  , .

184: 252–257 (1998)

SV40-LIKE DNA SEQUENCES IN PLEURAL

MESOTHELIOMA, BRONCHOPULMONARY
CARCINOMA, AND NON-MALIGNANT PULMONARY
DISEASES
. -1, . 2, . 3, . 2, . 3, . 4, . . 3, . 3,
. 5, . . 3  . 2*
1
Department of Pathology, CHRU Caen, France
Department of Human and Molecular Virology, CHRU Caen, France
2
3
INSERM U139, University of Medicine, Creteil, France
4
Department of Occupational Medicine, CHRU Caen, France
5
University Bordeaux II, Bordeaux, France

SUMMARY
Pleural and pulmonary malignancies are usually associated with well-known carcinogen exposure. Recently, the presence of simian
virus 40 (SV40)-like DNA sequences has been detected in brain and bone-related human cancers and in pleural mesothelioma. In order
to determine whether SV40-like DNA sequences are also present in bronchopulmonary carcinoma and non-malignant lung samples, 125
frozen pleural and pulmonary samples (including 21 mesotheliomas, 63 bronchopulmonary carcinomas, 8 other tumours, and 33
non-malignant samples) and 38 additional samples distant from tumours were studied for the occurrence of SV40-like DNA sequences
by polymerase chain reaction (PCR) amplification followed by hybridization with specific probes. Sequences related to SV40 large T
antigen (Tag) were present in 28·6 per cent of bronchopulmonary carcinomas, 47·6 per cent of mesotheliomas, and 16·0 per cent of cases
with non-neoplastic pleural and pulmonary disease. No statistically significant difference in the occurrence of these DNA sequences was
found between malignant mesothelioma and bronchopulmonary carcinoma, but a significantly higher number of mesothelioma cases
exhibited SV40-like DNA sequences in comparison with cases of non-malignant pleural or pulmonary disease (P<0·04). Among cases
positive for SV40-like DNA sequences, a history of asbestos exposure was found in 3 out of 12 bronchopulmonary carcinomas and 8 out
of 10 mesotheliomas. Immunohistochemistry using monoclonal antibodies directed against Tag did not demonstrate nuclear staining. The
DNA sequences were not related to BK virus sequences, but three samples were positive with probes hybridizing with JC virus DNA
sequences. In conclusion, this study demonstrates the presence of SV40-like DNA sequences in pulmonary neoplasms and in
non-malignant lung tissues. It appears that the presence of SV40-like DNA is not unique to cancer. ? 1998 John Wiley & Sons, Ltd.

