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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3825-3832
Demonstration of Kaposi's Sarcoma-Associated Herpes Virus Cyclin D
Homolog in Cutaneous Kaposi's Sarcoma by Colorimetric In Situ
Hybridization Using a Catalyzed Signal Amplification System
By
Jon A. Reed,
Roland G. Nador,
David Spaulding,
Yoichi Tani,
Ethel Cesarman, and
Daniel M. Knowles
From the Department of Pathology, The New York Hospital-Cornell
Medical Center, New York, NY; and Dako Corp, Carpinteria, CA.
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ABSTRACT |
Kaposi's sarcoma-associated herpes virus (KSHV)/human herpes virus
8 (HHV8) DNA sequences have been demonstrated in Kaposi's sarcoma
(KS), as well as in some acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (NHL) and in multicentric Castleman's disease. Although KSHV DNA generally is abundant in KSHV-associated lymphomas, few copies of the virus are present in KS, a
property that confounds detection by in situ methods. Previous in situ
studies, which identified KSHV in lesions of KS, relied on the use of
polymerase chain reaction (PCR) to amplify target DNA sequences before
in situ hybridization (ISH) for localization or used ISH with
radioactively-labeled probes to obtain adequate levels of detection
sensitivity. In this study, a novel nonisotopic nucleic acid ISH method
using catalyzed signal amplification and colorimetric detection without
PCR-dependent target amplification was used to identify KSHV-specific
sequences. The level of sensitivity was increased further by using a
probe that detects viral cyclin D homolog transcripts, which are
expressed at significant levels during latent viral infection. Thirty
cutaneous lesions of KS (25 AIDS-related and five classical European
type) were evaluated. AIDS-related NHL and cell lines derived from
patients with AIDS-related NHL, all of which were known to harbor KSHV
by Southern blot analysis, were used as positive controls. NHL and
benign cutaneous vascular lesions not associated with AIDS were used as
negative controls. For each of the 30 KS lesions studied, hybridization
signals were detected in most of the spindle cells surrounding the
atypical slit-like vascular channels and also were detected in some
endothelial cells in well-formed blood vessels in the perilesional
dermis. Plaque and nodular lesions generally contained more labeled
cells than did early patch lesions. All AIDS-related NHL and cell lines contained KSHV-specific sequences; however, the non-AIDS-related NHLs
and benign vascular lesions were negative. These results confirm the
presence of KSHV sequences in cutaneous KS and provide in situ evidence
of infection by this virus in early patch-stage lesions. This study
also defines the in situ expression of the KSHV cyclin D homolog viral
oncogene in cutaneous KS. The use of this sensitive nonisotopic ISH
method should allow detection of other KSHV-specific gene products,
further defining the pathobiology of this virus.
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INTRODUCTION |
KAPOSI'S SARCOMA-ASSOCIATED herpes virus
(KSHV) is a recently-discovered gammaherpes virus1 also
called human herpes virus 8 (HHV8).2 KSHV DNA sequences
have been demonstrated in Kaposi's sarcoma (KS),1 in an
unusual form of acquired immunodeficiency syndrome (AIDS)-related
non-Hodgkin's lymphoma (NHL), which usually presents as an
effusion3-5 and in a significant percentage of multicentric
Castleman's disease.6 Furthermore, KSHV has been identified in all forms of KS including AIDS-related (epidemic), classical European, endemic, and posttransplant
(iatrogenic),1,7-13 suggesting a specific etiologic role
for this virus in the pathogenesis of KS. To better define this role,
the stage of tumor progression during which specific virally-encoded
genes are expressed must be determined. For KS, histologic evidence of
tumor progression is recognized as the evolution of early patch-stage
lesions into larger plaques and ultimately into nodules and
tumors.14 Several in situ hybridization (ISH) studies have
demonstrated KSHV viral DNA sequences in lesions of
KS15-17; however, these studies evaluated later plaque- and
nodular/tumor-stage lesions exclusively and relied on labor-intensive
procedures using the polymerase chain reaction (PCR) ISH15
or ISH using radioactively labeled probes.16,17 These
highly sensitive ISH techniques were necessary because very few copies of the virus are present in KS lesions,1 thus confounding
detection by other less sensitive ISH methods. However, despite the
enhanced sensitivity of detection conferred by radiolabeled probes,
KSHV was not detected in all of the KS lesions in some of these ISH studies.16,17 Greater sensitivity was achieved by Boshoff
et al15 who successfully identified KSHV in all of the
plaque- and nodular/tumor-stage lesions studied by PCR-ISH and more
recently, Staskus et al18 who used radioactively labeled
probes to identify KSHV-specific sequences in all of their KS specimens
including one very early patch-stage lesion.
