1. No category

From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
1996 87: 3903-3909
Human herpesvirus-8 DNA sequences in human immunodeficiency
virus- negative angioimmunoblastic lymphadenopathy and benign
lymphadenopathy with giant germinal center hyperplasia and
increased vascularity
M Luppi, P Barozzi, A Maiorana, T Artusi, R Trovato, R Marasca, M Savarino, L Ceccherini-Nelli
and G Torelli
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Copyright 2011 by The American Society of Hematology; all rights reserved.
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Human Herpesvirus-8 DNA Sequences in Human Immunodeficiency
Virus -Negative Angioimmunoblastic Lymphadenopathy and Benign
Lymphadenopathy With Giant Germinal Center Hyperplasia
and Increased Vascularity
By Mario Luppi, Patrizia Barozzi, Antonio Maiorana, Tullio Artusi, Raffaella Trovato, Roberto Marasca,
Marco Savarino, Luca Ceccherini-Nelli, and Giuseppe Torelli
Human herpesvirus-8 (HHV-8) DNA sequences have been reported t o be strictly associated not only with various forms
of Kaposi’s sarcoma but also with an unusual subgroup of
acquired immunodeficiency syndrome (AIDS)-related B-cell
lymphomas. A possible relation of this putative virus also
with multicentric Castleman’s disease (MCD) has been recently suggested. We used polymerase chain reaction t o
look for the presence of HHV-8 sequences in a well characterized series of benign, atypical, and malignant lymphoid tissues from 45 Hodgkin’s disease and 43 non-Hodgkin’s
lymphoma (NHL) cases, as well as from 5 MCD, 15 angioimmunoblastic lymphadenopathy (AILD), and 23 benign lymphadenopathy cases. Among the 38 AIDS-related lymphoid
lesions, only 7 NHL and 1 persistent generalized lymphadenopathy (PGL) case were positive. Furthermore, among the
92 non- AIDS-related lymphoproliferative disorders, HHV-8
sequences were detected in 3 classic AILD cases and in 4
reactive lymphadenopathies. Six of 9 HHV-8 positive
lymphoid lesions (I NHL, 1 PGL, 1 AILD, and 3 reactive
lymphadenopathy cases) were also positive for Epstein-Barr
viral sequences. The four human immunodeficiency virus
(HIV) negative lymphadenopathies positive for HHV-8 sequences showed an almost identical histology, characterized by a predominantly follicular lesion, with giant germinal
center hyperplasia, and increased vascularity, resembling
HIV-related lymphadenopathy and MCD. Our results, while
providing the first evidence of the presence of HHV-8 sequences in AILD cases, suggest a possible association of
these herpesviral sequences with a distinct hystologic type
of non-neoplastic lymphadenopathy, not associated with
other common herpes infections.
0 1996 by The American Society of Hematology.
N
clear cells (PBMCs) of KS patients with and without AIDS”
notably in CD19’ B-cell fraction.”
In the attempt to investigate the possible occurrence of
HHV-8 in proliferative lesions other than KS, we judged it
appropriate to use PCR to look for HHV-8 sequences in a
large and well characterized series of benign, atypical, and
malignant lymphoid disorders both in immunocompetent and
in HIV positive subjects.
OVEL UNIQUE and specific DNA sequences were first
identified in Kaposi’s sarcoma (KS) tissues from patients with acquired immunodeficiency syndrome (AIDS).’
