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Journal of Clinical Microbiology, August 1998, p. 2220-2222, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection and Semiquantitative Analysis of Human Herpesvirus 8 DNA in Specimens from Patients with Kaposi's Sarcoma
J. C.
Mendez,1
G. W.
Procop,2
M. J.
Espy,2
C. V.
Paya,1 and
T. F.
Smith2,*
Divisions of Infectious
Diseases1 and
Clinical
Microbiology,2 Mayo Clinic, Rochester,
Minnesota 55905
Received 30 January 1998/Returned for modification 3 March
1998/Accepted 27 April 1998
 |
ABSTRACT |
Kaposi's sarcoma (KS) is the most common neoplasm in patients with
AIDS. Epidemiologic evidence and the recent identification of
herpesvirus-like DNA sequences in patients with KS have suggested a
role for viral agents in the etiopathogenesis of this disease. It is
unclear if these sequences are present in all types of KS and if the
copy number of these sequences has a correlation with disease severity
(staging). In order to clarify these issues, we retrospectively
analyzed, by PCR and Southern blotting, formalin-fixed, paraffin-embedded biopsy specimens from 12 patients previously diagnosed with KS by histopathologic examination of these specimens between the years of 1977 and 1996. We also analyzed tissue samples from these patients taken from dermal sites without KS lesions and
control tissues from healthy subjects. Of the 12 patients, 6 had
classic KS, 5 had AIDS-associated KS, and 1 had the endemic type of KS.
We tested the specimens for other herpesviruses, including cytomegalovirus, human herpesvirus 6 (HHV-6), HHV-7, HHV-8, and Epstein-Barr virus. Of 20 biopsy specimens from patients previously diagnosed with KS, 19 were positive for HHV-8 sequences (95%), while
PCR for the DNAs of other herpesvirus agents was negative. Uninvolved
tissue from these patients and control tissue from healthy subjects
gave negative results for all viruses. Semiquantitative analysis of
Southern blots showed higher levels of HHV-8 DNA in those patients with
multicentric and visceral involvement than in those patients with
localized involvement. In addition, in patients with localized skin
disease, the nodular stage had higher levels of HHV-8 DNA than the
patch or plaque stages. Our data confirmed that HHV-8 is involved in
the etiopathogenesis of all types of KS and that there is a correlation
between HHV-8 DNA load and the severity and staging of this disease.
| |
INTRODUCTION |
Kaposi's sarcoma (KS) is the most
common neoplasm in patients with AIDS (4). Described in 1872 by Moritz Kaposi, KS is a complex tumor of uncertain histogenesis with
a spindle-shaped cell proliferation and an inflammatory component.
There is controversy whether this is a true neoplasm versus a
hyperplastic lesion. Epidemiologic studies and behavorial and
geographic risk factors suggest an infectious cause of KS; a sexually
transmitted agent is probably the most likely etiologic factor involved
in this tumor (14).
In 1972, Giraldo and coworkers (10) found herpesvirus-like
particles in KS lesions. Nine years later (1981), the Centers for
Disease Control (6) reported 26 cases of KS in homosexual men, which is now recognized as the beginning of the human
immunodeficiency virus (HIV) epidemic. More recently, in 1994, Chang
and colleagues (7) isolated herpesvirus-like DNA sequences
from KS lesions from patients with AIDS (7). These sequences
corresponded to a new herpesvirus that was called KSHV and that was
later defined as human herpesvirus 8 (HHV-8). HHV-8, a rhadinovirus,
shares close homology with other lymphotropic oncogenic gamma
herpesviruses (Epstein-Barr virus [EBV] and herpesvirus saimiri).
It is not known whether these sequences are present in all types of KS
and if the viral load in tissue specimens has a correlation with
clinical severity and histologic staging of KS. In order to clarify
these issues, we retrospectively analyzed by PCR and Southern blotting
formalin-fixed, paraffin-embedded tissues from patients with KS. Twelve
patients with biopsy-proven KS and whose KS was diagnosed at the Mayo
Clinic between the years of 1977 and 1996 were included in this study.
The performance of these assays was evaluated, and the results were
correlated with histopathologic findings for biopsy specimens to assess
the severity and staging of this disease.
| |
MATERIALS AND METHODS |
Clinical samples.
Formalin-fixed, paraffin-embedded biopsy
specimens were obtained from 12 patients diagnosed with KS by
histopathologic examination between 1977 and 1996. Histopathologic
staging was performed by using a previously described classification
(2). Of the 12 patients, 6 had classic KS, 5 had
AIDS-associated KS, and 1 had the endemic type of KS. As positive
controls, we used strains of cytomegalovirus (CMV), EBV, HHV-6, and
HHV-7 obtained from the American Type Culture Collection, Rockville,
Md., and HHV-8 from an infected BCBL-1 cell line (15).
Several negative controls were used: biopsy specimens from healthy skin
or skin with other clinical conditions (psoriasis, dermatitis) from
study patients with KS, similar tissue specimens from healthy patients,
and liver and lung tissue from patients with no recognized infectious
disease.
Extraction of nucleic acids, oligonucleotides, and PCR.
Nucleic acids were extracted from biopsy specimens by using a universal
DNA isolation technique described previously (16).
