(Chest. 1999;116:470S-473S.)
© 1999
American College of Chest Physicians
Simian Virus 40 Is Present in Most United States Human Mesotheliomas, but It Is Rarely Present in Non-Hodgkin’s Lymphoma*
Paola Rizzo, MS;
Michele Carbone, MD, PhD;
Susan G. Fisher, PhD;
Christine Matker, BS;
Lode J. Swinnen, MD;
Amy Powers, BS;
Ilaria Di Resta, MS;
Serhan Alkan, MD;
Harvey I. Pass, MD, FCCP and
Richard I. Fisher, MD
*
From Cardinal Bernardin Cancer Center, Loyola University Chicago (Drs. Rizzo, Carbone, Fisher, Matker, Swinnen, Powers, Di Resta, Alkan, and Fisher), Maywood, IL; and Wayne State University, Harper Hospital (Dr. Pass), Detroit, MI.
Correspondence to: Michele Carbone, MD, PhD, Cardinal Bernardin Cancer Center, Room 205, 2160 South First Ave, Maywood, IL 60153; e-mail: mcarbon{at}orion.it.luc.edu
 |
Abstract
|
|---|
Simian virus 40 (SV40) causes mesotheliomas, osteosarcomas,
ependymomas, choroid plexus tumors, and lymphomas in hamsters. In
humans, SV40 has been detected in tumors of the first four types. Using
the polymerase chain reaction (PCR), we tested 29 non-Hodgkin’s
lymphomas (intermediate and high-grade), 25 posttransplant
lymphoproliferative disorders, and 5 AIDS lymphomas for SV40 DNA. PCR
analysis revealed that 3 of 29 lymphomas, 6 of 25 posttransplant
lymphoproliferative disorders, and 2 of 5 AIDS lymphomas contained SV40
sequences corresponding to the retinoblastoma (RB)-pocket binding
domain of SV40 tumor antigen (Tag). However, among positive
samples, only one posttransplant lymphoproliferative disorder and one
AIDS lymphoma contained the SV40 regulatory region, which suggest a
higher viral load in these patients. In parallel experiments, 8 of 12
mesotheliomas tested positive for SV40 for both the RB-pocket binding
domain of Tag and the SV40 regulatory region. These data confirm the
presence of SV40 in most United States mesotheliomas and indicate that
in human non-Hodgkin’s lymphomas, the prevalence of SV40 is
low.
|
Introduction
|
|---|
In
the last half century, there has been an enormous increase in the
incidence of lymphomas and mesotheliomas. A link between SV40 and
mesothelioma was established when the tumors were found to develop in
hamsters injected intrapleurally, intracardially, and intraperitoneally
with simian virus 40 (SV40).1
In addition, through
polymerase chain reaction (PCR) analysis, RNA in situ
hybridization, Western blotting, and immunohistochemistry, the majority
of human mesotheliomas were found to contain and express
SV40.2
3
4
5
6
7
8
9
10
Aside from mesothelioma, SV40 has been found to
cause specific tumor types, mainly lymphomas, and also osteosarcomas,
ependymomas, and choroid plexus tumors when injected into
hamsters.2
Weanling hamsters injected IV with the virus
were found to develop lymphomas, lymphocytic leukemias, and
osteosarcomas at sites distant from the point of
injection.11
In addition, intracardiac injection of
wild-type SV40 caused lymphomas and osteosarcomas in 33% and 10% of
hamsters, respectively (the remaining 60% developed
mesothelioma).1
Of interest, the deletion of the SV40
small t antigen, which removes the ability of SV40 to inhibit the
activity of cellular PP2A, leads to a mutated SV40 (SV40 small t
mutant), which caused lymphomas in 100% of the injected
hamsters.12
Similarly to mesothelioma, several studies
demonstrated that human ependymomas, choroid plexus tumors, and
osteosarcomas contain and express the SV40 genome.2
13
The
prevalence of SV40 in human lymphomas, however, has not been thoroughly
investigated.
In immunodeficient rhesus monkeys, the natural host of the virus, SV40
infection results in a variety of diseases, including
encephalomyelitis, pneumonia, and astrocytomas.13
In a
recent study of rhesus monkeys, simian immunodeficiency
virus-positive primates were found to exhibit SV40-induced
lesions and SV40 sequences in their kidneys and brains, while lesions
associated with SV40 or the presence of SV40 sequences were not found
in simian immunodeficiency virus-negative monkeys.14
These
data suggested that immunosuppressed individuals might also be at
higher risk for SV40 infection.