In this study, cutaneous lesions of KS representing early patch-stage,
plaque-stage, and late nodular/tumor-stage lesions were evaluated by
ISH for the presence of KSHV-specific nucleic acid sequences. A novel
ISH method based on colorimetric detection of a biotinylated
KSHV-specific probe after catalyzed signal
amplification19-21 was used. This ISH method is based on
the catalyzed amplification of a detection system that does not require
PCR amplification of nucleic acid target sequences or the use of
radioactively labeled detection probes. Recently, this technique has
been used to identify human papillomavirus-specific sequences in
infected cell lines and in human tissues.22 Because it is
likely that KSHV primarily exists in a latent state in KS lesional
tissues,18,23,24 a latently-expressed gene was targeted in
this ISH study to increase the sensitivity of viral detection. Large
segments of the KSHV genome have been mapped and
sequenced.2,25,26 Among the open reading frames (ORF)
identified for KSHV is ORF72, a gene that encodes a protein with 257 amino acids and which shares homology with a number of human cellular
cyclins including cyclin D125,27 and which is now
designated v-cyc. Cyclins are a class of proteins, which are
required for normal cellular proliferation,28 and cyclin D1
is now recognized as the PRAD1 oncogene implicated in a number of human
malignancies.29-32. In vitro studies on cell lines
harboring KSHV33 have shown that v-cyc is a
latently-transcribed gene,34 which is expressed at
significant levels in KS tissues.25 This gene
encodes a protein, which is functional in its ability to phosphorylate
and thus inactivate the retinoblastoma tumor suppressor
protein.27,35 The in situ demonstration of KSHV cyclin D
RNA sequences in early lesions of KS would, in addition to confirming
the presence of the virus, provide further evidence linking KSHV to
specific early steps in the pathogenesis of these lesions.
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MATERIALS AND METHODS |
Tissues and cell lines.
Formalin-fixed, paraffin-embedded tissues used in these experiments
were obtained from the files of the Department of Pathology, The New
York Hospital-Cornell Medical Center. Thirty lesions of cutaneous KS
including 25 from patients with AIDS and five classical-type lesions
from human immunodeficiency virus (HIV)-negative patients of European
descent were included in the study (Table 1). The 30 specimens included 10 early patch-stage, 10 plaque-stage, and 10 nodular/tumor-stage lesions. As a positive control, three AIDS-related NHL, which had solid tissue involvement and which were known to contain
KSHV sequences by Southern blot analysis, also were studied. As
negative controls, five NHL not associated with AIDS, one cutaneous lymph node demonstrating reactive follicular hyperplasia, and five
benign cutaneous hemangiomas were studied. All tissues were cut 5 µm
thick on positively-charged glass slides (ProbeOn Plus, Fisher
Scientific, Pittsburgh, PA).
Cell lines established from three patients with AIDS-related primary
effusion lymphomas (PEL) and previously shown to harbor KSHV33,36 also were used as positive controls for ISH.
These cell lines (BC-1, BC-2, and BC-3) were grown to a density of 0.3 to 0.5 × 106 cells/mL in RPMI 1640 supplemented with
2 mmol/L glutamine and 20% fetal bovine serum, 37°C in the
presence of 5% CO2. The cells were harvested, washed in
1× phosphate-buffered saline (PBS), fixed in 10% phosphate buffered
formalin for 1 hour, mixed with an equal volume of molten agar (final
concentration, 2% wt/vol), allowed to solidify, and then routinely
processed to produce a cell block from which paraffin sections were
cut. In addition, a B-cell line (BJAB) known to lack KSHV, was used as
a negative control for the mixing control described below.
High temperature target retrieval.