J. Pathol. 184: 252–257, 1998.

KEY WORDS—malignant mesothelioma; bronchopulmonary carcinoma; SV40; PCR

INTRODUCTION been found to contain papilloma virus sequences by

Pleuropulmonary malignancies are aggressive
tumours, mostly resulting from exposure to environ-
mental factors. Bronchopulmonary carcinomas, thought
to be generated by tobacco smoking and various
occupational exposures, are the most common
malignant tumours and are the major cause of cancer
death.1,2 Mesothelioma is mostly associated with
asbestos exposure and is expected to increase during
the next 20 years,3 but 20 per cent of patients with
malignant mesothelioma have no detectable evidence of
asbestos exposure;4,5 on the other hand, as few as 10 per
cent of highly exposed patients will develop mesothelio-
mas. Mesotheliomas have been diagnosed in patients
suffering from AIDS, without occupational or domestic
asbestos exposure, suggesting a possible viral connec-
tion.6 At the same time, squamous cell carcinomas have
*Correspondence to: Professor F. Freymuth, Department of
Human and Molecular Virology, CHU Cote de Nacre, 14033 Caen
Cedex, France.
Contract grant sponsor: French Ministry of Health.
CCC 0022–3417/98/030252–06 $17.50
? 1998 John Wiley & Sons, Ltd.
PCR,7 suggesting a possible role for viral infection in
lung carcinogenesis. Simian virus 40 (SV40) is another
virus that might play a role in cancer.
SV40 induces mesotheliomas in hamsters following
intrapericardial inoculation.8 SV40-like DNA sequences
have been detected in mesotheliomas,9 in brain tumours
(ependymomas, choroid plexus tumours, and neuro-
blastomas),10 as well as in bone-related tumours (osteo-
sarcomas, chondrosarcomas, and Ewing’s sarcomas).11
SV40 is a simian virus isolated from rhesus monkey
cell cultures and is able to play a role in the transfor-
mation of several cell types, in cell culture. The onco-
genic properties of SV40 are mainly mediated by large
T viral protein (Tag), which is able to bind to and
inactivate pRb and p53.12–14
SV40 may have contaminated polio vaccine.15 It is not
known to infect man naturally, but can replicate in and
immortalize human cells.16,17 SV40 is closely related
to the two human polyomaviruses BK virus (BKV) and
JC virus (JCV), which are endemic in human adult
populations.18
Received 26 March 1997
Accepted 9 September 1997
 SV40-LIKE DNA SEQUENCES IN PULMONARY NEOPLASMS AND NON-MALIGNANT LUNG TISSUES
These data prompted us to determine whether SV40-
like sequences could be found in bronchopulmonary
carcinomas and pleuroparietal sarcomas, in addition to
mesotheliomas. As asbestos exposure is known to be a
risk factor for bronchopulmonary carcinoma, we also
studied the association of SV40-like DNA sequences and
asbestos exposure in patients with bronchopulmonary
carcinomas.
We used a PCR hybridization technique, with primers
and probes previously described by Bergsagel et al.10
We also studied SV40 large Tag nuclear expression
in mesotheliomas and bronchopulmonary carcinomas
using immunoperoxide staining techniques.
MATERIALS AND METHODS
Materials
Frozen samples were collected from the files of the
Department of Pathology of the CHU de Caen. These
cases had been diagnosed between 1992 and 1994 and
were not selected. Six additional epithelial malignant
mesotheliomas were cultured according to conditions
described elsewhere.19 This study included 21 meso-
theliomas (15 tissue samples and 6 cell lines), 63
bronchopulmonary carcinomas, 8 other tumours, 33
non-malignant samples, and 38 additional lung and
pleural samples distant from tumours. In total, 163
samples were studied.
The mesothelioma samples were included according
to the accepted criteria of the U.S./Canadian meso-
thelioma panel20 and were certified by the French
mesothelioma panel (Mesopath group). Epithelial
mesotheliomas had a tubulopapillary, epithelioid or
pseudoglandular pattern. Two mesotheliomas demon-
strated a desmoplastic pattern. The bronchopulmonary
samples included the four main types of broncho-
pulmonary carcinoma according to the World Health
Organization classification (WHO, 1982).21 The 33
non-malignant samples included emphysema (9), sero-
fibrinous pleuritis (3), organizing pleuritis (8), bron-
chiolitis obliterans-organizing pneumonia (BOOP) (3),
cystic bronchiolectasis (1), hamartochondroma (1),
aspergilloma (1), iatrogenic alveolar haemorrhage (1),
inflammatory pseudotumour (1), pulmonary granulo-
matosis (1), and traumatic normal pleura (2).