In this study, we investigated non-Hodgkin’s human lymphomas obtained
from nonimmunocompromised patients to determine if they contained SV40
DNA sequences. Because viruses may spread more efficiently and be more
pathogenic in immunocompromised individuals, we also tested for SV40
lymphoproliferative disorders that had developed in immunocompromised
patients.
|
Materials and Methods
|
|---|
DNA Detection
Frozen tumor samples were obtained from 29 non-Hodgkin’s
lymphoma patients who were classified according to the Revised
European-American Lymphoma classification15
: 26
large cell, B lineage; 2 large cell high-grade T-cell lymphoma, and 1
large cell B, Burkitt’s-like lymphoma. In the immunocompromised group,
we studied 25 patients with posttransplant lymphoproliferative
disorders and 5 AIDS patients with large cell lymphoma, B-cell origin.
DNA was extracted and purified from frozen lymphoma or mesothelioma
tumor tissue as previously described in detail.16
All of
the DNA samples were tested for suitability of amplification with
primers specific for a 268-base pair (bp) fragment of the
ß-globin gene; all DNA samples could be amplified. PCR reactions were
performed using the hot-start technique as described.16
The GeneAmp PCR reagent kit containing Amplitaq polymerase
(Perkin-Elmer Biosystems; Norwalk, CT) and the Ampliwax PCR gems
(Perkin-Elmer Biosystems) were utilized for the analysis. DNA
samples from tumors were amplified using the SV5 and SV6 set of primers
which amplifies the retinoblastoma (RB)-pocket binding domain of SV40
tumor antigen (Tag), as previously described.16
The
samples were further analyzed with primers R1/R2. This set of primers
amplifies the regulatory region and the origin of replication of the
virus.16
The total volume for each PCR reaction was 100
µL; the concentrations of MgCl2 and that of the
primers were 2.5 mM and 0.5 µM, respectively (the concentration of
each primer was calculated from the equation 1 OD = 20
µg/mL). One µg of DNA was used per each PCR. All other
reagents were used according to the recommendations of the
manufacturer. Negative controls were included in each PCR experiment to
test for PCR contamination. Thermocycling was performed by denaturation
for 3 min at 94°C followed by 45 cycles. Each cycle consisted of 1'
at 94°C, 1' at 60°C, and 1' at 72°C.
For Southern blot analysis, 20 µL of the PCR reaction was loaded onto
and run in a 2% agarose gel for 3 h at 100 V. The gel was
stained with ethidium bromide to visualize the PCR products, and then
the DNA samples were transferred overnight in 0.4 M NaOH to a nylon
membrane (Hybond N+, Amersham Pharmacia Biotech; Uppsala,
Sweden). Hybridization was carried out using a SV40-specific
32P-end-labeled internal
oligoprobe.16
Hybridization was performed overnight in 10
mL of hybridization solution containing the following:
5 x Denhardt’s solution, 0.5% sodium dodecyl sulfate, and
100 µg/mL of salmon sperm DNA at 52°C. Filters were washed at
52°C with a final stringency of 0.5 x sodium saline
citrate, 0.1% sodium dodecyl sulfate and exposed to radiograph
film at room temperature for 
30 min.
DNA Sequencing
The DNA products obtained with the primers SV5 and SV6 were
always sequenced to confirm their identity. The PCR product was
purified using a commercially available kit (QIAquick gel extraction
kit; Qiagen; Santa Clarita, CA) according to the suggested
protocol of the manufacturer. Both strands of the PCR product were then
sent to a sequencing facility for final identification (Kimmel Cancer
Institute, Jefferson University; Philadelphia, PA).
|
Results
|
|---|
In the initial PCR screening of the DNA samples isolated from
lymphomas, we used the set of primers SV5/SV6 that amplifies the
RB-pocket binding domain of SV40 Tag. In our experience, this is a very
sensitive set of primers for the detection of SV40 in human
tumors.16
As indicated in Table 1
and shown in Figures 1
and 2
,
top
, 3 of 29 lymphomas in the nonimmunocompromised group, 6 posttransplant
lymphoproliferative disorders, and 2 AIDS lymphomas tested positive.
All of the positive samples were of B-cell lineage.
View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 1. Top, A and bottom,
B: Southern blot of the PCR products obtained from 29
non-Hodgkin’s lymphoma DNA samples using the SV5/SV6 set of primers.
