mesothelioma cancer

Over the past 50 years epidemiology has been involved in the field of cancer
research. By studying the association between risk factors and cancer
occurrence, epidemiologists have contributed to the identifica¬tion of the most
important determinants of cancer in humans. In recent years, epidemiologists
have concentrated on the link between genetic and environment in carcinogenesis,
by focusing interests on low-level exposures.

Indeed, traditional epidemiology, called “black box epidemiology,” is unable to
study the mechanistic aspects of a disease (1). Therefore, the design of
epidemiologic studies has been enriched by introducing biologic markers (Fig.
14.1), and step-by-step molecular epidemiology has been created. This new
research, Perera (2) states, “seeks to combine the precision of laboratory
methods to quantify carcinogenic dose or preclinical response in humans, with
the relevance and rigor of analytic epidemiology.” This new research philosophy
is based on properly designed epidemiologic studies that take into account the
control of confounding factors, the selection of appropriate control groups, the
power of the studies, and the extent to which a biologic marker can predict
cancer occurrence (3,4).

The aim of molecular epidemiology is to assess individual exposures to
carcinogens and to quantify genetic damages linked to individ¬ual
susceptibility, in order to estimate cancer risk at the individual level.
Studies of genetically susceptible subgroups can detect high-risk subjects and
can implement new methodologies to prevent cancer at the primary
(chemoprevention) or secondary (screening programs) prevention level. Moreover,
a better understanding of the natural history of cancer may also improve cancer
treatment, by selecting the patients who will be able to benefit from specific
therapies. In addition, a multidisciplinary approach between molecular
biologists and epidemiologists, as well as physicians and biostatisticians, is
needed.

The outcome of malignant mesothelioma (MM) is poor, and the therapy of the
disease has not progressed in the past decade (5). But MM is largely preventable
because the causative factors are mostly of

environmental origin. Nevertheless, genetic factors appear to be impor¬tant by
affecting individual susceptibility to carcinogens.

Many years ago traditional epidemiology highlighted the carcino-genetic effect
of asbestos (6–8). Today, the molecular epidemiology approach has a great
capability to study MM, by assessing suscepti¬bility factors that might
predispose to cancer and by detecting markers to monitor cancer risk in
individuals exposed to carcinogens and to improve patient management.

The molecular events that underlie the development of this neo¬plasm have not
yet been completely elucidated, such as the possible relationship between
asbestos and simian virus 40 (SV40), and the intrinsic predisposition of
mesothelial cells to accumulate genetic damages. It is clear that multiple
genetic alterations are required for malignant transformation of mesothelium, as
suggested from the long latency period between exposure and the onset of disease
(9). Differ¬ent mechanisms are involved in the etiology of MM, such as modified
gene expression, gene silencing, gene amplification or rearrangement, complete
gene loss, modified expression of their protein products, or combinations of
multiple mechanisms. Other mechanisms include genomic instability, reduced DNA
repair capacity and individual susceptibility (10).

Figure 14.1. Paradigm of molecular epidemiology.

In Figure 14.2 a multistep process of risk is hypothesized for MM. Environmental
exposure, which may be monitored with specific expo¬sure markers, such as
asbestos bodies in the lung or SV40 antibodies, increases the risk of damaging
DNA at the individual level. This event may be monitored through different
markers of DNA damage and of susceptibility to DNA damage. Therefore, the
presence of damage itself increases the risk of gene impairment. It is supposed
that asbestos fibers interact with mesothelial cells, generating reactive free
radicals (11), which may influence the activation of some oncogenes or the
inhi¬bition of suppressor genes by interfering with mechanisms of cell growth
and with the expression of cytokines and growth factors or mutated proteins
(11,12). The biologic features of the involved genes affect the aggressiveness
and, consequently, the prognosis of disease.

This chapter summarizes the principal ideas and methodologies used in the field
of molecular epidemiology that can be applied to increase our understanding of
the prevention and surveillance of malignant mesothelioma.

Biomarkers

Biomarkers are measurable biologic indicators of exposure, effect,
sus-ceptibility, or disease state that are used to understand the mechanisms of
cancer progression (13). Even organs or tissues that are not con¬sidered
directly involved in the carcinogenetic process can show a response proportional
to the effective biologic dose. These tissues are commonly known as “surrogate
cell populations.” Chromosomal aber¬rations (CA) (14) or micronuclei (MN) in
lymphocytes can be consid¬ered indirect indicators of response that takes place
in target organs (e.g., pleura) and can therefore be considered as “surrogate”
markers. Overall, this aspect is relevant in biomonitoring studies performed in
living patients, since target tissues are often obtained by invasive procedures.