DNA extraction
All specimens were blindly analysed following the
same procedure. Fresh frozen biopsies were cut on ice,
under sterile conditions, into pieces of about 2 mg and
incubated at 56)C with proteinase K (final concentration
130 mg/l) in 750 ìl of lysis buffer (50 m Tris–HCl, pH
8·5; 1 m EDTA, pH 8; 1  NaCl; 0·5 per cent SDS)
until completely digested. DNA was extracted by the
phenol/chloroform method, precipitated in ethanol at
"20)C for 18 h, and resuspended in 150 ìl of sterile
distilled water. DNA was extracted from cell cultures
according to similar procedures, except that the
proteinase K concentration was 500 mg/l. Prior to DNA
? 1998 John Wiley & Sons, Ltd.
253
extraction, cells in culture were detached with a mixture
of 0·25 per cent trypsin/0·02 per cent EDTA and pelleted
by centrifugation at 350 g for 10 min.
PCR assay
DNA amplification was performed with the primers
designed by Bergsagel et al.:10 PYV.for (5*-TAG-GTG-
CCA-ACC-TAT-GGA-ACA-GA-3*) and PYV.rev
(5*-GGA-AAG-TCT-TTA-GGG-TCT-TCT-ACC-3*).
These primers amplify a conserved sequence of the
large Tag gene in the polyomaviruses SV40 (173 bp),
JCV (179 bp), and BKV (182 bp). PCRs were per-
formed in 100 ìl containing 2·5 U of Taq polymerase
(Amplitaq>, Perkin Elmer) in the corresponding buffer
(GeneAmp>, Perkin Elmer), 100 pmol of each primer
(PYV.for and PYV.rev), 200 ì of each of the four
deoxynucleotides (Boehringer Mannheim), and 10 ìl of
DNA extract. Reactions were performed in a thermal
cycler (Hybaid>, Omnigen). After 5 min of denaturation
at 94)C, 40 cycles were performed (94)C for 5 s, 52)C
for 30 s, 72)C for 30 s) with a final extension for 10 min
at 72)C.
Amplified DNA was visualized by UV light on 2·4 per
cent agarose gel after ethidium bromide staining.
Each PCR series was carried out with one negative
and one positive control. The positive controls consisted
of DNA extracted from ALSV cells for SV40, VERO
cells infected with BKV (kindly provided by Professor P.
Lebon, Virology Department, Hôpital Saint Vincent de
Paul, Paris, France), and MAD-1 strain genome inserted
in P Br 322 plasmid grown in Escherichia coli HB 101 for
BK and JC, respectively (provided by Professor D.
Ingrand, Microbiology Department, Hôpital Robert
Debré, Reims, France).
Hybridization
Hybridization was performed with specific SV40, BK
and JC biotinylated probes, using a GEN-ETI-K>
DEIA (DNA enzyme immunoassay revealed with
peroxidase staining) commercial kit (Sorin Biomedica).
The probes were those used by Bergsagel et al.:10
BK probe:
5*-CAG-AAT-CTG-CTG-TTG-GTT-CTT-3*
JC probe:
5*-GTT-GGG-ATC-CTG-TGT-TTT-CAT-3*
SV40 probe:
5*-ATG-TTG-AGA-GTC-AGC-AGT-AGC-C-3*
(All probes and primers were provided by the unit of
organic chemistry URA CNRS 487, Institut Pasteur,
Paris.) For DEIA, the specific probe is immobilized on
the wells of a microtitre plate by a streptavidin–biotin
bridge. Sample DNA is then denatured by heat and
added to the wells. After 1 h incubation at 50)C and
removal of excess reagents, the addition of an anti-
double strand DNA monoclonal antibody identifies the
wells where hybridization has occurred. The addition of
peroxidase conjugated with anti-globulin is followed,
after washing, by enzymatic reaction with chromogen.
The sample OD was determined by subtracting the
  , . 184: 252–257 (1998)
254 F. GALATEAU-SALLE ET AL.
absorbance at 630 nm from that at 450 nm. The OD
index was defined as the sample OD/(0·150+mean
negative control OD value), as recommended by the
manufacturer. An index greater than 1·5 is indicative of
a positive hybridization. The means of OD in negative
controls were 0·026 for SV40, 0·0620 for JC, and 0·049
for BK.
Contamination was rigidly controlled by performing
specimen extractions and DNA preparation, and PCR
in separate laboratory rooms. Following PCR, no
sample yielded a visible band of the expected size on
agarose gel electrophoresis, but a tiny band appeared at
the expected place after reamplification of some SV40
hybridization-positive amplified products. Thus, all the
results given are based on specific hybridization with the
SV40 probe.
All the positive samples and all the SV40-negative
mesotheliomas and bronchopulmonary carcinomas were
tested twice. To control the extraction procedure and
to rule out the presence of any inhibitory factor, all
the mesothelioma extracts and the negative extracts of
formerly positive samples were tested with betaglobin
PCR: the cellular gene was always easily amplified.
Immunohistochemistry