The reported sequences of the apparently KS-specific DNA
segments showed partial similarity to two capsid-protein
coding genes of known y-herpesviruses, namely EpsteinBarr virus (EBV) and herpesvirus Saimiri (HVS).’ The presence of these sequences argued in favor of the existence of
a new human herpesvirus called KS virus (KSHV) or human
herpesvirus-8 (HHV-8).* These herpesvirus-like sequences
could be amplified by polymerase chain reaction (PCR) in
the vast majority of KS biopsies, not only AIDS-associated,’
but also endemic3and
suggesting that this putative
new human herpesvirus is not solely an opportunistic infectious agent, but is possibly an essential agent involved in
all the different forms of KS. Of interest, recently HHV-8
sequences have been detected in various proliferative nonKS skin lesions affecting immunocompromised organ transplant patients,’ and in an unusual subgroup of AIDS-related
body-cavity-based B-cell lymphomas.*Recently, the KS-associated sequence has been identified in a significant number
of human immunodeficiency virus (HIV)-positive and negative cases of multicentric Castleman’s disease (MCD), which
is an atypical lymphoproliferation with a known increased
risk of developing KS.9.’”The presence of HHV-8 in lesions
other than KS suggests that, like other human herpesviruses,
HHV-8 might be harbored in latently infected cells and may
sometimes be activated not only by AIDS-related condit i o n ~ ? but
~ also by iatrogenic immunesuppressive conditions,’ although the natural reservoir of this virus in the
general population is unknown. However, the assumption
that the tropism of this as yet unisolated virus is not restricted
to KS tissues has been supported not only by the high sequence homology with EBV and HVS, which are mainly
lymphotropic, but also, importantly, by the recent identification of HHV-8 sequences in the peripheral blood mononu-
Btood, Vol 87, No 9 (May 1). 1996: pp 3903-3909
MATERIALS AND METHODS
Patient samples. We retrospectively analyzed pathologic tissues
obtained from a group of 131 benign and malignant lymphoid lesions
including 45 Hodgkin’s disease (HD) (42 non-AIDS- and 3 AIDSrelated), 43 non-Hodgkin’s lymphoma (NHL) (10 non-AIDS- and
33 AIDS-related), 20 HIV negative atypical lymphoproliferative disorder, and 23 benign lymphadenopathy (21 non-AIDS- and 2 AIDSrelated) cases (Table I). The distribution of HD cases according to
subtype was as follows: 23 mixed cellularity, I 1 nodular sclerosis,
From the Department of Medical Sciences, Section of Haematology, and the Department of Pathology, University of Modena, Italy;
and the Department of Biomedicine, Section of Virology, University
of Pisa, Italy.
Submitted September 19, 1995; accepted December 29, 1995.
Supported by grants from Associazione Italiana per la Ricerca
sul Cancro (AIRC) Rome, Italy; from Regione Emilia Romagna; and
from the Minister0 della Sanita. Istituto Superiore di Sanita, Progetto AIDS, to P. B.
Address reprint requests to Mario Luppi, MD, Dipartimento di
Scienze Mediche, Sezione di Ematologia, Policlinico- Via del
Pozzo, 71, 41100 Modena, Italy.
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.
0 1996 by The American Society of Hematology.
0006-4971/96/8709-0020$3.00/0
3903
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LUPPl ET AL
3904
Table 1. Screening for HHV-8 Sequences by PCR in
Lymphoproliferative Disorders in Non-AIDS and AIDS Patients
No. of
Diagnosis
Non-AIDS patients
Hodgkin's disease
mixed cellularity
nodular sclerosis
lymphocyte prevalence
lymphocyte depletion
Subtotal
Non-Hodgkin's lymphoma
B cell
T cell
Subtotal
Atypical lymphoproliferative disorders
Multicentric Castleman's disease
Angioimmunoblastic lymphadenopathy
Subtotal
Benign lymphadenopathies
Florid germinal center hyperplasia
Castleman's disease (localized, hyalinvascular type)
Paracortical lesion
Cat scratch disease
Toxoplasmosis
Subtotal
AIDS patients
Hodgkin's disease (mixed cellularity)
Non-Hodgkin's lymphoma
Persistent generalized lymphadenopathy
All patients
Cases
No. Positive
for HHVd
20
11
5
6
42
0
0
0
0
0
6
4
10
0
0
0
5
15
20
0
3
3
5
4
7
0
0
0
0
4
6
2
1
21
3
33
2
131
0
1
1
5 lymphocyte prevalence (diffuse), and 6 lymphocyte depletion. The
non-AIDS-related NHL cases were six of B- and four of T-cell
origin. Cellular pellets from three pleural and one peritoneal effusion
from four of these six HIV negative patients with B-NHL were also
available for molecular studies. The AIDS-related NHL cases were
all of B-cell origin and also included 20 cases of primary cerebral
lymphoma. The atypical lymphoproliferative disorders included I5
angioimmunoblastic lymphadenopathy with dysproteinaemia (AILD),
and 5 MCD cases. The series of 23 benign lymphadenopathies consisted of 12 cases with a predominantly follicular lesion, including
5 cases with florid germinal center hyperplasia and 7 cases of Castleman's disease of localized, hyalin-vascular type: 6 cases with a
predominantly paracortical lesion ( 2 of which with serologically
confirmed EBV induced infectious mononucleosis); 2 cases of persistent generalized lymphadenopathy (PGL); 2 cases with histologic
features consistent with cat scratch disease; and 1 case of serologically confirmed toxoplasmosis. Representative portions of each tissue specimen were routinely fixed in buffered formalin and embedded in paraffin; sections were stained with hematoxylin and eosin.