Studies with biomarkers may contribute to clarifying the etiology of cancer and
improving risk estimates, leading to better preventive strategies.

Biomarkers of Susceptibility

Biomarkers indicate whether an individual is particularly sensitive in relation
to the events produced by exposure. They highlight the dif¬ferences among
individuals or populations that may affect the response to asbestos, SV40, or
other environmental agents. These differences depend on genetic or other
individual features influencing the response of the target tissue. We have
divided these markers into two main groups: metabolic susceptibility markers and
DNA repair markers.

Metabolic Susceptibility Markers

The activity of the enzymes involved in the metabolism of carcinogens has great
variability among individuals, due to the existence of a poly¬morphism in the
genes coding for these enzymes. These differences may be inherited and can lead
to conspicuous differences in individ¬ual sensitivity to the effects of chemical
exposure, modifying the ability of a chemical to interact with proteins, RNA, or
DNA.

Metabolic susceptibility markers can assess interindividual varia¬tions in the
activities of metabolizing enzymes responsible for activation (phase I
reactions) or deactivation (phase II reactions) of environmental or endogenous
toxicants (16,17). Some metabolic sus¬ceptibility genes have been considered in
recent years as risk factors for lung cancer and for human MM.

The most important polymorphic genes involved in respiratory cancer risk, as
reported in the literature, are:

1. the genes of the cytochrome P450 (CYP) family, which mediate the phase I
reactions of metabolic activation;

2. the phase II genes glutathione S-transferases (GSTs) and N-acetyltransferases
(NATs);

3. the microsomal epoxide hydrolase (mEH) gene, which plays a dual role in
bioactivation and detoxification of procarcinogens.

Many P450 genes are polymorphic, including CYP1A1, whose product metabolizes
polycyclic aromatic hydrocarbons (PAHs) such as ben-zopyrene (BP). The higher
lung cancer risk from the “susceptible” CYP1A1 genotype was seen in light
smokers, whereas heavy smokers with this genotype had less than twice the risk
of heavy smokers without the genotype (18,19). The gene GST has a basic role in
phase II reactions to deactivate carcinogens. About 40% to 50% of Caucasians
possess a GSTM1 null genotype (20). The state of acetylation is con¬trolled by
two autosomal alleles in a single locus; rapid acetylation is dominating,
whereas slow acetylation is recessive. Approximately half of the Western
population is slow acetylating (NAT).

Microsomal epoxide hydrolase (mEPHX) is a critical metabolic enzyme involved in
the activation and subsequent detoxification of procarcinogens and plays a role
in the metabolic activation of the PAHs.

Metabolic genes encoding for enzymes involved in conjugation and detoxification
are likely to be implicated in the MM carcinogenetic pathway, due to the
presence of free radicals generated from asbestos exposure.

One study showed that nonmalignant asbestos-related diseases develop more
frequently in occupationally asbestos-exposed subjects carrying a homozygous
deletion (null genotype) of GSTM1 gene (21). A second study reported that
“individuals with homozygous deletion of the GSTM1 gene and a NAT2
slow-acetylator genotype who are exposed to high levels of asbestos appear to
have enhanced suscepti¬bility to asbestos-related pulmonary disorders” (22). On
the contrary, the GSTM1 genotype did not prove to interact with asbestos
exposure in the risk of lung cancer (23).

A paper on MM was published in 1995 (24) that reported that indi¬viduals with
combined GSTM1 and NAT2 defects had about a fourfold risk of developing MM
compared to those with the GSTM1 gene and NAT2 fast acetylator genotype [odds
ratio (OR) = 3.6; 95% confidence interval (CI) = 1.3–9.6]. Moreover, the risk
among subjects highly exposed to asbestos with the double at-risk genotype was
more than sevenfold greater than those with the more beneficial genotypes of
both GSTM1 and NAT2 genes (OR = 7.4; 95% CI = 1.6–34.0).

We have carried out a preliminary study by analyzing the distribu¬tion of
CYP1A1, mEH, GSTM1, GSTT1, and NAT2 genotypes in an Italian study population
consisting of 55MM patients and 200 popula¬tion controls. The combination of the
NAT2 fast acetylator and the GSTM1 null genotype posed a significantly increased
risk of MM com¬pared to the combination of the NAT2 slow acetylator and the
func¬tional GSTM1 genotype. A combined effect was also observed for the NAT2
fast acetylator and the mEH low-activity genotypes compared with the NAT2 slow
acetylator and the high mEH activity genotype combination.

When the MM patients were stratified according to the degree of asbestos
exposure, the putative mEH high-activity genotypes appeared to be totally absent
among the patients with low or unlikely exposure to asbestos. The association
reached the statistical significance when cases with high activity were compared
with cases of intermediate or low activity, or intermediate plus low activity
cases combined.