Before ISH, the tissue sections were deparaffinized, rehydrated through
a graded series of alcohols, and incubated for 40 minutes in Target
Retrieval Buffer (Dako Corp, Carpinteria, CA), which had been preheated
and maintained at 95°C. The slides were allowed to cool at room
temperature (RT) for 20 minutes before removal from the target
retrieval buffer. The tissue sections next were incubated for 20 minutes in 0.2 N HCl, RT followed by a 3-minute wash in distilled
H2O (dH2O). Endogenous peroxidase activity was
blocked in the tissue sections by incubation in a 3% aqueous solution
of H2O2 for 20 minutes, RT followed by a
3-minute wash in dH2O. After blocking endogenous peroxidase
activity, tissue sections were used immediately for ISH.
Southern blot analyses.
For the three AIDS-related NHL and the three cell lines used in this
study, fresh samples were available for DNA extraction and subsequent
Southern blot analyses. Genomic DNA was extracted from cryopreserved
tissue blocks or cell pellets using a salting-out procedure.37 Five-microgram aliquots of genomic DNA were
digested with BamH I or Hind III restriction
endonucleases according to the manufacturer's instructions
(Boehringer-Mannheim, Indianapolis, IN). DNA fragments were separated
by electrophoresis in 0.8% agarose gels, denatured with alkali,
neutralized, and transferred to nitrocellulose filters according to the
method of Southern.38 The filters were hybridized as
previously described39 with 32P random
primer-labeled (PrimeIt; Stratagene, La Jolla, CA) KS330Bam and
KS631Bam probes25 to detect the presence of
KSHV-specific sequences.
ISH probes.
A 115-bp DNA fragment of the KSHV/HHV8 ORF 72 (cyclin D homolog) was
amplified by PCR from DNA of the BC-3 cell line using previously
published primers25 and PCR conditions.1 The
amplification product was purified (QIAquick Spin, Qiagen Inc,
Chatsworth, CA), quantified, and an aliquot was run on a 0.8% agarose
gel (Fig 1) to verify fragment size and
uniformity. The remaining PCR product was biotinylated (Fasttag kit,
Vector Laboratories, Burlingame, CA) using the manufacturer's
protocol. Biotinylated probes were purified by two cycles of gel
filtration using Sephadex G50-packed columns (Pharmacia Biotech Inc,
Piscataway, NJ) equilibrated with TNE buffer (100 mmol/L NaCl, 10 mmol/L Tris, pH 7.5, 1 mmol/L EDTA).
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| Fig 1.
PCR-generated KSHV cyclin D probes derived from BC-3
(lanes 3 to 6). Each lane represents a PCR reaction product. Reaction products were pooled before biotinylation. Hind III-digested
lambda phage DNA and Hae III-digested  X174 DNA mixture used
as a molecular weight standard (lane 1). The Hae III-digested
X174 DNA was labeled and used as the irrelevant specificity probe
mixture, as these fragments were of similar size to the KSHV probe.
H2O control (lane 2). PCR-generated probes were prepared
and labeled as described in the text.
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Irrelevant specificity probes were prepared by biotinylation of an
aliquot of a cocktail of linearized Hae III-digested  X174 DNA routinely used as DNA molecular weight markers in agarose gels
(Life Technologies, Gaithersburg, MD). This probe mixture was labeled
and purified using the same protocols used for the KSHV cyclin
D-specific probe.
ISH.
All tissues used for ISH were deparaffinized, rehydrated through a
graded series of ethanol, submitted to high temperature target
retrieval, and blocked of endogenous peroxidase activity as described
above. A step using enzymatic digestion of the tissue before
hybridization was not required in this protocol. Rehydrated tissue
sections then were prehybridized with probe cocktail: final concentration 2× SSC (1× SSC = 150 mmol/L NaCl, 15 mmol/L sodium citrate); 10× Denhardt's solution40; 25% vol/vol
ultrapure formamide (Life Technologies), 30% vol/vol low viscosity
probe diluent41 (Research Genetics, Huntsville, AL), and
0.8 mg/mL sheared herring sperm DNA. For prehybridization, 30 µL of
probe cocktail was placed directly onto the tissue section and a glass
coverslip was applied to evenly distribute the cocktail making sure
that bubbles were not introduced. The slides were heated for 5 minutes
on the surface of a 95°C hotplate then incubated for 30 minutes,
37°C in a humidified chamber. After the prehybridization step, the
coverslips were gently removed by immersion of the slides in Coplin
jars containing Tris-buffered saline (TBST) (150 mmol/L NaCl, 10 mmol/L
Tris, pH 7.5, 0.1% vol/vol Tween 20) for 5 minutes, RT. Excess buffer
was carefully removed by wiping the slide around the tissue sections
with absorbent paper. A 30-µL aliquot of probe cocktail containing
the biotinylated KSHV Cyclin-specific probe (0.1 µg/mL) was then
applied to the tissue sections and covered with a glass coverslip. The
slides were heated for 5 minutes, 95°C on a hotplate to denature
both probe and target sequences and then incubated for 1 hour, 37°C
in a humidified chamber to allow hybridization. Coverslips were gently
removed by soaking in TBST for 5 minutes, and the sections were washed
twice for 5 minutes in 1× SSC, 55°C and then twice under
conditions of greater stringency, 0.01× SSC, 55°C, 10 minutes.