Samples 11, 22, and 26 are positive. (-) = H2O, negative
control; (+) = SV40 DNA, positive control.
|
|
View larger version (44K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 2. Top, A: Southern blot hybridization
of the PCR products obtained from lymphoproliferative posttransplant
disorder DNA samples (samples 1–23, 29, and 30) and from AIDS lymphoma
DNA samples (samples 24–28) using the SV5/SV6 set of primers. Samples
4, 11, 12, 15, 24, 26, and 29 are positive. Prolonged exposure of this
autoradiography revealed a faint reproducible positive signal in sample
17. Bottom, B: Southern blot hybridization of the PCR
products obtained from DNA samples of selected immunocompromised
patients using the R1/R2 set of primers. Samples 7, 15, and 26 are
positive. (-) = H2O, negative control; (+) = SV40 DNA,
positive control.
|
|
Because of the high homology that exists between the DNA of SV40 and
the DNA of the JC or BK viruses, the identity of the band obtained by
PCR and visualized by ethidium bromide staining was confirmed by both
Southern blot hybridization and DNA sequencing. The results of
these analyses confirmed that authentic SV40 was amplified from these
tumors. All of the positive samples were further analyzed with the
primers R1/R2 for the SV40 regulatory region (Table 1)
. All of the
lymphomas in the nonimmunocompromised group tested negative. In the
immunocompromised group, one specimen from posttransplant
lymphoproliferative disorders and one AIDS lymphoma also tested
positive for the SV40 regulatory region. The other specimens
from immunocompromised individuals that had tested positive with the
primers SV5/SV6 tested negative with primers for the regulatory region
(Table 1
, and Fig 2
, bottom, B). Primers for the RB-pocket
binding domain are more sensitive than primers for other regions of the
SV40 genome.16
Therefore, the positivity for the
regulatory region in these two specimens suggested that a larger
percentage of the cells contained SV40. It should be noted, however,
that one additional specimen from an immunocompromised individual
(sample 7, Fig 2 , bottom, B) tested positive for the
regulatory region and negative with primers for the RB-pocket of Tag.
This unusual result could be related to deletions or mutations that
occasionally occur in the Tag DNA sequence in some human
tumors.2
In parallel, we tested 12 mesothelioma samples for SV40. Eight of 12
mesotheliomas tested positive with the primers SV5/SV6 for the
RB-pocket binding domain of Tag (Fig. 3
, top, A). The same
samples tested positive for the regulatory region and the origin of
replication of the SV40 virus (primers R1 and R2) (Fig. 3
,
bottom, B).
View larger version (43K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 3. Top, A: Southern blot hybridization
of the PCR products obtained from mesothelioma DNA samples using the
SV5/SV6 set of primers. Bottom, B: Southern blot
hybridization of the PCR products obtained from mesothelioma DNA
samples using the R1/R2 set of primers. Samples 1, 3–6, 8, 11, and 12
are positive with both sets of primers. (-) = H2O,
negative control; (+) = SV40 DNA, positive control.
|
|
|
Discussion
|
|---|
We found that, overall, SV40 DNA sequences are rarely present in
lymphomas from both immunocompetent and immunodeficient patients, and
we further confirmed the presence of SV40 in most human mesotheliomas.
Thus, SV40 does not appear to be related to the increased incidence of
lymphomas observed in the second half of this century, but may be
related to mesothelioma development.17
Some of the
lymphomas tested positive for SV40 only when we used the very sensitive
SV5/SV6 primers for the RB-pocket binding domain of Tag, and negative
when tested with primers for the SV40 regulatory region. This suggests
that, in these samples, only a few cells were SV40 positive. SV40
sequences have been detected in peripheral blood lymphocytes and
granulocytes from normal individuals.18
19
Furthermore, it
has been suggested that circulating mononuclear phagocytes may
represent a reservoir for SV40 in humans.2
18
Thus, it is
possible that the positive results obtained for tumors with the very
sensitive SV5/SV6 set of primers are related to the presence of
SV40-positive mononuclear phagocytes within the tumor tissue, rather
than representing tumor-associated virus. If this hypothesis is
correct, all of the non-Hodgkin’s lymphomas that developed in the
nonimmunocompromised group we tested should be considered SV40
negative. The rare association of SV40 with human lymphomas and
leukemias is supported by a recent study in which SV40 was found in
only 6 of 15 B-cell immunoblastic lymphomas, and SV40 was not detected
in 20 myeloid leukemias and 9 large cell lymphomas (Carl W. Miller,
PhD; personal communication; May 1998). However, it has also
been suggested that SV40 could be present in lymphoma cells in the
early phase of lymphomagenesis to promote transformation, remaining
present afterwards only in a fraction of the tumor cells and eventually
in none of the tumor cells during tumor progression.18
In
support of this hypothesis, Salewski et al20
demonstrated
very recently that SV40 is required to induce the transformed phenotype
but it is not required for the maintenance of the transformed
phenotype. Actually, following SV40-mediated transformation, cells that
lost SV40 became more aggressive and oncogenic, possibly because the
immune system did not react against the SV40 tumor antigens that are
highly immunogenic.20
It is unknown how frequently SV40
transformed cells lose SV40, but it is possible that the number of
SV40-positive tumors could be underestimated when advanced malignancies
are tested.