The most remarkable combined effect of borderline significance was observed for
the concurrent presence of the NAT2 fast acetylator geno¬type and the mEH
low-activity genotype, compared to the combination of the NAT2 slow acetylator
genotype and the high mEH activity genotype.

Our preliminary results strengthen the hypothesis that metabolic gene
polymorphisms involved in oxidation processes have a role in modulating
individual susceptibility to MM in subjects with different degrees of asbestos
exposure.

The lack of any association between CYP1A1 genotypes and MM cor-roborates the
hypothesis that PAHs, the main metabolic substrates of this enzyme, have no
direct effect in the development of malignant mesothelioma.

In an immunohistochemistry experiment, expression of the GST sub¬classes alpha,
mu, and pi in 20 patients with nonneoplastic mesothe-lium and in 57 patients
with malignant mesothelioma was studied. The expression of GST pi was reported
to be positively correlated with increased survival in MM. Therefore, the
authors concluded that GST and P-170 glycoprotein may contribute to the
resistance to cytotoxic drugs frequently observed in these tumors, but no
correlation was demonstrated between GST and P-170 expression (25).

DNA Repair Markers

The repair of DNA damage, i.e., base excision repair and nucleotide excision
repair, protects the cell from the injuries of mutagens and it is necessary for
the maintenance of genomic stability. Failure of this

system, originally demonstrated in individuals with xeroderma pig-mentosum, can
lead to cancer. It has been estimated that inherited defects in the DNA repair
system can account for 15% to 25% cases— and even more—of sporadic cancers of
different organs (26).

The assays of DNA repair capacity have been recently grouped into four
categories on the basis of the evaluation of DNA damage induced with chemicals
or physical agents. The evaluation tests are the mutagen sensitivity assay,
induced micronuclei, and the comet assay. Moreover, there are more accurate
measures of repair kinetics, such as the host cell reactivation assay, measures
of genetic variation associated with DNA repair, and indirect tests of DNA
repair, such as unscheduled DNA synthesis (27).

There is no direct information available on DNA damage and MM. Here some data
related to asbestos and SV40, the two major determi¬nants of MM are reported.
Asbestos does not significantly induce gene mutations in bacterial and mammalian
systems but causes structural and numerical chromosome aberrations in cultured
mammalian cells. It has been found that asbestos fibers produce a cell
transformation and genotoxicity characterized by the formation of aneuploid
cells, abnor¬mal anaphases, chromosomal aberrations, DNA single-strand breaks,
and DNA repair in human mesothelial cells (28–35).

In SV40 immortalized cell lines an interference of tumor (T) antigen with DNA
repair has been reported (28). Simian Virus 40 large-T antigen (SVLTAg) has been
widely used to immortalize cells. It was hypothesized that DNA mismatch repair
(MMR) activity is important during SVLTAg-induced immortalization and that the
immortalized cells are deficient in repairing G:T, A:C, and G:G mispairs in
bacte-riophage M13mp2 (29). In addition, the p53 tumor suppressor gene is able
to activate excision repair that is ultraviolet (UV) induced in human cells. The
SVLTAg binds p53 protein and can interfere with its function. Simian virus 40
transformation was shown to reduce the levels of DNA repair, most likely because
of the inhibition of normal p53 function by LTAg (30). Tag expression in
mesothelial cells might have both adverse and beneficial effects by impairing
the control of DNA integrity and enhancing apoptosis, respectively (31). As SV40
appears to play a possible role in the impairment of DNA repair mech¬anisms in
mesothelial cells, an additive effect with asbestos fibers may be hypothesized.

In consideration of the DNA damages generated by asbestos or SV40 exposure, all
markers reported in the above-mentioned four categories could be useful to
estimate DNA repair capability at the individual level. The DNA repair
capability in MM patients is still overlooked in the literature.

Micronucleus Test as an Index of Susceptibility to Malignant Mesothelioma

The decrease in DNA repair leads to increased genetic damage as mea¬sured by
cytogenetic damage, including formation of micronuclei.

The micronucleus assay is the method we have chosen for assessing chromosome
damage because it enables both chromosome loss and chromosome breakage to be
measured reliably (36). The micronucleus test (MT) in peripheral blood
lymphocytes (PBLs) seems to be a useful method for monitoring individuals with
genetic instability (37), and recent evidence suggests the usefulness of MT as a
screening test for carriers of specific mutations in evaluating cancer
susceptibility (38). Micronuclei (MN) are small amounts of DNA that arise in the
cyto¬plasm when chromatid/chromosomal fragments are not incorporated into
daughter nuclei during mitosis, often because these fragments do not possess a
centromere. Acentric fragments remain behind at anaphase, whereas chromosomal
elements with centromeres are drawn toward the spindle poles (39). Therefore,
the formation of micronuclei requires a dividing cell population. Micronuclei
are about 1/20 to 1/5 the size of the main nucleus. Usually there is only one
micronucleus formed per cell (40). The frequency of micronuclei is usually
reported as the number of cells containing micronuclei per total cells counted.

