bp) and
molecular-weight markers (100-bp DNA ladder; GIBCO-BRL, Gaithersburg,
MD) are indicated on the left.
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HHV-8 sequence analysis.
Sequencing of ORF26 was successfully performed in 9 samples from 6 HHV-8+ patients with POEMS syndrome, of whom 5 had MCD.
Each sequence was aligned and compared with the others and with the
sequences from 8 HHV-8+ African blood donors, from 1 HIV-infected individual with both KS and MCD, and from 12 sequences
reported in previous studies.3,14,43,45,55 Genetic
variation within ORF26 was not random. Eleven variable positions were
identified: they included the 10 variable positions previously
identified in ORF26 by Zong et al,14 and one in position 1160. Sequences were identical in all tissues from all POEMS patients. The nucleotide pattern of variability consisted of A (position 926), C
(981), C (989), A (1032), T (1033), T (1055), C (1086), G (1094), G
(1132), C (1139), and A (1160) (Fig 4). The
pattern found in POEMS patients was different from the patterns found in African blood donors and in the HIV-infected individual with KS and
MCD (Fig 4).
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| Fig 4.
Sequence variability and similarity between the
nucleotides 893 to 1226 of the ORF26 of HHV-8 genome among
HHV-8+ samples from 6 patients with POEMS syndrome, 1 HIV-infected patient with Kaposi's sarcoma (KS) and multicentric
Castleman's disease (MCD) (patient KS/MCD), and 8 healthy African
blood donors (ABD). Only nucleotides of the 11 identified variable
positions are included. All nucleotides at the other unvariable
positions are similar to those reported in the original sequence KSHV
AIDS (GenBank U40377) initially published by Chang et al.3
The positional nomenclature used for ORF26 follows that of Chang et
al.3 Bold and underlined nucleotide substitutions at
positions 1032, 1033, and 1132 (patients with POEMS syndrome) and 981 (patient KS/MCD) lead to amino acids changes. BM, bone marrow; LN,
lymph node; Myel, myeloma.
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Phylogenetic analysis was performed on 334 bp of HHV-8 ORF26 obtained
from 6 patients with POEMS syndrome and controls, and 12 representative
sequences previously used to define three subgroups of HHV-8
variability in ORF26.14 The phylogram showed that
individual HHV-8 DNA sequences were distributed into three subgroups
(Fig 5). These subgroups could not be
recognized as distinct clades, because bootstrap values were not
significant. However, the three branches of the phylogram corresponded
to subgroups A, B, and C of ORF26 variability, defined by Zong et
al.14 All HHV-8 sequences from POEMS patients belonged to
the subgroup B when only 4 of the 22 sequences used for comparison
belonged to this subgroup.
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| Fig 5.
Phylogram generated by the Fitch and Margoliash method,
based on 334 nucleotides of the ORF26 of HHV-8 detected in tissue
samples from 6 patients with POEMS syndrome, 1 HIV-infected patient
with Kaposi's sarcoma and multicentric Castleman's disease (KS/MCD),
8 healthy African blood donors (ABD), and 12 representative sequences
previously used to define 3 subgroups of HHV-8 variability in ORF26
(from patients with HIV-related Kaposi's sarcoma: KSHV AIDS, ST1 AIDS
KS, ST2 AIDS KS, ST3 AIDS KS, C282 AIDS KS, ASM70 Lung KS, and AKS1
AIDS KS; from patients with non-HIV-related Kaposi's sarcoma: 431 KAP
Endemic KS and EKS1 non-AIDS KS; and from patients with HIV-associated
body-cavity-based lymphoma: BCBL-1, BCBL-2, and BCBLR AIDS
Lym).14 KSHV AIDS is from the original sequence published
by Chang et al,3 including the KS330233Bam
fragment; BCBL-1 and BCBL-2 have been previously reported by Cesarman
et al.56 The 31 examined 334-bp ORF26 sequences of HHV-8
genome fell into three distinct but very narrow subgroupings,
corresponding to variants of the subgroups A, B, and C defined by Zong
et al.14 However, distinct clades among the HHV-8 strains
were not supported by significant bootstrap values. All ORF26 sequences
from patients with POEMS syndrome belonged to the subgroup B. Homologous BDLF1 gene of EBV (GenBank: VO1555) and ORF26 of herpesvirus
Saimiri (HVS) (GenBank: AF005370) were used as outgroups. Vertical
branches are for clarity only; the lengths of the horizontal branches
are proportional to the single base changes. Numbers at nodes represent
the percentage of bootstrap samples for 100 replications, for which the
corresponding cluster is depicted to the right. BM, bone marrow; KS,
Kaposi's sarcoma; Lym, lymphoma; LN, lymph node; Myel, myeloma.