Immunohistochemistry. Monoclonal antibodies, including L26
(cluster of differentiation-CD20: DAKO Glostrup, Denmark) and
4KB5 (CD45 RA, DAKO) for B cells; polyclonal CD3 (CD3,
DAKO) and monoclonal UCHLI (CD45 RO, DAKO) for T cells:
polyclonal factor V11I-related antigen (DAKO) for endothelial cells:
monoclonal KP1 (CD 68, DAKO) for macrophages; and monoclonal
CD21 (DAKO) for follicular dendritic cells, were obtained commercially and used for standard immunohistochemical examination (avidin biotin method). Staining with the lectin Ulex europaeus lectin
agglutinin 1 (UEA-I; Vector, Burlingame, CA) was also performed.
Polymerase chain reaction (PCR) analysis. DNA was extracted
from cellular pellets from pleural and peritoneal effusions with a
modification of the technique described by Enrietto et al.': Crude
extracts were prepared from single 5 pm formalin-tixed and paraftinembedded tissue sections, as we previously reported.' ' Aliquots of
5 pL were submitted to PCR using a pair of specific primers. derived
from the HHV-8 sequences (KS330,,?), amplifying a fragincnt of
233 base pairs (bp) as described.'." Each 100 pL reaction contains
30 pmol of each primer, 2.5 U of Taq polymerase, 200 pmol/L each
of deoxynucleotide triphosphate, I O mmol/L Tris-HCI. 1.5 mmol/L
MgCl?, and 50 mmol/L KCI (pH 8.3). PCR conditions were as
follows: 94°C for 1 minute followed by 45 cycles of 94°C for 30
seconds, 58°C for 1 minute. and 72°C for 90 seconds. Reactions
were terminated by a 7-minute extension at 72°C. For each PCR
test the positive controls were represented by the crude extracts
obtained from I O HHV-8 positive AIDS- and non-AIDS-KS biopsies. All standard recommended procedures were performed in order
to avoid false positive results." PCR analysis was invariably performed on tissue sections derived from different paraffin blocks for
each patient. Negative controls, consisting of all reagents except
sample DNA, were also present during the crude extract preparation
and equalled or exceeded the number of the assayed samples. Primers
for the DQa gene were used as a positive amplification control."
The PCR products were analyzed on 1.5% ethidium bromide-stained
agarose gel, which allowed the direct visualization of the predicted
233 bp band. In order to confirm the viral origin of the amplified
DNA template, one-fifth of the PCR product was subjected to electrophoresis on a I .5% agarose gel blotted on Zeta-Probe GT Menibrane (Bio-Rad Laboratories, Hercules. CA) and hybridized with an
internal oligonucleotide probe specific for HHV-8,' end labeled with
y-32 P-ATP (Amersham-USB, Cleveland, OH)." To exclude amplification of other types of herpes virus, virus preparations of EBV,
herpes simplex virus I and 2 (HSV I , 2), cytomegalovirus (CMV),
and human herpesvirus 6 (HHV-6) were examined by PCR. using
the same HHV-8 primers, but no PCR products were obtained with
any of these virus strains.
Specific DNA sequences of HIV gag/pol genes were searched by
PCR as described.' B-cell clonality was investigated by PCR according to the procedure described by Trainor et al." The PCR
procedure we used for EBV detection and subtype characterization
has been previously described by Borisch et al."
Seyuerzcing o f PCR products. The HHV-8 PCR products were
subjected to direct sequence analysis by "cycling sequencing,"" a
variation of the classic dideoxy chain termination technique.Ix
Briefly, DNA bands were cut from a I% low-melting agarose gel and
further purified by a commercial kit (PCR Preps DNA Purification
System: Promega, Madison, WI). The cycle sequencing reaction was
performed in a total volume of 8 pL containing SO mmol/L KCI:
50 mmollL Tris-HCI, pH 8.3; 2.5 mmol/L MgCl?; I O pmol/L each
of dATP, dTTP, and dCTP: 20 pmol/L 7-deaza-dGTP: 0.4 pmol
"P-labeled primer: 0.2 U of Taq polymerase; 100 fmol of purified
PCR products: and 60 pmollL ddGTP, 400 pmol/L ddATP. 600
pmol/L ddTTP, or 200 pmollL ddCTP. The DNA was amplified in
20 cycles of I minute at 95"C, I minute at SYC, and 1 minute at
72°C. The reactions were stopped with 4 p L of 95% formamide. 20
mmol/L EDTA, 0.05% bromophenol blue, and 0.02% xylene cyanol.