Washes and subsequent signal amplification and detection system
reagents (see below) were applied to the tissue sections by capillary
action.42
As a control to evaluate the specificity of hybridization,
representative tissue sections were hybridized with a cocktail of
biotinylated probes of irrelevant specificity. The irrelevant specificity probes were prepared as described above and were used at
the same concentration as the KSHV-specific probe. In addition, selected tissues containing KSHV sequences as determined by ISH and/or Southern blot hybridizations were digested with
DNAse-free Ribonuclease A (Boehringer Mannheim), 1 mg/mL, for 1 hour,
37°C before the high temperature target retrieval step. This
control was performed to demonstrate that hybridizations were
predominantly between the probe and cytoplasmic mRNA sequences in these
tissues.
An additional control was performed to ensure that there was no
diffusion of hybridization signals between cells. For this control, a
B-cell line known to harbor KSHV (BC-3) was mixed in varying
percentages with another B-cell line, which does not contain the virus
(BJAB). Cell blocks and paraffin sections were prepared as described
previously from the cell suspension mixures, followed by ISH with the
KSHV cyclin probe. As a further test of labeling fidelity, the brown
DAB colorimetric signal was further intensified (rendering it dark
brown-to-black) by a 15-second incubation in NiCl2 (Vector
Laboratories).
Catalyzed signal amplification.
A nonisotopic, colorimetric signal amplification system (GenPoint kit,
Dako Corp) was used in these experiments to visualize specific
hybridization signals. The procedure was followed according to the
manufacturer's recommendations. For this procedure, after the
stringent washes, tissue sections first were incubated with a
streptavidin-horseradish peroxidase (SA-HRP) reagent for 15 minutes, RT
and washed three times with TBST. After this step, the
sections were incubated with a solution containing
H2O2 and biotinyl tyramide19,20 for
15 minutes, RT and washed three times with TBST. This step
results in the catalyzed deposition of additional biotins at the site
of probe hybridization. The sections were then incubated a second time
with SA-HRP for 15 minutes, RT and finally washed three
times in TBST. Colorimetric signals were localized after
incubation in 3,3'diaminobenzidine (DAB) for 5 minutes, RT. Signal
development was stopped by placing the slides in dH2O.
Hybridizations were identified as punctate brown colorimetric signals
in the cellular cytoplasm and/or nucleus. Nuclear signals were
considered to represent probe-target DNA hybridizations, whereas
cytoplasmic signals were considered to represent primarily probe-target
mRNA hybridizations. Tissue sections were counterstained with
hematoxylin and permanently mounted. For KS lesions, sections also were
counterstained with toluidine blue so that melanin in epidermal
keratinocytes and dermal melanophages would turn green and be easily
distinguishable from the brown DAB reaction product. Mounted tissue
sections were viewed with a standard light microscope using bright
field optics. All slides were interpreted by four pathologists (J.A.R.,
R.G.N., E.C., and D.M.K.).
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RESULTS |
Results of the ISH for KS tissues are shown in
Figs 2 and
3. All 30 KS lesions contained
colorimetric hybridization signals. Signals typically were located in
the spindle cell component surrounding the characteristic slit-like
vascular spaces of the lesions, in the endothelial cells of well-formed
blood vessels in the surrounding dermis, and in small mononuclear cells
scattered throughout the lesions. The lesional spindle cells usually
contained many cytoplasmic hybridization signals, but only one or very
few nuclear signals. Early patch-stage lesions generally had fewer
signals than plaque-stage or nodular lesions, as these also had fewer
spindle cells (Figs 2A and B and 3A). The intensity of labeling per
cell did not differ significantly between patch-stage and later-stage
lesions. Endothelial cells within well-formed vessels in the
surrounding dermis typically contained abundant cytoplasmic and nuclear
hybridization signals (Fig 2C). Of interest, not all of the blood
vessels in the surrounding dermis contained labeled endothelial cells.