A study was carried out to evaluate, by the modified cytokinesis-blocked method
of Fenech and Morley (41), the MN frequency in PBLs of patients with pleural MM
compared to lung cancer (LC) patients and two control groups [patients with
BRDs, such as chronic obstruc¬tive pulmonary disease, asbestosis and silicosis,
and healthy controls (HCs)] in order to ascertain the relevance of this
biomarker to express the susceptibility of individuals to develop
pleuropulmonary tumors. Analysis was performed blindly of the subjects’ status
only on binu-cleated (BN) lymphocytes with preserved cytoplasm. An average of
2000 cells were analyzed for each subject, and all reported MN counts were the
mean of duplicate determinations. Means, medians, and stan-dard deviations were
calculated in terms of BN cells with MN: (BN -MN)/1000 BN cells. Nonparametric
tests were used to check the dif¬ferences among the groups. A significant
increased MN frequency in PBLs was observed only in patients with MM in
comparison with all other groups. No difference was observed between LC patients
and HCs, or among the different types of BRD subjects (Fig. 14.3).

Asbestos exposure has never been associated with a high frequency of MN and DNA
alterations. Moreover, numerical and structural chromosomal aberrations and an
increase in MN frequency were also reported in human cells in a number of
studies (42–44). Since PBLs are not the direct target for asbestos fibers, an
increase in the cumulative genetic damage in this surrogate tissue supplies an
index of the cumu¬lative genetic damage occurring during the PBLs’ life span
(45).

A study on breast cancer has indicated a close relationship between the presence
of a BRCA1 mutation and sensitivity for the induction of micronuclei. The
authors, in contrast to the results with the micronu-cleus assay, found no
significant difference between women with and without a BRCA1 mutation with
respect to the induction and repair of DNA damage in the comet assay. The
results suggested a normal rate of damage removal and points to a disturbed
fidelity of DNA repair (46). The increase of micronucleated PBLs in MM, patients
together

with the report of an association of this event with genetic characteris¬tics in
other neoplasia, is consistent with the existence of genetic factors
predisposing to the development of MM related to defects in DNA repair systems.

Biomarkers of Exposure, Diagnosis, and Prognosis

The alterations observed in tumor suppressor genes and dominant oncogenes may be
found in applications to molecular epidemiology and to clinical work. The
impairment of the genes is persistent, so it may reflect etiologic exposure. It
may provide a useful tool for diag¬nostic tests, and even for early detection,
since the expression of certain markers may occur prior to the development of
some overt malignan¬cies (47). Moreover, the impairment may act as a prognostic
marker, providing a good estimate of tumor aggressiveness and, eventually, it
may be able to influence therapy decisions, because therapies can be addressed
to the oncogenes or their protein products (48).

Oncoproteins have access to the extracellular environment and are detectable in
peripheral fluids such as serum or plasma, so the measure of their circulating
level provides an interesting opportunity to assess the diagnostic and
prognostic value of a marker overexpression in patients with tumors that are not
easily accessible to biopsy (49).

We briefly describe some markers of genes impairment, according to their
possible utility in the molecular epidemiology of MM.

Tumor Suppressor Genes

A critical role in the development and progression of MM is played by the loss
or inactivation of tumor suppressor (50). p16INK4 is a protein product of the
CDKN2 tumor suppressor gene and is being studied as one of the most interesting
markers in MM, from a diagnostic and a therapeutic point of view (51,52). It has
been reported that deletion of

p16INK4a occurs in from 22% to 70% of primary mesotheliomas (10,53). Since the
prevalence of asbestos fibers was found to be lower in MM patients with any p16
alteration than in those with no p16 alteration, it is possible that deletion of
p16 occurs in a relatively susceptible subset of MM (54). Homozygous CDKN2A
deletion may be a diagnos¬tic marker to characterize malignant mesothelial cells
from benign reac¬tive cells (55). Loss of p16INK4 protein expression can result
also from epigenetic mechanisms, such as an abnormal DNA hypermethylation. The
inhibition of methylation in mesothelioma may provide a poten¬tial treatment
target in some MM (56,57).