The sequence products were analyzed on a 7 mol/L urea, 6%~polyacrylamide sequencing gel. The two strands and two independent
PCR products were sequenced to exclude mismatches due to polymerase mistakes.
RESULTS
The amplification of DQa gene was positive in all samples, suggesting the absence of major PCR inhibitors in crude
-
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HHV-8 IN AlLD AND BENIGN LYMPHADENOPATHIES
1
2
3
4 N C 5
6
7
3905
8 K S
-233 nt
9
10
11
NC 12 13 14 15 NC 16
m
17
18 19 NC 20 21
-233 nt
22 23 NC 24
0 -23313
25
26 27 28 NC 29 30 31 NC 32
-233 nt
KS 330233
Fig 1. Southern blot hybridization of the HHV-8 PCR products
(KS330233)with isotope-labeled KS330233sequence specific oligoprobe. Lanes: NC, negative controls; KS, Kaposi’s sarcoma specimen,
as positive control; 1-32, representative benign and malignant
lymphoid tissue samples, including all the HHV-8 positive reactive
lymphadenopathy (6, 10,14, 161, the AlLD (25, 27,28), the PGL (211,
and the AIDS-related NHL (24) cases.
extracts from tissue biopsy specimens. Crude extracts from
all the 131 benign, atypical. and malignant lymphoid lesions
shown in Table I were analyzed for the presence of HHV-8
(KS33OZTT)
sequences by PCR. Among the 38 AIDS-related
lymphoid lesions, agarose-gel electrophoresis of the PCR
products and subsequent Southern blot hybridization revealed the presence of the specific viral 233 bp fragment in
I of the 2 PGL and in 1 of the 33 B-NHL cases. while the
3 HD were negative. Unfortunately. clinical records were
not available, so we could not assess if the clinical features
of the positive B-NHL patient were similar to those reported
by Cesarman et al.’ Neither the 52 HIV negative lymphoma
specimens ( I O NHL and 42 HD), nor the 5 non-AIDSrelated MCD showed an amplified product. even after hybridization with an internal oligonucleotide probe (Table 1 ).
In particular, the lymphomatous effusions from four HIV
negative B-NHL patients did not harbor HHV-8 sequences.
Of interest. 3 lymph node biopsies of 15 HIV negative cases
with confirmed clinical and histologic diagnosis of AlLD
and, unexpectedly, 4 of 21 benign non-AIDS-related lymphadenopathies were clearly positive for HHV-8 sequences
(Fig I). In these HHV-8 positive specimens no HIV-I provirus DNA was detected by PCR. Moreover, we could not
document B-cell clonality in any of the 4 HHV-8 positive
reactive lymphadenopathies by PCR. thus confirming the
benign nature of the lymphoid hyperplasia in these cases.
To further characterize the relative amounts of HHV-8 DNA
present. we amplified serial dilutions of the crude extracts
from both the HHV-8 positive formalin-fixed lymph node
samples and the formalin-fixed KS biopsies used a s controls.
The load of HHV-8 DNA resulted in roughly similar results
in both the AIDS- ( I PGL and I NHL) and the non-AIDSrelated (3 AILD and 4 reactive lymphadenopathy cases)
lymphoid lesions as well as in the 10 KS biopsies examined
(data not shown).
Moreover. PCR products from all 9 HHV-8 positive samples were sequenced (Fig 2) and compared with the prototypic sequence originally derived from a genomic library
made from a Kaposi’s sarcoma lesion.’ PCR products from
three reactive lymphadenopathies (cases 6. 10, and 16)
showed five base-pair substitutions (at positions 46. 47. 69,
146. and 153). Base-pair changes at positions 46 and 47
code for a proline-to-isoleucine substitution and a base-pair
change at position 146 codes for an aspartate-to-glycine substitution, as compared with the prototypic sequence. The
remaining base-pair changes do not involve amino acid substitutions. PCR products from one reactive lymphadenopathy
(case 14), from the three AlLD (cases 25, 27, 28). from thc
PGL (case 21). and from the AIDS-related NHL (case 24)
samples revealed three base-pair substitutions (at positions
47, 100, and 153). The base-pair change at position 47 codes
for a proline-to-leucine substitution. while the other two base
changes are isocoding alterations from the prototypic coding
sequence. The high degree of sequence conservation among
PCR products suggests that the sequences obtained by PCR
from our four reactive lymphadenopathy cases. three AILD.
one PGL. and one AIDS-related NHL case correspond to
the same putative agent previously identified in Kaposi’s
sarcoma.’