In a few plaque-stage and nodular lesions, scattered epidermal
keratinocytes or eccrine ductular epithelial cells contained
hybridization signals as well. Only a few epidermal keratinocytes,
limited to small foci within the epidermis, were labeled, and these
usually contained only a few hybridization signals (Fig 2D). Some of
the labeled keratinocytes were surrounded by nonlabeled cells
indicating specific localization of the hybridization signal without
diffusion. Rare eccrine ductular epithelial cells, which were labeled,
typically had larger numbers of signals both in a nuclear and
cytoplasmic distribution (Fig 2E), but often were surrounded by
nonlabeled cells. The small labeled mononuclear cells scattered
throughout the KS lesions contained abundant nuclear and cytoplasmic
hybridization signals (Fig 2F).
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| Fig 2.
Expression of the KSHV cyclin D gene in cutaneous KS
lesions. (A) Early patch-stage lesion. Note hybridization signals in spindle cells forming the narrow vascular spaces in the dermis. (B)
Nodular/tumor-stage lesion showing large numbers of hybridization signals in lesional spindle cells. (C) Well-formed blood vessel in the
dermis adjacent to nodular lesion of KS. Note abundant hybridization
signals in the vascular endothelial cells. (D) Plaque-stage lesion of
KS with scattered keratinocytes in the overlying epidermis containing a
few hybridization signals. Some of the labeled keratinocytes (arrows)
are isolated and are surrounded by nonlabeled cells indicating that
diffusion of label between cells did not occur. (E) Eccrine ductular
epithelial cells containing hybridization signals. Only some of the
eccrine epithelial cells are labeled in this plaque-stage KS lesion.
Some of the labeled cells (arrows) are isolated and are surrounded by
nonlabeled epithelial cells. (F) Nodular KS lesion containing scattered
mononuclear cells, morphologically consistent with lymphocytes or
histiocytes, which are labeled. All panels, ISH with catalyzed signal
amplification, DAB chromogen, toluidine blue and hematoxylin
counterstain. (A through F) Original magnification × 270.
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| Fig 3.
Expression of KSHV cyclin D. (A) Higher magnification,
same nodular-stage lesion of KS as shown in Fig 2B. Note abundant
signal in the cytoplasm of lesional spindle cells; fewer nuclear
signals are present. (B) Same nodular-stage KS tissue hybridized with the biotinylated probe of irrelevant specificity. Hybridization signals
are not detected. (C) Nodular-stage lesion of KS hybridized with the
KSHV-specific probe after RNAse digestion of the tissue. Only a few
nuclear signals remain. (D) Benign cutaneous hemangioma, which did not
contain any labeled cells. (E) BC-3 positive control cell line showing
abundant cytoplasmic and nuclear hybridization signals. (F)
Non-AIDS-related cutaneous NHL, which did not contain any labeled
cells. All panels, ISH with catalyzed signal amplification, DAB
chromogen, hematoxylin counterstain. (A, B, C, and E) Original magnification × 660. (D and F) Original magnification × 270.
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ISH performed on KS lesions with biotinylated probes of irrelevant
specificity did not contain hybridization signals (Fig 3B) indicating
that the hybridizations with the KSHV probe were specific. KS tissues,
which had been digested with RNAse before ISH with the KSHV
cyclin-specific probe, did not contain or contained significantly fewer
cytoplasmic hybridization signals than did the matched tissue sections,
which did not receive RNAse treatment (Fig 3A and C) indicating that
the cytoplasmic hybridizations were RNA-specific. The five benign
cutaneous hemangiomas evaluated in this study did not contain
KSHV-specific sequences (Fig 3D).