The commonest genetic alterations observed in human cancer are linked to
mutations in the p53 tumor suppressor gene. p53 Antibodies (p53-Abs) have been
indicated as possible biomarkers for early diag¬nosis and prognosis in various
neoplasms (49,58), and sometimes have been associated with a poor prognosis and
poor survival (59). In addi¬tion, serum p53-Abs could be useful in identifying
individuals at high cancer risk. This has been suggested by studies on a group
of workers with occupational exposure to vinyl chloride who were found to be
seropositive even more than 11 years before the clinical detection of
angiosarcoma of the liver (60), or on patients with chronic obstructive
pulmonary disease (COPD) who had elevated serum p53-Abs about 7 months before
the diagnosis of lung cancer (61). The p53 gene is rarely mutated in MM
(52,62–64), but p53 immunoreactivity in tumor tissue was shown in a proportion
ranging from 25% to 86% (65,66). There are few data in the literature on p53-Abs
in MM, and the results are not encouraging concerning their value as diagnostic
or prognostic indicators (67,68).

We analyzed the anti-p53 autoantibody level in 30MM patients, 48 LC patients, 55
subjects with benign lung diseases (BLDs), and in 51 HCs. In our investigation
7.4% of the MMs, 17% of the LCs, 3.6% of BLDs, and none of the HCs had elevated
serum levels of the anti-p53 autoantibodies (69). According to these data, the
presence of detectable p53-Abs in serum of patients with MM appears to be
occasional and does not seem to serve as either a diagnostic or a prognostic
indicator. Nevertheless, the presence of two positives among patients at high
risk of developing pleuropulmonary malignancies underlines the need for further
investigation with prolonged follow-up and an increased num¬ber of subjects.

Some genes have been recognized to be hallmarks of MM and suitable for
differentiating MM from other tumors. Among these, the Wilms’ tumor 1
susceptibility (WT1) is selectively expressed in MM (70). The detection of WT1
messenger RNA (mRNA) and of the WT1 protein is particularly useful in
differentiating MM from adenocarci-noma in tissue sections of pleural tumors
(71).

Oncogenes

The role of ras in the development of MM is uncertain. No mutation of H-ras has
been found in MM tissues by Cristaudo et al (72), and results from other studies
indicate that the K-ras proto-oncogene cannot play a critical role in the
induction of mesothelioma by asbestos, either in

humans or in rats (73). On the contrary, positivity for p21 was found in 35% of
MM tissues examined by Isik et al (68). In this study, a relationship between
asbestos exposure and p21 was found, since immunopositivity for p21 was higher
for patients with environmental asbestos exposure and was correlated to exposure
times (68). Interest¬ing results come from the determination of p21 expression
in serum. A cohort study on serum samples of 46 pneumoconiosis patients revealed
two MM patients, both positive for p21. In this case, the protein seemed to
support the role of an early diagnostic marker, since the positivity of the MM
patients preceded by 11 and 26 months the clinical evidence of the tumor (74).
The role of p21 as a prognostic marker is controver-sial (68,75).

Growth Factors

In recent years, attention has been focused on oncogenes’ causing the production
of growth factors or their receptors that have intrinsic tyro-sine kinase
activity. Receptor tyrosine kinases have become therapeu¬tic targets for
molecularly aimed therapies, and it is possible that treatment of MM patients
may benefit from this research (31–33).

In a pilot study on 62MMs, 35 LCs, 51 nonneoplastic subjects exposed to asbestos
and assumed to be at-risk controls (RCs), and 25 HCs, we investigated the role
of platelet-derived growth factor (PDGF), which is involved in the pathogenesis
of MM (76–78). Only a few studies have investigated PDGF levels in the blood of
neoplastic patients (74,79,80). Brandt-Rauf et al (74) reported a higher serum
level of PDGF in advanced pneumoconiosis cases than in patients with disease at
an earlier stage. According to this study, patients with higher PDGF levels had
an increased probability of having disease progres¬sion, suggesting that serum
PDGF levels may be a marker for the devel¬opment of severe and progressive
asbestos-related diseases. We found positive values in 42% of MMs, 8% of RCs, 3%
of LCs, and in 4% of HCs (preliminary data, unpublished). These results indicate
that high serum PDGF-AB levels could be used as a suggestive indicator of MM.
This hypothesis, and the fact that PDGF is thought to be an autocrine growth
factor for mesothelioma, support the trials that are testing a highly selective
inhibitor of the PDGF receptor tyrosine kinase as a therapeutic agent (81).

The epidermal growth factor receptor (EGFR) family is a group of four
structurally similar tyrosine kinases, among which there are the EGFR and
HER2/neu protein, encoded by the HER-2/neu gene. Immunoreactivity for HER2/neu
was found in 97% of MM patients (82). The protein has been detected at increased
serologic levels in sub¬jects at risk of cancer, such as in asbestosis patients,
who later devel¬oped lung cancer (83). Thus, it may constitute a marker of
cancer risk (83), and possibly of exposure. In fact, an association between
occupa¬tional exposures, mainly asbestos, and enhanced secretion of the pro¬tein
was found among healthy asbestos workers without asbestosis or cancer (84,85).