A high prevalence of virally associated neoplasm has been documented in
acquired or congenital immunodeficiency states, specifically
Epstein-Barr virus-, human papilloma virus-, and human herpesvirus
8-related tumors. SV40-related lymphomagenesis might, therefore,
be more prevalent or easier to detect in such patients. We found a
slightly higher percentage of SV40-positive lymphomas in
immunocompromised patients than in immunocompetent individuals.
Furthermore, specimens from one lymphoproliferative disorder and one
AIDS lymphoma were positive with primers for both the RB-pocket binding
domain of Tag and the regulatory region. Similar results were
obtained in 8 of 12 mesotheliomas and indicate a higher viral load
and/or the presence of extensive portions of the SV40 genome. However,
the relatively higher prevalence of SV40-positive tumors in
immunocompromised patients that we observed in this study may be due to
the small sample size tested, and/or to the fact that most of the
patients were organ transplant recipients, who have significant blood
product exposure and exposure to the donor organ. This hypothesis is
supported by recent studies suggesting that HIV-positive individuals do
not have a higher frequency of SV40-positive tumors.18
In
these studies, PCR analysis for SV40 sequences in HIV-negative and
HIV-positive individuals revealed that SV40 prevalence and load was
similar in immunocompromised and nonimmunocompromised
patients.18
Finally, the detection of SV40 DNA sequences in 8 of 12 mesothelioma
samples and the detection of the virus in only a small number of
lymphomas samples through DNA extractions and PCR reactions that were
carried out at the same time by the same investigators rule out the
possibilities that either the technical procedure used was not
sufficiently sensitive to detect SV40 in lymphomas or that the high
percent of positive results obtained with mesotheliomas might be
related to PCR contamination.
In conclusion, we find no evidence to support a role for SV40 in the
increased incidence of lymphomas in the second half of this century,
and we confirm the strong association of SV40 with mesotheliomas.
|
Footnotes
|
|---|
Abbreviations: bp = base pair; PCR = polymerase
chain reaction; RB = retinoblastoma; SV40 = simian virus 40;
Tag = tumor antigen
The work was supported by NIH grant CA77220–01 to Michele Carbone.
|
References
|
|---|
-
Cicala, C, Pompetti, F, Carbone, M (1993) SV40 induces mesotheliomas in hamsters. Am J Pathol 142,1524-1533[Abstract]
-
Carbone, M, Rizzo, P, Pass, HI (1997) Simian virus 40, poliovaccines and human tumors: a review of recent developments. Oncogene 15,1877-1888[CrossRef][ISI][Medline]
-
Carbone, M, Pass, HI, Rizzo, P, et al (1994) Simian virus 40-like DNA sequences in human mesothelioma. Oncogene 9,1781-1790[ISI][Medline]
-
Cristaudo, A, Vivaldi, A, Sensales, G, et al (1995) Molecular biology studies on mesothelioma tumor samples: preliminary data on H-ras, p21, and SV40. J Environ Pathol Toxicol Oncol 14,29-34[Medline]
-
Pepper, C, Jasani, B, Navabi, H, et al (1996) Simian virus 40 large T antigen (SV40LTag) primer specific DNA amplification in human pleural mesothelioma tissue. Thorax 51,1074-1076[Abstract]
-
Carbone, M, Rizzo, P, Grimley, PM, et al (1997) Simian virus-40 large-T antigen binds p53 in human mesotheliomas. Nat Med 3,908-912[CrossRef][ISI][Medline]
-
De Luca, A, Baldi, A, Esposito, V, et al (1997) The retinoblastoma gene family pRb/p105, p107, pRb2/p130 and simian virus-40 large T-antigen in human mesotheliomas. Nat Med 3,913-916[CrossRef][ISI][Medline]
-
Griffiths, DJ, Nicholson, AG, Weiss, R (1998) Detection of SV40 sequences in human mesotheliomas. Dev Biol Stand 94,127-136[ISI][Medline]
-
Galateau-Salle, F, Bidet, P, Iwatsubo, Y, et al (1998) SV40-like DNA in pleural mesothelioma, bronchopulmonary carcinoma, and non-malignant pulmonary diseases. J Pathol 184,252-257[CrossRef][ISI][Medline]