|
|
The 32-amino acid sequences corresponding to nucleotide sequences from
all patients with POEMS syndrome and controls, and 14 sequences
previously published,3,14,43,45,56 were aligned and an
arbitrary consensus sequence of 111 amino acids of ORF26 was
established (Fig 6). By comparison with
this consensus sequence, it appears that base changes of HHV-8 in POEMS
syndrome at positions 1032 and 1033 encode a lysine to isoleucine
substitution in codon 134, and the base change at position 1132 encodes
an aspartate to glycine substitution in codon 167 (Fig 4). The
remaining base changes do not result in amino acid substitutions. Amino
acids encoded by base substitutions at positions 1032, 1033, and 1132 also appear different than those reported in the original sequence commonly used as a reference.3
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| Fig 6.
Multiple alignments of inferred amino acid sequences from
ORF26 nucleotides sequences of HHV-8 detected in tissue samples from 6 patients with POEMS syndrome, 1 HIV-infected patient with Kaposi's
sarcoma and multicentric Castleman's disease (KS/MCD), 8 healthy
African blood donors (ABD), and 14 sequences previously published,
including the 12 precedently used for phylogenetic analysis (KSHV AIDS,
ST1 AIDS KS, ST2 AIDS KS, ST3 AIDS KS, C282 AIDS KS, ASM70 Lung KS,
AKS1 AIDS KS, 431 KAP Endemic KS, EKS1 non-AIDS KS, BCBL-1, BCBL2, and
BCBLR AIDS Lym), 1 sequence from an HIV patient with
monocentric Castleman's disease (case 9),43 and sequences
of HHV-8 detected in three cases of reactive lymphadenopathy (cases 6, 10, and 16).45 By comparison with the 111 amino acid
consensus sequence of ORF26 deduced from these latter sequences, the
base changes of HHV-8 in patients with POEMS syndrome at positions 1032 and 1033 encode a lysine to isoleucine substitution in codon 134, and
the base change at position 1132 encodes an aspartate to glycine
substitution in codon 167. The positional nomenclature used for ORF26
amino acid sequences follows that of Chang et al.3 Hyphens
(-) indicate sequence homology, and dots (·) indicate gaps introduced
for optimal alignment. Single-letter abbreviations for the amino acid
residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His;
I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser;
T, Thr; V, Val; W, Trp; and Y, Tyr. BM, bone marrow; KS, Kaposi's
sarcoma; Lym, lymphoma; LN, lymph node; Myel, myeloma.
|
|
Sequencing of the 494 bp in ORF75 was successfully performed in 4 of 7 evaluated patients with POEMS syndrome (POEMS-1, POEMS-2, POEMS-7, and
POEMS-11) and in the KS/MCD patient. Sequences were aligned, and
nucleotide position was established by reference to the ORF75 sequence
provided by Chang et al.3 Variability in ORF75 was observed
at positions 150, 417, and 462, delineating four different variants
(POEMS-1 and POEMS-7: A,T,A; POEMS-2: G,T,A; POEMS 11: G,T,G; KS/MCD:
A,C,A). The genetic variations in ORF75 confirmed the lack of cross
contamination between samples. The finding of T in position 417 in
ORF75 of HHV-8 variants found in POEMS patients further assessed their
belonging to the subgroup B.14
HHV-8 serology.
Antibodies to HHV-8 were found in all (10 of 10) patients with
Kaposi's sarcoma, in 78% (7 of 9) of patients with POEMS syndrome and
MCD, in 22% (2 of 9) of patients with POEMS syndrome without MCD, in
none (0 of 15) of patients with multiple myeloma, and in none (0 of
15) of healthy controls. Among the 13 POEMS patients who had both
antibody testing and N-PCR for HHV-8, 6 had both tests positive
(POEMS-1, -3, -4, -6, -7 and -11), 2 had undetectable HHV-8 antibodies
despite DNA sequences in tissues (POEMS-2 and -5), 1 had
HHV-8 antibodies without DNA sequences (POEMS-9), and 4 had
undetectable HHV-8 antibodies without DNA sequences (POEMS -8, -10, -12, and -13). HHV-8 antibody levels were higher in HIV-infected patients with Kaposi's sarcoma (mean, 3.0+; range,
2+ to 4+) than in patients with POEMS syndrome
seropositive for HHV-8 (mean, 1.3+; range, 1+
to 2+).