Each of the three AIDS-related NHL and three cell line positive
controls studied contained KSHV-specific sequences (Fig 3E). The three
positive control cell lines showed hybridization signals in virtually
all of the cells with some cells having many nuclear signals. In the
AIDS-related NHL tissues, lymphoma cells typically contained many
nuclear and cytoplasmic signals. These tissues also contained scattered
smaller mononuclear cells, which were labeled. Five primary cutaneous
non-AIDS-related NHL and one cutaneous lymph node with reactive
follicular hyperplasia did not contain KSHV-labeled cells (Fig 3F). All
of these AIDS-related NHL and cell lines used as positive controls in
the ISH experiments contained KSHV by Southern blot analyses
(Fig 4).
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| Fig 4.
Southern blot analysis identifying KSHV in AIDS-related
NHL used as positive controls for the ISH experiments. Lanes 2, 6, 8:
Hind III-digested DNA from AIDS-associated NHL containing
KSHV-specific sequences identified by the KS631 probe. Lanes 3 to 5, 7, 9: AIDS-associated NHL lacking KSHV-specific sequences. Lane 1, Control
DNA extracted from HL-60 promyelocytic leukemia cell line. Southern
blots showing KSHV-specific sequences in the BC-1, BC-2, and BC-3 cell
lines, which also were used as positive controls for ISH have been
published previously.33,36
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Control sections prepared from mixtures of BC-3 (KSHV-positive) and
BJAB (KSHV-negative) cell suspensions contained appropriate percentages
of cells labeled for KSHVv-cyc (Fig
5).
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| Fig 5.
KSHV cyclin expression in BC-3 and BJAB cell suspension
mixtures. (A) 80% BC-3 (KSHV-positive), 20% BJAB (KSHV-negative); (B)
20% BC-3, 80% BJAB. These results show appropriate percentages of
cells with punctate labeling indicating that diffusion of the label
between cells, a possible artifact of PCR-ISH-based assays, does not
occur in this procedure.
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DISCUSSION |
Based on epidemiological studies, it has been suggested that KS is an
infectious, sexually-transmitted disease independent of HIV
infection.43 In the US, KS is much more prevalent in homosexual and bisexual men than in other groups at high risk for HIV
infection.44 For women who develop KS, there is a
correlation with prior sexual contact with bisexual men.45
Recently, DNA sequences from a novel human herpes virus KSHV/HHV8 were
identified in lesions of KS.1 Since this discovery, many
studies have confirmed the presence of KSHV in all subtypes of KS,
strongly suggesting an etiologic role for this virus.7-13
Previous ISH studies identified KSHV in plaque and nodular lesions of
KS.15-18 In these tissues, KSHV DNA sequences were
identified in the spindle cells surrounding the atypical vascular
spaces. Many other PCR-based studies have identified the virus in early
patch stage lesions of KS, but did not use in situ methods to define
the specific cell types harboring the virus. Recently, however, Staskus
et al18 showed three different KSHV-specific transcripts in
lesions of KS; one of these transcripts was present in one very early patch-stage lesion included in their study. Another of these
transcripts encodes the major capsid protein (MCP) of KSHV
(ORF25).26 MCP-specific sequences were identified in low
levels in lesional spindle cells in only a few later-stage lesions
indicating that KSHV exists primarily in a latent state or undergoes an
abortive replication cycle in these lesions.18 In the
present study, KSHV-specific sequences were identified in each of the
stages of progression recognized for KS. For all 10 early patch-stage
lesions, KSHV-specific sequences consistently were identified in the
fusiform, spindle-shaped cells surrounding the atypical vascular
spaces, as well as in some endothelial cells of well-formed blood
vessels in the adjacent dermis. This represents the first in situ
demonstration of KSHV cyclin D mRNA in the earliest
histopathologically-recognizable lesion of KS. In our study, a similar
distribution of KSHV-infected cells was seen in later plaque-stage and
nodular lesions, corroborating results of previous ISH-based studies,
which defined the cellular distribution of KSHV.15-18 In
addition to the previously described cellular distribution of KSHV, it
also was observed that small mononuclear cells containing the virus and
morphologically consistent with lymphocytes and/or histiocytes
were present within the tissues. In addition, a few later-stage lesions
contained keratinocytes in the epidermis and/or epithelial
cells in eccrine structures harboring KSHV cyclin sequences. The in
situ demonstration of transcriptional activity of KSHV v-cyc in
early cutaneous lesions of KS provides further evidence for an
etiologic role of this virus.