Screening for Tag nuclear expression was carried out
on all mesothelioma samples and on 16 out of 63
bronchopulmonary carcinoma samples using an avidin–
biotin–peroxidase complex (ABC) method on 5 ìm-
thick formalin-fixed, paraffin-embedded tissue sections.
SV40 Tag mouse monoclonal antibody (Ab-1 Oncogene
Sciences) was applied at the commercial predilution
of 1/20 and incubated for 2 h at room temperature.
Staining was performed with the LSAB> kit (Dako
Ltd.).
Positive controls (SV40 immortalized rat pleural
mesothelial cells infected with a Tag recombinant retro-
virus) were obtained from collaborative research with
W. H. Gunzburg, B. Salmon, Munich, Germany. Rats
were prepared on Tek slides (Nunc, Naperville, U.S.A.).
Negative controls consisted of omission of the primary
antibody from the staining protocol. Nuclear staining
was considered as positive and cytoplasmic staining as
negative.
Evaluation of asbestos exposure from occupational
questionnaire
Complete occupational histories were available for
all mesothelioma patients and for 40 patients with
bronchopulmonary carcinoma. Occupational exposure
to asbestos was then evaluated by two of us (JCP, ML),
on the basis of data collected in the occupational
questionnaire and knowledge of occupational exposure
in different settings.
Analysis of asbestos bodies in lung tissue
The lung tissue samples were collected in cleaned
flasks containing 10 ml of 10 per cent formalin (Prolabo,
Paris, France). Specimens were prepared as described
? 1998 John Wiley & Sons, Ltd.
elsewhere.22 Typical ferruginous bodies, as defined
by Churg and Warnock,23 were quantified by light
microscopy. According to previously published
threshold values,22 significant retention of asbestos
bodies in the lung was defined as more than 1000
asbestos bodies per g of dry tissue. Non-neoplastic lung
tissue was available for mineralogical analysis from 33
subjects with bronchopulmonary carcinoma.
Statistical analysis
Statistical analysis of the results was performed by
use of the ÷2 test. All calculations were made with
the SAS package. When both a tumour sample and
non-tumoural tissue were available in cancer cases,
data obtained in the tumour sample were taken into
consideration unless specified.
RESULTS
SV40-like DNA sequences were detected in both
malignant mesotheliomas and bronchopulmonary car-
cinomas, as well as in non-malignant lung samples.
Table I summarizes the results on the detection of
SV40-like DNA sequences according to the pathology.
SV40-like DNA sequences were detected in 35/105 men
and in 6/20 women. The mean age of subjects exhibiting
SV40-like DNA sequences in their tissue samples was
63 years (range 41–74 years). Ten mesotheliomas and
18 out of 63 bronchopulmonary carcinomas yielded a
positive hybridization (OD indices ranging respectively
from 1·9 to 5·9 and from 2·3 to 9·7). Analysing the
presence of SV40-like DNA sequences according to
the histological bronchopulmonary subtypes, 47·0 per
cent of adenocarcinoma, 24·2 per cent of squamous
cell carcinoma, and 40·0 per cent of neuroendocrine
carcinoma samples revealed the presence of those
sequences. In the other tumour group, only a primitive
chest wall osteosarcoma yielded a positive hybridization
for SV40-like DNA sequences (OD index 7·9).
Among the non-malignant samples, SV40-like DNA
sequences were detected in 4 out of 25 samples from
subjects having non-malignant disease and in 3 out of 8
organizing pleuritis. The OD index in positive organiz-
ing pleuritis was quite similar to that observed in
positive malignant mesothelioma. We did not find any
significant difference in the detection of SV40-like DNA
sequences, either between malignant mesothelioma
and bronchopulmonary carcinoma, or between malig-
nant mesothelioma and non-malignant disease. How-
ever, a significantly higher frequency of SV40-like DNA
sequences was observed between mesothelioma samples
and non-tumoural samples (Table II). Nevertheless, no
statistical difference was found when the data obtained
in mesothelioma cases were compared with those of
subjects with non-malignant pleural disease.
SV40-like DNA sequences were detected in 5 out of 38
non-malignant lung samples taken from patients with
bronchopulmonary carcinoma and providing both
tumour tissue and uninvolved lung tissue; in two of
them, the SV40 sequences were also detected in the
  , . 184: 252–257 (1998)
 SV40-LIKE DNA SEQUENCES IN PULMONARY NEOPLASMS AND NON-MALIGNANT LUNG TISSUES 255