| |
DISCUSSION |
In the present study, 7 of 13 patients with POEMS syndrome had HHV-8
DNA sequences in their tissues, and 9 of 18 had circulating HHV-8
antibodies. Presence of HHV-8 DNA sequences was assessed by three N-PCR
targeting nonoverlaping regions in ORF25 and ORF26. HHV-8 was mainly
detected in the subset of POEMS patients with MCD (6 of 7 for DNA
sequences ; 7 of 9 for antibodies); the percentage of positive N-PCR
was higher in lymph nodes than in bone marrow and spleen; HHV-8 DNA
sequences showed a restricted variability in the ORF26 region
characteristic of the subgroup B defined by Zong et al.14
Including the patient reported by Soulier et al,9 the
proportion of patients with POEMS and MCD in whom HHV-8 DNA sequences were found in lymphoid tissues or PBMC is 88% (7 of 8). HHV-8 DNA
sequences were detected in lymphoid tissues and PBMC of 100% (22 of
22) of HIV-infected patients with MCD,8,9,11,12 33% (11 of
33) of non-HIV-infected, non-POEMS patients with
MCD,9-11,45 and less than 10% of HIV
individuals used as normal controls in the present study (0 of 21 with
three positive N-PCR; 2 of 21 with one positive N-PCR). In our
patients, HHV-8 DNA sequences were detected in only 2 of 6 lymph nodes
with Castleman's disease when the classical single-step KS330233 PCR was used, and in 5 of 6 when HHV-8 DNA
sequences were detected by the most sensitive N-PCR procedure,
suggesting a rather low HHV-8 viral load. In the same way, Southern
blot detection of HHV-8 in patients with MCD and positive PCR detection of HHV-8 is constantly positive in case of HIV-infection and is usually
negative in the absence of HIV infection.9 In our POEMS patients positive for HHV-8, the viral load in tissues, indirectly estimated by the amount of HHV-8 PCR products obtained with a linear
amplification procedure, and the anti-HHV-8 antibody response in
blood, were significantly lower than in HIV-infected controls with
Kaposi's sarcoma. These data point to the fact that HHV-8 may be
easily detected only in patients with strong immunodepression, probably
because of higher viral loads.
In patients with Kaposi's sarcoma, the HHV-8 viral load is higher in
the tumor than elsewhere in the body, although HHV-8 may usually be
detected in blood,50 lymphoid organs, prostatic tissue, and
unaffected skin.10 The higher percentage of positive N-PCR
found in lymph nodes of our patients with MCD, compared with bone
marrow and spleen, could suggest a higher viral load in this tissue. We
confirmed that the estimated HHV-8 load in lymph nodes from POEMS
patients was higher than that present in their other HHV-8+
tissues. These findings point to the lymph node as a candidate target
tissue of HHV-8 in POEMS syndrome. Consistently, a longitudinal study
of three HIV-infected patients with MCD showed a strong positive
correlation between circulating HHV-8 viral load, lymphadenopathy, and
systemic manifestations.12 MCD is characterized by
angiofollicular lymph node hyperplasia and high IL-6 expression in
affected lymph nodes.17,18,38-40 It is possible that the
viral homolog to human IL-6 encoded by HHV-8 genome15,16
accounts for plasma cell accumulation of MCD60 through the
potent B-cell growth factor activity of IL-6. The presence of an
homolog to the human molecule bcl2 in HHV-8 genome61 may
protect infected cells against apoptosis and, therefore, maintain
protracted viral IL-6 (vIL-6) production. Vascular proliferation is not
a direct effect of IL-6. However, IL-6 can promote angiogenesis
indirectly by inducing expression of vascular endothelial growth factor
(VEGF), a potent mitogen for endothelial cells.62
Consistently, increased circulating levels of VEGF have been recently
reported in POEMS syndrome.36,37
The HHV-8 variants in POEMS patients were of the ORF26 subgroup B of
Zong et al,14 unlike HHV-8 variants found in the patient with Kaposi's sarcoma and MCD (subgroup A), and in African blood donors (subgroup C). The subgroup B is a minority subgroup accounting for 13% of HHV-8 sequences reported from previous studies involving patients from the United States, Europe, and Africa, and for 24% of
HHV-8 variants in European HIV-infected individuals without Kaposi's
sarcoma.63
HHV-8 variants in patients with POEMS showed intra- and
inter-individual homogeneity. The restricted variability in ORF26 was
responsible for isoleucine and glycine substitutions at amino acid
positions 134 and 167, respectively. Interestingly, a variability at
positions 989 to 1160 similar to that observed in POEMS patients has
been previously reported in patients with lymph node pathology, including one case of monocentric Castleman's disease,43
and three cases of reactive lymphadenopathy unrelated to HIV
infection.45 Further studies are needed to determine to
what extent this particular genotype in ORF26 is associated with
nonneoplastic lymphoproliferative disorders.