Histologically, the three AlLD cases positive for HHV-8
sequences showed classic features. including complete effacement of the lymph node architecture. abundance of small
arborizing vessels, and a polymorphous infiltrate. in strict
adherence to the original criteria of Frizzera et al for the
diagnosis of AILD.’” In two cases the predominant cell population was represented by CD3 positive peripheral T cells,
with very few B cells in a scattered pattern. but with particularly abundant plasma cells. In one case the portion of CD20
positive B cells was particularly elevated. forming the 35%of the mononuclear component.
It should be noted that careful examination of the four
positive cases of reactive lymphadenopathy showed identical
histologic features. In details. follicles of different shape and
dimensions occupied the whole cross-sectional area of each
lymph node. pushing residual sinuses at the periphery. The
majority of follicles appeared hyperplastic with giant germinal centers. surrounded by markedly attenuated mantle
zones. often partially lost. Adjacent germinal centers tended
to coalesce one with another, acquiring bizarre, “geographic” shapes (Figs 3A and 4A). Tangible body macrophages were numerous within germinal centers, giving them
a “starry sky” appearance (Fig 4A and B). In addition, small
involuted follicles were also present, constituting approximately 30% to 40% of
follicular population. They were
:!I.%
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
3906
LUPPl ET AL
50
TTGACCCCGT
TTGAWCGT
TTGAWCGT
TTGACCTCGT
TTGACUCGT
TTGACCTCGT
TTGACCTCGT
TTGACCTCGT
TTGACCTCGT
TTGACCTCGT
70
GTCGTGCCGC
GTCGTGCCTC
GTCGTGCCTC
GTCGTGCCGC
GTCGTGCCTC
GTCGTGCCGC
GTCGTGCCGC
GTCGTGCCGC
GTCGTGCCGC
GTCGTGCCGC
AGCAACTGGG
AGCAACTGGG
AGCAACTGGG
AGCAACTGGG
AGCAACTGGG
AGCAACTGGG
AGCAACTGGG
AGCAACTGGG
AGCAACTGGG
AGCAACTGGG
CGGCCGGGGC
CGGCCGGGGC
CGGCCGGGGC
CGGCCGGGGC
CGGCCGGGGC
CGGCCGGGGC
CGGCCGGGGC
CGGCCGGGGC
CGGCCGGGGC
CGGCCGGGGC
150
CCCGGATGAT
CCCGGBTGAT
CCCGGBTGAT
CCCGGATGAT
CCCGGaTGAT
CCCGGATGAT
CCCGGATGAT
CCCGGATGAT
CCCGGATGAT
CCCGGATGAT
GTAAATATGG
GTCAATATGG
GTCAATATGG
GTCAATATGG
GTCAATATGG
GTCAATATGG
GTCAATATGG
GTCAATATGG
GTCAATATGG
GTCAATATGG
220
CGTCTGGACG
CGTCTGGACG
CGTCTGGACG
CGTCTGGACG
CGTCTGGACG
CGTCTGGACG
CGTCTGGACG
CGTCTGGACG
CGTCTGGACG
CGTCTGGACG
TAGACAACAC
TAGACAACAC
TAGACAACAC
TAGACAACAC
TAGACAACAC
TAGACAACAC
TAGACAACAC
TAGACAACAC
TAGACAACAC
TAGACAACAC
10
20
30
40
HHV-8
CASE 6
CASE 10
CASE 14
CASE 16
CASE 21
CASE 24
CASE 25
CASE 27
CASE 28
AGCCGAAAGG
AGCCGAAAGG
AGCCGAAAGG
AGCCGAAAGG
AGCCGAAAGG
AGCCGAAAGG
AGCCGAAAGG
AGCCGAAAGG
AGCCGAAAGG
AGCCGAAAGG
ATTCCACCAT
ATTCCACCAT
ATTCCACCAT
ATTCCACCAT
ATTCCACCAT
ATTCCACCAT
ATTCCACCAT