Suggestions for the utility of circulating HER-2/neu protein as an independent
biologic prognostic factor come from a study on patients

with advanced non–small-cell lung cancer (NSCLC) who submitted to standard
combination chemotherapy. In this study elevated serum level of HER-2/neu
protein were associated with a poor survival outcome, even if no significant
differences were observed in HER-2/ neu serum concentration in lung cancer
patients and a group of matched healthy controls (86,87).

Several studies have demonstrated that both EGF and its receptor (EGFR) are
involved in the development and progression of MM (88,89), and are correlated
with poor prognosis in some types of tumors. Autophosphorylation of EGFR occurs
in mesothelial cells after exposure to asbestos, and may initiate cell-signaling
cascades that are important in asbestos-induced carcinogenesis (90).
Modification of phosphorylation provides a rationale for the preventive and
therapeu¬tic approaches to lung cancers and mesothelioma (91). Other studies in
vitro or on animal models have shown that an agent that significantly inhibits
EGFR may be an effective therapeutic option for patients with MM (81,92).

The data in the literature support the diagnostic importance of the hepatocyte
growth factor/scatter factor (HGF/SF) and its receptor in MM (82,93,94).
Moreover, studies on lung cancer showed that when c-met is mutated or
overexpressed in malignant cells it serves as an important therapeutic
indication (95), while elevated HGF is associated with a poor prognosis and may
be useful as a marker of risk in early-stage tumors (96).

In our experience, the concentration of both HGF and EGF markers in MM was
double the concentration in HC. In this case, positivity was found in 60% of MM
patients and in none of the HCs for HGF, and in 50% of MMs and 18% of HCs for
EGF. In addition, a significantly cor¬relation existed between the two markers.
Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor
(bFGF) are potent angiogenic factors that promote vessel formation. An effective
thera¬peutic approach for MM (97,99) may derive from VEGF. The concen¬tration of
this factor was significantly higher in pleural effusions of MM patients than in
those from patients with nonmalignant pleural disease, and an inverse
correlation between serum VEGF levels and MM outcome was found (100).

The survival of MM patients seems to be affected also by bFGF, even if it is
expressed more in nonmalignant than in malignant pleural effusions (101).

References

1. Skrabanek P. The emptiness of the black box. Epidemiology 1994;5:553– 555.

2. Perera FP. Molecular cancer epidemiology: a new tool in cancer preven¬tion. J
Natl Cancer Inst 1987;78:887–898.

3. Hulka BS, Wilcosky T. Biological markers in epidemiologic research. Arch
Environ Health 1988;43:83–89.

4. Wogan GN. Molecular epidemiology in cancer risk. Assessment and pre¬vention:
recent progress and avenues for future research. Environ Health Perspect
1992;98:167–178.

5. Waller DA. The role of the surgery in diagnosis and treatment of malig¬nant
pleural mesothelioma. Curr Opin Oncol 2003;15:139–143.

6. Selikoff IJ, Churg J, Hammond EC. Asbestos exposure and neoplasia. JAMA
1964;188:22–26.

7. Wagner JC, Gilson JC, Berry G, Timbrell V. Epidemiology of asbestos cancer. B
Med Bull 1971;27:71–76.

8. Puntoni R, Vercelli M, Merlo F, Valerio F, Santi L. Mortality among ship¬yard
workers in Genoa, Italy. Ann NY Acad Sci 1979;330:353–377.

9. Huncharek M. Genetic factors in the aetiology of malignant mesothe-lioma. Eur
J Cancer 1995;31A:1741–1747.

10. Lechner JF, Tesfaiqzi J, Gerwin BI. Oncogenes and tumor-suppressor genes in
mesothelioma—a synopsis. Environ Health Perspect 1997;105: 1061–1067.

11. Kamp DW, Weitzman SA. The molecular basis of asbestos induced lung injury.
Thorax 1999;54:638–652.

12. Heintz NH, Janssen YM, Mossmann BT. Persistent induction of c-fos and c-jun
expression by asbestos. Proc Natl Acad Sci USA 1993;90:3299–3303.

13. Committee on Biological Markers of the National Research Council: biological
markers in environmental health research. Environ Health Perspect 1987;74:3–9.

14. Bonassi S, Hagmar L, Stromberg U, et al, for the European Study Group on
Cytogenetic Biomarkers and Health (ESCH). Chromosomal aberra¬tions in
lymphocytes predict human cancer independently of exposure to carcinogens.
Cancer Res 2000;60:1619–1625.