It has been suggested that KSHV may progress from a latent phase to a
lytic phase in some cell types.46,47 This is especially true for the B lymphocytes of AIDS-related PEL and the cell lines derived from them, both of which may evolve a lytic phase yielding assembled viral particles.3,36 Other studies have shown
that the spindle cells of KS are likely latently infected with
KSHV.18,23,24 Our observation that the nuclei of lesional
spindle cells generally contain only few hybridization signals, but
many cytoplasmic signals, is consistent with that conclusion.
Furthermore, prior treatment of the spindle cells with RNAse eliminated
or greatly reduced the amount of cytoplasmic signals generated after
ISH, indicating that the majority of cytoplasmic labeling was due to
hybridization of the probe to cytoplasmic mRNA and not to viral DNA
sequences. Nodular/tumor-stage lesions of KS also showed a marked
reduction in hybridization signal intensity with prior RNAse digestion. This would further support the argument advanced by Staskus et al18 that a lytic phase of viral replication does not occur or is abortive during the progression of KS lesions. Additional in situ
experiments are in progress to identify other latently-expressed genes
in early patch-stage lesions of KS.
Because some cells infected with KSHV (including B lymphocytes) can
progress to a lytic phase of viral replication, it is possible that KS
lesional tissues may contain spindle cells, which are latently infected
together with other types of cells capable of productive viral
replication. Thus, it seems possible that lesions of KS may arise in
the setting of a lytic infection of B lymphocytes. It has been shown
that some patients have detectable KSHV in the peripheral blood or
KSHV-specific antibody titers before the development of KS
lesions.48,49 Further elucidation of the pathobiology of
the virus will rely on identifying specific viral genes, which are
transcriptionally active at different stages of KS lesions, as well as
in other cell populations from patients who subsequently develop KS.
In this study, a novel colorimetric ISH procedure using a catalyzed
signal amplification system was used to identify KSHV-specific sequences. This amplification system was adapted for use from a similar
system designed for solid-phase immunocytochemical assays such as
Western blot procedures.19,20 Recently, Zehbe et
al22 used a variation of this catalyzed signal
amplification technique using streptavidin-nanogold with silver
enhancement to identify with high specificity and sensitivity human
papillomavirus nucleic acid sequences in infected cell lines and human
tissues. The system used in the current study allowed the sensitive and
specific identification of KSHV cyclin sequences in 100% of the
routinely processed, formalin-fixed, paraffin-embedded archival tissue
samples of KS studied. As such, the level of sensitivity of this method
appears to be equal to that of previous studies that used PCR
ISH15 or ISH with radiolabeled probes,18 both
of which also identified KSHV in 100% of the KS lesions studied.
Furthermore, this method allowed the sensitive detection of KSHV even
in the earliest recognizable patch-stage lesions of KS, an observation
that has been documented in only one specimen so far using radiolabeled
probes.18 Other ISH methods using radioactively-labeled
probes identified KSHV-specific sequences in a more variable 25% to
67% of the lesions evaluated.16,17 Because the procedures
described here did not require use of radioactively labeled probes or
the in situ amplification of specific target sequences by PCR before
ISH, the problems inherent to those techniques such as the requirement
for radioactivity precautions, long emulsion development times, or for
avoidance of sample contamination by extraneous DNA were avoided. This
ISH procedure allowed direct visualization of the specific cell types
harboring KSHV with preservation of cellular morphology and without the
diffusion of amplified DNA products, which may occur in PCR ISH-based
assays. The complete ISH procedure required approximately 5 hours to
perform, which is significantly less time that that required for
detection of radioactively labeled probes after ISH and comparable to
the time needed for PCR ISH. This procedure should be useful with other types of probes designed for specifically targeting low copy numbers of
mRNA and DNA sequences. For KSHV, recognition of the specific sequence
of transcriptional events and of the specific cells infected will
further define the pathobiology of this virus.
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FOOTNOTES |
Submitted January 24, 1997;
accepted December 29, 1997.
Supported in part by US Public Health Service Grants No.
CA73531 and CA68939 from the National Cancer Institute, National Institutes of Health.
Address reprint requests to Jon A. Reed, MD, Department of Pathology,
The New York Hospital-Cornell Medical Center, 1300 York Ave, Room
F-309, New York, NY 10021.
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.
| |
REFERENCES |
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Chang Y,
Cesarman E,
Pessin MS,
Lee F,
Culpepper J,
Knowles DM,
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Abstract
Introduction
Abstract