ATTCCACCAT
ATTCCACCAT
ATTCCACCAT
TGTGCTCGAA
TGTGCTCGAA
TGTGCTCGAA
TGTGCTCGAA
TGTGCTCGAA
TGTGCTCGAA
TGTGCTCGAA
TGTGCTCGAA
TGTGCTCGAA
TGTGCTCGAA
TCCAACGGAT
TCCAACGGAT
TCCAACGGAT
TCCAACGGAT
TCCAACGGAT
TCCAACGGAT
TCCAACGGAT
TCCAACGGAT
TCCAACGGAT
TCCAACGGAT
90
100
110
120
130
140
HHV-8
CASE 6
CASE 10
CASE 14
CASE 16
CASE 21
CASE 24
CASE 25
CASE 27
CASE 28
GCACGCTATT
GCACGCTATT
GCACGCTATT
GCACGCTATT
GCACGCTATT
GCACGCTATT
GCACGCTATT
GCACGCTATT
GCACGCTATT
GCACGCTATT
CTGCAGCAGC
CTGCAGCAGC
CTGCAGCAGC
CTGCAGCAGT
CTGCAGCAGC
CTGCAGCAGT
CTGCAGCAGT
CTGCAGCAGT
CTGCAGCAGT
CTGCAGCAGT
TGTTGGTGTA
TGTTGGTGTA
TGTTGGTGTA
TGTTGGTGTA
TGTTGGTGTA
TGTTGGTGTA
TGTTGGTGTA
TGTTGGTGTA
TGTTGGTGTA
TGTTGGTGTA
CCACATCTAC
CCACATCTAC
CCACATCTAC
CCACATCTAC
CCACATCTAC
CCACATCTAC
CCACATCTAC
CCACATCTAC
CCACATCTAC
CCACATCTAC
TCCAAAATAT
TCCAAAATAT
TCCAAAATAT
TCCAAAATAT
TCCAAAATAT
TCCAAAATAT
TCCAAAATAT
TCCAAAATAT
TCCAAAATAT
TCCAAAATAT
170
180
190
200
210
HHV-8
CASE 6
CASE 10
CASE 14
CASE 16
CASE 21
CASE 24
CASE 25
CASE 27
CASE 28
CGGAACTTGA
CGGAACTTGA
CGGAACTTGA
CGGAACTTGA
CGGAACTTGA
CGGAACTTGA
CGGAACTTGA
CGGAACTTGA
CGGAACTTGA
CGGAACTTGA
TCTATATACC
TCTATATACC
TCTATATACC
TCTATATACC
TCTATATACC
TCTATATACC
TCTATATACC
TCTATATACC
TCTATATACC
TCTATATACC
ACCAATGTGT
ACCAATGTGT
ACCAATGTGT
ACCAATGTGT
ACCAATGTGT
ACCAATGTGT
ACCAATGTGT
ACCAATGTGT
ACCAATGTGT
ACCAATGTGT
CATTTATGGG
CATTTATGGG
CATTTATGGG
CATTTATGGG
CATTTATGGG
CATTTATGGG
CATTTATGGG
CATTTATGGG
CATTTATGGG
CATTTATGGG
GCGCACATAT
GCGCACATAT
GCGCACATAT
GCGCACATAT
GCGCACATAT
GCGCACATAT
GCGCACATAT
GCGCACATAT
GCGCACATAT
GCGCACATAT
60
GTTCCCCATG
GTTCCCCATG
GTTCCCCATG
GTTCCCCATG
GTTCCCCATG
GTTCCCCATG
GTTCCCCATG
GTTCCCCATG
GTTCCCCATG
GTTCCCCATG
80
160
230
GGA
GGA
GGA
GGA
GGA
GGA
GGA
GGA
GGA
GGA
Fig 2. DNA sequences for KS33OPs PCR products amplified from all the HHV-8 positive cases. Two genotypes were found that differed
from the prototypic sequence of HHV-8. PCR products from three reactive lymphadenopathycases (6, IO, 16) had five base-pair substitutions,
while products from one reactive lymphadenopathy(14). three AILD (25,27,28), one PGL (21). and one AIDS-related NHL (24) case had three
base-pair substitutions (substitutionsare bold and underlined).
mainly composed of CD2 1-positive dendritic reticulum cells
and residual large lymphoid cells, frequently devoid of a rim
of mantle lymphocytes. Numerous small blood vessels were
invariably present in interfollicular areas and particularly
abundant in one case (Fig 3B). Focal collections of cells
exhibiting abundant, pale cytoplasm and oval, indented nuclei with small nucleoli were clearly recognizable; such cells
expressed CD20 and were interpreted as “monocytoid B
cells” (Fig 4A and B).