Table I—Percentage of samples exhibiting SV40-like DNA sequences and value of the OD index according
to disease

Percentage of
samples with SV40-like Range of
Histology DNA sequences (%) OD index

Mesothelioma (n=21) 47·6 1·9–5·9

Primary bronchopulmonary carcinoma (n=63) 28·6 2·3–9·7

Adenocarcinoma (n=15) 47·0 6·6–9·7
Squamous cell carcinoma (n=33) 24·2 2·8–4·7
Large cell carcinoma (n=1) 0·0 0·0
Neuroendocrine carcinoma (n=5) 40·0 2·5–5·1
Pleomorphic carcinoma (n=9) 11·1 2·3

Other tumours (n=8) 13 7·9

Normal tissues distant from tumours (at least 5 cm) (n=38) 13 1·9–5·9

Non-malignant lung/pleura samples (n=25) 16 5·2–10

Organizing pleuritis (n=8) 38 2·2–4·7

Total (n=163) 23 1·9–10

n=number of cases.

Table II—Summary of statistical analyses performed with data Table III—Comparison of the occurrence of SV40-like DNA
on the occurrence of SV40-like DNA sequences in tissue sequences in tissue samples from cancer cases providing both
samples from different diseases tumour tissue and uninvolved lung tissue (number of cases)

P value, In tumour tissue

Disease ÷2 test
SV40-like DNA sequences Present Absent Total

Non-malignant disease (n=33) versus

All cancers (n=92) 0·26 In uninvolved tissue Present 2 3 5
Bronchogenic carcinoma (n=63) 0·44 Absent 5 28 33
Mesothelioma (n=21) 0·04 Total 7 31 38
Other tumours (n=8) 0·50