15. Bartsch H, Aitio A, Camus AM, et al. Carcinogen-metabolizing enzymes and
susceptibility to chemical carcinogenesis. IARC Sci Publ 1982;39:337– 350.

16. Hanke JZ. Genetic susceptibility to toxic substances and its relationship to
carcinogenesis. IARC Sci Publ 1984;59:99–106.

17. Dejmek A, Brockstedt U, Hjerpe A. Immunoreactivity of pleural malig¬nant
mesotheliomas to glutathione S-transferases. APMIS 1998;06:489– 494.

18. Segers K, Kumar-Singh S, Weyler J, et al. Glutathione S-transferase
expres¬sion in malignant mesothelioma and non-neoplastic mesothelium: an
immunohistochemical study. Cancer Res Clin Oncol 1996;122:619–624.

19. Seidegard PG, Vorachek VR, Pero RW, Pearson WR. Hereditary differ¬ences in
the expression of human glutathione trasferase active in trans-stilbene oxide
are due to a gene deletion. Proc Natl Acad Sci USA 1988; 85:7293–7297.

20. Zhao H, Spitz MR, Gwyn KM, Wu X. Microsomal epoxide hydrolase poly-morphisms
and lung cancer risk in non-Hispanic whites. Mol Carcinog 2002;33,2:99–101.

21. Smith CM, Kelsey KT, Wiencke JK, Leyden K, Stephen L, Christiani DC.
Inherited glutathione S-transferase deficiency is a risk factor for pul¬monary
asbestosis. Cancer Epidemiol Biomarkers Prev 1994;3:471–477.

22. Hirvonen A, Saarikoski ST, Linnainmaa K, et al. Glutathione S-transferase
and N-acetyltransferase genotypes and asbestos-associated pulmonary disorders. J
Natl Cancer Inst 1996;88:1853–1856.

23. Stucker I, Boffetta P, Antilla S, et al. Lack of interaction between
asbestos exposure and glutathione S-transferase M1 and T1 genotypes in lung
car-cinogenesis. Cancer Epidemiol Biomarkers Prev 2001;10:1253–1258.

24. Hirvonen A, Pelin K, Tammilehto L, Karjalainen A, Mattson K, Linnainmaa K.
Inherited GSTM1 and NAT2 defects as concurrent risk modifiers in
asbestos-related human malignant mesothelioma. Cancer Res 1995;55:2981–2983.

25. Segers K, Kumar-Singh S, Weyler J, et al. Glutathione S-transferase
expres¬sion in malignant mesothelioma and non-neoplastic mesothelium: an
immunohistochemical study. J Cancer Res Clin Oncol 1996;122:619–624.

26. Peltomaki P. Role of DNA mismatch repair defects in the pathogenesis of
human cancer. J Clin Oncol 2003;21:1174–1179.

27. Berwick M, Vineis P. Markers of DNA repair and susceptibility to cancer in
humans: an epidemiologic review. J Natl Cancer Inst 2000;92:874–897.

28. Renier A, Yegles M, Buard A, et al. Use of mesothelial cell cultures to
assess the carcinogenic potency of mineral or man made fibers. Cell Biol Toxicol
1992;8:133–139.

29. Ollikainen T, Linnainmaa K, Kinnula VL. DNA single strand breaks induced by
asbestos fibers in human pleural mesothelial cells in vitro. Environ Mol Mutagen
1999;33:153–160.

30. Okayasu R, Takahash S, Yamada S, Hei TK, Ullrich RL. Asbestos and DNA double
strand breaks. Cancer Res 1999;59:298–300.

31. Liu W, Ernst JD, Courtney Broaddus V. Phagocytosis of crocidolite asbestos
induces oxidative stress, DNA damage, and apoptosis in mesothelial cells. Am J
Respir Cell Mol Biol 2000;23:371–378.

32. Digweed M, Demuth I, Rothe S, et al. SV40 large T-antigen disturbs the
formation of nuclear DNA-repair foci containing MRE11. Oncogene 2002;
21:4873–4878.

33. Yeh CC, Lee C, Huang MC, Dahiya R. Loss of mismatch repair activity in
simian virus 40 large T antigen-immortalized BPH-1 human prostatic epithelial
cell line. Mol Carcinog 2001;31:145–151.

34. Bowman KK, Sicard DM, Ford JM, Hanawalt PC. Reduced global genomic repair of
ultraviolet light-induced cyclobutane pyrimidine dimers in simian virus
40-transformed human cells. Mol Carcinog 2000;29:17–24.