Examination of the clinical records from the four patients
(18, 32, 41, and 30 years old, respectively, 3 male and 1
female) with HHV-8 positive lymphadenopathies revealed
that in all cases lymph node enlargement was localized (in
axillary region in 1 patient and in neck region in the others)
and not accompanied by hepatosplenomegaly. Fever or constitutional symptoms were absent, while no laboratory abnormalities indicative of viral or other infections and inflammation were documented. Serology for common herpes viruses
like EBV, CMV, HSV 1 and 2 and for common hepatitis
viruses was consistently negative. Seronegativity for HIV
infection was repeatedly confirmed at 6-month intervals by
ELISA, for at least 2 years. The patients did not have a
history of KS at the time of lymph node enlargement and
have not developed KS, after a 4 to 6 year follow-up. In two
patients marked thrombocytopenia was present at the time
of lymph node enlargement: in one patient thrombocytopenia
persisted and the diagnosis of idiopathic thrombocytopenic
purpura was finally made; in the other patient the platelet
count became normal in 2 months, without therapy.
Our series of non- AIDS-related benign lymphadenopathies included a further case with similar histologic features
but negative for HHV-8. Of interest, these herpesviral sequences were absent in all the other 16 benign lymph node
biopsies examined, which showed significantly different histologic features, including 7 cases of Castleman’s disease of
localized type, 6 cases with predominantly paracortical lesions typically seen in viral infections, 2 cases with histologic features consistent with cat scratch disease, and 1 case
of serologically confirmed toxoplasmosis.
EBV sequences were identified in 6 of the 9 HHV-8 positive cases, in particular in both the AIDS-related lesions (1
NHL and 1 PGL), in 1 of 3 AILD, and in 3 of 4 benign
lymphadenopathy cases. EBV subtype characterization could
be performed only in three cases. It showed B type in one
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
HHV-8 IN AlLD AND BENIGN LYMPHADENOPATHIES
.
L;-.-*Srr,
3907
.....
or not, but also in a large series of HD cases, representative
of all histologic subtypes. In fact, only one AIDS-related
NHL, typed histologically as diffuse large B-cell lymphoma,
was positive for HHV-8. The pleural and peritoneal lymphomatous effusions obtained from four HIV negative B-cell
NHL patients were negative, confirming the apparent restriction of the presence of HHV-8 sequences to lymphomatous
effusions occurring in the rare body-cavity-based B-NHL of
HIV positive patients.
Fig 3. Reactive lymphadenopathy positive for HHV-8 sequences.
Case 14. (AI Germinal centers are enlarged and irregularly shaped,
while mantle zones are partially lost (hematoxylin-eosin, original
magnification x201. (Bl Numerous small blood vessels are present in
interfollicular areas (hematoxylin-eosin, original magnification x200).
benign lymphadenopathy and in the NHL case, while a mixture of A and B types was present in the PGL case (data not
shown).
DISCUSSION
In a recent study, Cesarman et a18 investigated the presence of HHV-8 sequences in a series of AIDS- and nonAIDS-related lymphoid lesions, which included only malignant lymphomas, mainly NHLs. Out of 193 cases examined,
they could identify these sequences only in an unusual subgroup of 8 HIV positive body-cavity-based lymphomas,
characteristically showing pleural, pericardial, or peritoneal
lymphomatous effusions, two of which also showed a history
of KS.ROur study confirmed the infrequent occurrence of
these herpesviral sequences not only in NHLs, AIDS-related
Fig 4. Reactive lymphadenopathy positive for HHV-8 sequences.
Case 16. (A) Giant, irregularly shaped germinal centers with "starry
sky" pattern. Clusters of monocytoid B cells are seen in interfollicular
areas (hematoxylin-eosin, original magnification x201. (Bl Enlarged
germinal center with focal accumulation of monocytoid B cells on
one side (hematoxylin-eosin, original magnification x 120).
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3908
The detection of these herpesviral sequences in pathologic
lymph node tissues from three patients with classic clinical
and histologic diagnosis of AILD is the first evidence of this
novel herpesvirus in this rare type of atypical lymphoproliferative disorder. Although the cause of AILD is unknown
and is likely to vary in different subjects, a common feature
is excessive immune activity, and much speculation has centered on the possibility that a viral infection could directly
or indirectly trigger this disorder. In particular, human herpesviruses like EBVZ0,”and HHV-6,I3 have been found to
be associated with AILD. Thus, it is not totally unexpected
that these new herpesviral sequences, belonging to the family
of lymphotropic y-herpesviruses, are found at least in some
cases of the disease. However, it is not possible to rule out
that HHV-8 sequences detected in these cases represent mere
passengers in AILD lesions, resulting either from reactivation or primary infection induced by the immunosuppression.