-
Testa, JR, Carbone, M, Hirvonen, A, et al (1998) A multi-institutional study confirms the presence and expression of simian virus 40 in human malignant mesotheliomas. Cancer Res 58,4505-4509[Abstract/Free Full Text]
-
Diamandopoulous, GT (1972) Leukemia, lymphoma and osteosarcoma induced in the Syrian Golden hamster by simian virus 40. Science 176,173-175[Abstract/Free Full Text]
-
Cicala, C, Pompetti, F, Nguyen, P, et al (1992) SV40 small t deletion mutants preferentially transform mononuclear phagocytes and B-lymphocytes in vivo. Virology 190,475-479[CrossRef][ISI][Medline]
-
Butel, JS, Lednicky, JA (1999) Cell and molecular biology of simian virus 40: implications for human infections and disease. J Natl Cancer Inst 91,119-134
-
Newman, JS, Baskin, GB, Frisque, RJ (1998) Identification of SV40 in brain, kidney and urine of healthy and SIV-infected rhesus monkeys. J Neurovirol 4,394-406[ISI][Medline]
-
Harris, NL, Jaffe, ES, Stein, H, et al (1994) A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 84,1361-1392[Free Full Text]
-
Rizzo, P, Di Resta, I, Powers, A, et al (1998) The detection of simian virus 40 in human tumors by polymerase chain reaction. Monaldi Arch Chest Dis 53,202-210[Medline]
-
Fisher, SG, Weber, L, Carbone, M (1999) Cancer risk associated with simian virus 40 contaminated polio vaccine. Anticancer Res 19,2173-2180[ISI][Medline]
-
Martini, F, Dolcetti, R, Gloghini, A, et al (1998) Simian virus-40 footprints in human lymphoproliferative disorders of HIV- and HIV+ patients. Int J Cancer 78,669-674[CrossRef][ISI][Medline]
-
Carbone, M, Rizzo, P, Giuliano, MT, et al (1996) SV40-like sequences in human bone tumors. Oncogene 13,527-535[ISI][Medline]
-
Salewski, H, Bayer, T, Eidhoff, U, et al (1999) Increased oncogenicity of subclones of SV40 large T-induced neuroectodermal tumor cell lines after loss of large T expression and concomitant mutation in p53. Cancer Res 59,1980-1986[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
D. L. Poulin and J. A. DeCaprio
Is There a Role for SV40 in Human Cancer?
J. Clin. Oncol.,
September 10, 2006;
24(26):
4356 - 4365.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. A. Engels, R. P. Viscidi, D. A. Galloway, J. J. Carter, J. R. Cerhan, S. Davis, W. Cozen, R. K. Severson, S. de Sanjose, J. S. Colt, et al.
Case-Control Study of Simian Virus 40 and Non-Hodgkin Lymphoma in the United States
J Natl Cancer Inst,
September 15, 2004;
96(18):
1368 - 1374.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. A. Vilchez and J. S. Butel
Emergent Human Pathogen Simian Virus 40 and Its Role in Cancer
Clin. Microbiol. Rev.,
July 1, 2004;
17(3):
495 - 508.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-i. Nakatsuka, A. Liu, Z. Dong, S. Nomura, T. Takakuwa, H. Miyazato, and K. Aozasa
Simian Virus 40 Sequences in Malignant Lymphomas in Japan
Cancer Res.,
November 15, 2003;
63(22):
7606 - 7608.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. MacKenzie, K. S. Wilson, J. Perry, A. Gallagher, and R. F. Jarrett
Association Between Simian Virus 40 DNA and Lymphoma in the United Kingdom
J Natl Cancer Inst,
July 2, 2003;
95(13):
1001 - 1003.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. D. Strickler, J. J. Goedert, S. S. Devesa, J. Lahey, J. F. Fraumeni Jr., and P. S. Rosenberg
Trends in U.S. Pleural Mesothelioma Incidence Rates Following Simian Virus 40 Contamination of Early Poliovirus Vaccines
J Natl Cancer Inst,
January 1, 2003;
95(1):
38 - 45.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. TOGNON, F. MARTINI, L. IACCHERI, R. CULTRERA, and C. CONTINI
Investigation of the simian polyomavirus SV40 as a potential causative agent of human neurological disorders in AIDS patients
J. Med. Microbiol.,
February 1, 2001;
50(2):
165 - 172.
[Abstract]
[Full Text]
[PDF]
|
|
|
TOP
Abstract
Introduction