Mesothelioma (n=21) versus

Non-malignant pleural disease (n=12) 0·28
Bronchopulmonary carcinoma (n=63) 0·11
Other tumours (n=8) 0·11
n=number of cases.
tumour tissue. No difference was found in the occur-
rence of SV40-like DNA sequences between samples
from tumours and those from tissue distant from the
tumour (Table III).
To make sure that there was no cross-reaction with
polyomavirus BK and JC, all three SV40, BK, and JC
positive controls were tested, after amplification, with
BK and JC probes: no cross-hybridization occurred. In
addition, all SV40-positive products amplified from
pleuropulmonary samples were hybridized with BK
and JC probes: no hybridization occurred with the
BK probe, but three samples hybridized with the JC
probe (one pulmonary papillary adenocarcinoma:
index=7·6; one pseudomesotheliomatous carcinoma:
index=3·1; and one cystic bronchiolectasis: index=1·5).
? 1998 John Wiley & Sons, Ltd.
We definitively interpret those DNA sequences as
different from BK and JC virus sequences.
Asbestos exposure was established in 19 out of 21
mesothelioma cases, and in 18 out of 40 broncho-
pulmonary carcinomas. No significant difference was
observed for the frequency of asbestos exposure in
relation to the detection of SV40-like DNA sequences in
any of these disease (Table IV). Similarly, there was no
difference for the lung retention of asbestos bodies (data
not shown).
SV40-like DNA sequences were detected in 8 out of
25 polio vaccinated patients with bronchopulmonary
carcinomas (born from 1921 to 1933). We did not find
SV40-like sequences in the eight patients born after 1963
(last year of possible vaccination with a contaminated
polio vaccine).
Immunohistochemistry performed on 15 meso-
thelioma and 16 bronchopulmonary carcinoma samples
demonstrated cytoplasmic staining and no nuclear stain-
ing, in contrast with the positive control cells which
showed nuclear staining.
  , . 184: 252–257 (1998)
256 F. GALATEAU-SALLE ET AL.
Table IV—Percentage of mesothelioma and bronchopulmo-
nary carcinoma cases exposed to asbestos in relation to the
occurrence of SV40-like DNA sequences (number of cases)
Bronchopulmonary
Mesothelioma carcinoma
Exposure Exposure
SV40-like DNA
sequences Yes No Yes No
Present 8 2 3 9
Absent 11 0 15 13
DISCUSSION
To our knowledge, this is the first study to demon-
strate the presence of SV40-like DNA sequences in
bronchopulmonary carcinoma as well as in non-
malignant pulmonary samples, but it also confirms the
presence of SV40-like DNA sequences in malignant
mesothelioma previously reported by others.9,10,24 The
occurrence of SV40-like DNA sequences in meso-
thelioma was not different from that in broncho-
pulmonary carcinoma, but was significantly enhanced in
comparison with non-malignant pulmonary samples.
SV40 positivity does not therefore argue for meso-
thelioma diagnosis. Positivity was not specific for any
one bronchopulmonary carcinoma phenotype, but
adenocarcinomas from distal lung exhibited a higher
percentage of SV40-like DNA sequences (47·0 per cent)
as well as higher OD indices (range 6·7–9·7) than
squamous or neuroendocrine carcinomas.
It is now well demonstrated that many but not all
mesotheliomas are associated with asbestos exposure.4,5
The search for other agents is therefore of great interest.
As we have already stated, both Cicala et al.8 and
Carbone et al.9 demonstrated the presence of SV40-like
DNA in mesotheliomas and thus suggested a potential
implication of SV40 in mesothelioma oncogenesis.9 In
our series, all but two mesothelioma cases showing
SV40-like DNA sequence were highly exposed to asbes-
tos, making it impossible to determine the exact role of
SV40 in the development of mesothelioma. Moreover,
no significant association was found between SV40-
like DNA detection and asbestos exposure, nor with
lung retention of asbestos bodies, in a subgroup of 40
bronchopulmonary carcinomas for which exposure data
were available.
As previously suggested, SV40 was a contaminant of
polio vaccines in the 1950s, explaining possible human
exposure.9,24 In our series, a history of polio vaccination
was obtained in 29 patients with bronchopulmonary
carcinoma. SV40-like DNA sequences were detected in 8
out of 25 tissue samples from vaccinated patients, while
no positivity was found in the four patients with a
definite absence of vaccination in the 1950s. All samples
exhibiting SV40-like DNA sequences originated from
patients born before 1963 (37 cases) who were old
enough and could have been infected by contaminated
polio vaccination. Moreover, SV40-like DNA sequences
? 1998 John Wiley & Sons, Ltd.
were not detected in patients born after 1963 and too
young to have received the vaccine. These data proved
difficult to interpret, because most of the patients did
not remember the precise date and route of polio
vaccination and because we could not perform sero-
logical studies as a control of SV40 contamination.
These observations do not prove any causal relationship
between SV40 and the occurrence of these sequences,
but agree with this hypothesis.
Controversial findings are reported in the literature on
the presence and role of SV40 in mesothelioma.9,25–27
Our data do not argue for or against a role of SV40 in
the neoplastic process, but confirm the presence of these
sequences in human samples. Despite the detection of
SV40-like DNA sequences, we found low hybridization
indices compared with positive controls, suggesting that
very few copies are present within the tissue samples, or
that the sequences had been diluted following the cell
division associated with tumour growth. To interact
with cell proliferation and exert its deleterious effects,
Tag should be expressed in the cell nucleus. In contrast
with positive control cells, cytoplasmic staining, but
no nuclear staining with Tag-specific antibodies was
found in both paraffin-embedded sections and cell lines
exhibiting SV40-like DNA sequences (data not shown).
These results differ from those previously reported9,25–27
and the discrepancies are difficult to explain.
The origin of these DNA sequences in human samples
remains enigmatic. Some viral sequences could have
been acquired during evolution and integrated into the
normal human genome, but in that case, they should be
found in all the cells of the biopsies and should be as
easily amplified as any other cellular gene. Several
authors have reported the presence of SV40 antibodies
in serum from patients having no history of vaccination
with the suspected vaccine,28–30 suggesting that SV40
can naturally infect human populations. Finally, it could
be due to an unknown polyomavirus, or a possible
recombinant polyomavirus adapted to the human
species.
The exact nature of these amplified sequences and
their oncogenic role remains to be determined. These
data have prompted us to propose future multicentric
studies with inter-laboratory controls.
ACKNOWLEDGEMENTS
This work was supported by grants from the French
Ministry of Health according to the PHRC 1994. We are
grateful to M. A. Billon-Galland from the Laboratoire
des Particules Inhalées, 13 Rue Georges Eastman, Paris,
France for performing mineralogical analysis in lung
tissue and to Brenda Gerwin from the NCI/NIH,
Bethesda, Maryland, U.S.A. for reviewing our
manuscript.
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