On the other hand, the disease etiologically related to the
presence of HHV-8 sequences, namely KS, is itself characterized by chronic immune activation and release of inflammatory cytokines, so that common immune dysfunctions underlying both disorders may be hypothesized.” Furthermore,
at least nine well-documented cases of KS have been reported in patients with AILD,” and vascular hyperplasia is
characteristically observed in both pathologic lesions. The
other atypical lymphoproliferative disorder known to be associated with an increased risk of developing KS is the multicentric giant lymph node hyperpla~ia,’~
now commonly defined as MCD.24Rather unexpectedly, the five cases of HIV
negative MCD available for this study were also negative
for the presence of HHV-8 sequences. The small number of
cases examined, all collected at the early stage of the disease,
might explain the discrepancy with the results reported by
Soulier et al: who found 7 positive out of 17 HIV negative
MCD cases examined.
The most unexpected finding is, however, the identification of these herpesviral sequences in four of five cases of
HIV negative reactive lymphadenopathies, all showing the
same histologic features, characterized by florid germinal
center hyperplasia (giant follicles) associated with various
degrees of condensation and regression of germinal centers
(follicular involution), in the presence of increased vascularity and of “monocytoid B cell” hyperplasia. The above
described lymphoid lesion harboring these sequences is predominantly a follicular lesion, which rarely occurs in the
course of herpes infections.” In fact, in EBV-induced infectious mononucleosis, as well as in other lymphadenitis
caused by cytomegalovirus or herpes simplex virus, the prototypical histologic lesion is predominantly a diffuse paracortical expansion, with an abundance of immunoblasts and
high endothelial venules, usually with small germinal cent e r ~ . The
* ~ six reactive lymphadenopathies with a predominantly paracortical lesion in our series were all negative for
HHV-8 sequences. Similarly, HHV-8 sequences were absent
in all seven cases of hyaline vascular, localized type CD,
showing a typical follicular lesion, which significantly differs from the giant germinal center hyperplasia.*’
The histologic features of florid germinal center hyperpla-
LUPPl ET AL
sia invariably recognized in the four HHV-8 positive and
HIV negative benign lymphadenopathies may be observed
in a spectrum of other clinical conditions, like autoimmune
disorders (especially rheumatoid arthritis, and Sjogren’s syndrome), MCD and HIV infe~tion.’~
In fact, one of two AIDSrelated cases of PGL included in our study was positive for
the same herpesviral sequences. Similarly, Chang et al’
found HHV-8 sequences in 3 of 12 benign HIV lymph nodes.
In addition, vascular hyperplasia is also a common feature
of all the HHV-8 positive lesions identified so far, including
KS, MCD, AILD, and the above described benign lymphadenopathies. In other words, the histologic features recognized
in these HHV-8 positive cases of reactive lymphadenopathy
represent a defined pattern of response of the lymph node,
possibly induced by a wide range of antigenic stimuli. HHV8 should now be considered in the differential diagnosis of
the possible causes for such a response. We are aware of
the fact that the assessment of a causal link between these
four cases of reactive lymphadenopathies and HHV-8 infection would require viral isolation and also serologic confirmation. However, the identification of these herpesviral sequences in four of five cases of apparently benign
lymphadenopathies with shared typical histologic features,
suggests that this distinct histologic pattern of lymphoid response induced by HHV-8 is likely to reflect the original
pathogenic potential of this herpesvirus in HIV negative subjects. It is worth mentioning that Soulier et al’ reported the
presence of HHV-8 in one reactive axillary lymph node,
with pathologic evidence of follicular hyperplasia, from a
clinically asymptomatic HIV negative subject, which seems
similar to our cases. These cases may thus reveal the possible
clinico-pathologic entity, benign in nature, which might occur during the primary HHV-8 infection in normal subjects;
similarly, infectious mononucleosis represents the most common clinical manifestation of EBV infection in the general
population.
The possibility still remains that HHV-8 may exhibit an
indirect transforming ability in conjunction with other factors, like EBV, as it has been proposed for the unusual
subgroup of AIDS-related body-cavity-based lymphomas.x
The only one AIDS-related HHV-8 positive NHL patient
in our series harbored EBV sequences, but we could also
document the coinfection with both herpesviruses in five
nonmalignant lymphoid lesions. These data clearly indicate
that the presence of EBV together with HHV-8 is not necessarily associated with the full neoplastic phenotype.
Finally, it also should be noted that all four patients with
HHV-8 positive lymphadenopathies are from the areas of
Italy with the highest incidence of classic KS (3 from the
Po valley and 1 from Si~ily).’~
Therefore, the possibility is
also raised that the unexpectedly higher frequency of HHV8 sequences in our series of reactive lymphadenopathies is
due, at least in part, to the different geographic distribution
of the putative KS-associated virus and to its possibly higher
frequency in the Italian population.
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