Among POEMS patients without MCD, one had both HHV-8 DNA sequences in
bone marrow and HHV-8 antibodies in blood, and another one had HHV-8
antibodies in blood. Because lymph node biopsy was not performed,
occult MCD cannot be excluded in these patients, but this is unlikely
because they had neither lymphadenopathy nor splenomegaly at physical
examination and abdominal echography. Our results in this group of
patients are in keeping with the negative detection of HHV-8 previously
reported in patients with multiple myeloma, using either PCR analysis
of bone marrow biopsy samples44,64 or serological
analysis.65,66 However, Said et al67 have
detected HHV-8 DNA sequences in fresh bone marrow biopsy samples from 6 of 7 myeloma patients, and Rettig et al13 have reported the
presence HHV-8 DNA sequences in bone marrow dendritic cells of patients
with multiple myeloma. In this study, HHV-8 sequences were found in 0 of 23 fresh samples of myeloma bone marrow mononuclear cells, and in 15 of 15 stromal cells obtained by separation of bone marrow aspirates by
Ficoll-Hypaque and culture.13 These observations may
indicate either very low HHV-8 viral load in bone marrows of myeloma
patients, or restriction of HHV-8 to cells not retrieved by bone marrow
aspirates, or the inhibitory effect of heparin on Taq DNA
polymerase. Archival material used for molecular biological analysis
was suboptimal for an ultrasensitive detection of HHV-8 DNA sequences
in POEMS patients. It cannot be excluded that the differences observed
between POEMS patients with and without MCD reflect a difference of
viral load in patients with and without MCD.
HHV-8 is a lymphotropic virus, and lymphoid organs are major sites of
viral latency.68 Latent HHV-8 in lymphoid organs can reactivate at time of immunosuppression in patients with
AIDS.69 MCD is associated with a type of immunodeficiency
resembling that of AIDS.42 If one considers the recent
evidence that myeloma patients may be infected by HHV-8 at very low
burdens, one can speculate that a subset of patients with
osteosclerotic myeloma may develop a specific immunodeficient state or
may be associated with an unknown cofactor capable of inducing
reactivation of HHV-8 in lymphoid tissues, with subsequent increase of
systemic viral load and local release of cytokines at the origin of MCD lesions.
We conclude that (1) HHV-8 DNA sequences and HHV-8 antibodies are
frequently detected of patients with POEMS syndrome; (2) HHV-8 is
mainly detected in POEMS patients with MCD and HHV-8 DNA sequences are
more easily detected in lymph nodes than in bone marrow; and (3) HHV-8
variants associated with POEMS syndrome show a restricted variability
in ORF26 and belong to a minority subgroup of HHV-8. These findings
strongly suggest an association of HHV-8 infection with POEMS
syndrome-associated MCD.
| |
ACKNOWLEDGMENT |
We are indebted to Dr Antoine Gessain and Dr Marina Karmotchkine for
providing clinical samples for analysis, Dr Xavier Jeunemaître for assistance with nucleotide sequencing, and Dr Michaela
Müller-Trutwin for helpful discussions.
| |
FOOTNOTES |
Submitted November 24, 1997; accepted January 24, 1999.
Supported by Association pour la Recherche contre le Cancer (ARC);
Projet Hospitalier de Recherche Clinique AP-HP 1996.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Romain K. Gherardi, MD, Département
de Pathologie, Hôpital Henri Mondor, F-94010 Créteil Cedex,
France.
| |
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