PCR for the detection of CMV, EBV, HHV-6, HHV-7, and HHV-8 was
performed by using the oligonucleotide primers described previously
(Table
1). The probe corresponding to a
region between these
oligonucleotide primers was synthesized and
labeled for chemiluminescence
by using the enhanced chemiluminescence
kit from Amersham (Arlington
Heights, Ill.).
As a control of internal consistency, for enzymatic amplification of
target sequences, and for semiquantitative analysis of
the PCR
products, five reference standards were included in each
PCR batch. For
HHV-8, these standards were prepared by using known
amounts of a
purified plasmid pCR 2.1 (Invitrogen, Carlsbad, Calif.)
constructed
with a fragment with the same sequence as the PCR
product and
equivalent to approximately 10 to 100,000 genome equivalents.
For
objective evaluation, Southern blots were analyzed by a computer
image
processing system (NIH Image, National Institutes of Health,
Bethesda,
Md.).
| |
RESULTS |
Of 20 biopsy specimens from 12 patients with KS, for 19 (95%)
HHV-8 target sequences were amplified by PCR (6 of 7 [86%] from patients with classic KS, 9 of 9 [100%] from patients with
AIDS-associated KS, and 4 of 4 [100%] from patients with the endemic
form of KS). All specimens were negative for CMV, EBV, HHV-6, and HHV-7
DNAs. Uninvolved tissue from these specimens and control tissues from healthy subjects (skin with other disorders [n = 9],
healthy skin [n = 10], and healthy liver and lung
specimens [n = 10]) gave negative PCR results for all
viral DNA targets.
Semiquantitative analysis of gel electrophoresis and subsequent
Southern blots showed higher levels of HHV-8 DNA in those patients with
multicentric and visceral involvement than in those with localized
disease (Table 2).
In the patients with localized skin disease, the nodular stage had
higher levels of HHV-8 DNA than the patch or the plaque stages (Table
2; Fig. 1).
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FIG. 1.
Molecular detection of HHV-8 DNA. (A) Gel
electrophoresis with dilutions of HHV-8 target DNA of plasmid (pCR 2.1)
representing 109 (lane 2) to 101 (lane 10)
copies of the viral genome. (B) Southern blotting results for samples
of tissue from KS lesions containing HHV-8 DNA. The dots are for the
patch, plaque, and nodule stages, from top to bottom, respectively.
|
|
| |
DISCUSSION |
Epidemiologic studies performed in the past suggested that both
the AIDS-associated and the non-AIDS-associated types of KS may be
caused by an infectious agent (14). For example, it was shown that homosexual and bisexual males with AIDS were approximately 20 times more likely than hemophiliacs with AIDS to develop KS (3). These and other studies (1) showed that KS
may be associated with behavioral and geographic risk factors most
consistent with the tumor being caused by a sexually transmitted
agent.
Application of novel molecular techniques revealed the strong
association of HHV-8 with AIDS-related KS (7). Those
investigators extended these findings to a search for HHV-8 DNA
sequences in other tissues from KS patients, as well as in KS lesions
from patients without HIV infection, and found that HHV-8 was
presumably not solely an opportunistic infection in patients with AIDS
but that the other three forms of KS seen in patients without HIV infection were caused by the same infectious agent (13).
At the Mayo Clinic, in archival biopsy specimens from patients with
clinically diagnosed and histologically confirmed KS, we found HHV-8
DNA in 19 of 20 KS specimens (95%) from patients with different types
of KS. Importantly, semiquantitative determination of viral load was
directly correlated with the staging and dissemination of this tumor.
These results have important clinical implications and diagnostic
relevance and emphasize more than ever the pathogenic role of HHV-8 in
this tumor. The HHV-8 DNA load could potentially have applications for
the early diagnosis, staging, and monitoring of this disease. The
identification of tumor growth under viral control, possibly a viral
promoter, may lead to novel therapeutic interventions.
Rapid progress in the understanding of the relationship between HHV-8
and KS has been made. Despite this progress, a number of fundamental
questions remain unanswered, such as those regarding the mechanisms of
transmission, risk factors that place certain population groups such as
homosexual men at an increased risk for KS compared to the risk for
other groups, and effective interventions through the use of
therapeutic agents and other interventions. Although recent studies
have shown that HHV-8 is resistant to acyclovir but susceptible to
ganciclovir, foscarnet, and cidofovir (11, 12), we still
need to develop better and easier antiviral drug susceptibility assays,
improve our ability to grow HHV-8 in cell cultures, and perform trials
to determine the correlation of these results in clinical practice.
In conclusion, this study has shown that (i) HHV-8 is associated with
all types of KS, (ii) higher levels of HHV-8 DNA are found in patients
with multicentric and visceral KS than in those with localized skin
disease, (iii) for mucocutaneous involvement, the nodular stage had
higher levels of HHV-8 DNA than the patch or plaque stages, and (iv)
there was an association between HHV-8 DNA load and the severity and
staging of this disease. Importantly, our study suggests that
understanding of the relationship between viral load and
histopathologic stage needs to be expanded by investigations involving
a large set of patient specimens examined by sophisticated quantitation
techniques.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Clinical Microbiology, Mayo Clinic, 200 First St. S.W., Rochester, MN
55905. Phone: (507) 284-8146. Fax: (507) 284-4272. E-mail:
TFSmith{at}mayo.edu.
| |
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Journal of Clinical Microbiology, August 1998, p. 2220-2222, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