35. Levresse V, Moritz S, Renier A, et al. Effect of simian virus large T
antigen expression on cell cycle control and apoptosis in rat pleural
mesothelial cells exposed to DNA damaging agents. Oncogene 1998;16:1041–1053.

36. Fenech M. The in vitro micronucleus technique. Mutat Res 2000;455:81– 95.

37. Maluf SW, Erdtmann B. Genomic instability in Down syndrome and Fanconi
anemia assessed by micronucleus analysis in single-cell gelelec-trophoresis.
Cancer Genet Cytogenet 2001;124:71–75.

38. Trenz K, Rothfuss A, Schutz P, Speit G. Mutagen sensitivity of peripheral
blood from women carrying a BRCA1 or BRCA2 mutation. Mutat Res 2002;500:89–96.

39. Schmid W. The micronucleous test. Mutat Res 1975;31:9–15.

40. Schlegel R, MacGregor JT, Everson RB. Assessment of cytogenetic damage by
quantification of micronuclei in human peripheral blood erythrocytes. Cancer Res
1986;46:3717–3721.

41. Fenech M, Morley AA. Cytokinesis-block micronucleus method in human
lymphocytes: effect of in vivo ageing and low dose X-irradiation. Mutat Res
1986;161:193–198.

42. Lechner JF, Tokiwa T, La Veck M, et al. Asbestos-associated chromosomal
changes in human mesothelial cells. Proc Natl Acad Sci USA 1985;82: 3884–3888.

43. Dopp E, Schuler M, Schiffmann D, Eastmond DA. Induction of micro-nuclei,
hyperploidy, and chromosomal breakage affecting the centric/

pericentric regions of chromosomes 1 and 9 in human amniotic fluid cells after
treatment with asbestos. Mutat Res 1977;377:77–87.

44. Keane MJ, Stephens JW, Zhong BZ, Miller WE, Ong TM, Wallace WA. A study of
the effect of chrysotile fiber surface composition on genotoxicity in vitro. J
Toxicol Environ Health 1999;57:529–541.

45. Bolognesi C, Filiberti R, Neri M, et al. High frequency of micronuclei in
peripheral blood lymphocytes as index of susceptibility to pleural mesothelioma.
Cancer Res 2002;62:5418–5419.

46. Rothfuss A, Schutz P, Bochum S, et al. Induced micronucleus frequencies in
peripheral lymphocytes as a screening test for carriers of a BRCA1 mutation in
breast cancer families. Cancer Res 2000;60:390–394.

47. Partanen R, Koskinen H, Oksa P, et al. Serum oncoproteins in asbestosis
patients. Clin Chem 1995;41:1844–1847.

48. Taylor JA. Oncogenes and their applications in epidemiologic studies. Am J
Epidemiol 1989;130:6–13.

49. Brandt-Rauf PW. Biomarkers of gene expression: growth factors and
onco-proteins. Environ Health Perspect 1997;105S4:807–816.

50. Lee WC, Testa JR. Somatic genetic alterations in human malignant
mesothelioma (review). Int J Oncol 1999;14:181–188.

51. Frizelle SP, Grim J, Zhou J, et al. Re-expression of p16INK4a in
mesothe-lioma cells results in cell cycle arrest, cell death, tumor suppression
and tumor regression. Oncogene 1998;16:3087–3095.

52. Papp T, Schipper H, Pemsel H, et al. Mutational analysis of N-ras, p53,
p16INK4a, p14ARF and CDK4 genes in primary human malignant mesotheliomas. Int J
Oncol 200;18:425–433.

53. Xio S, Li D, Vijg J, et al. Codeletion of p15 and p16 in primary malignant
mesothelioma. Oncogene 1995;11(3):511–515.

54. Hirao T, Bueno R, Chen CJ, et al. Alterations of the p16(INK4) locus in
human malignant mesothelial tumors. Carcinogenesis 2002;23:1127– 1130.

55. Illei PB, Ladanyi M, Rusch VW, et al. The use of CDKN2A deletion as a
diagnostic marker for alignant mesothelioma in body cavity effusions. Cancer
2003;99:51–56.

56. Kratzke RA, Otterson GA, Lincoln CE, et al. Immunohistochemical analy¬sis of
the p16INK4 cyclin-dependent kinase inhibitor in malignant mesothelioma. J Natl
Cancer Inst 1995;87:1870–1875.

57. Wong L, Zhou J, Anderson D, et al. Inactivation of p16(INK4a) expression in
malignant mesothelioma by methylation. Lung Cancer 2002;38:131– 136.

58. Lubin R, Zalcman G, Bouchet, et al. Serum p53 antibodies as early markers of
lung cancer. Nature Med 1995;1:701–702.

59. Zalcman G, Tredaniel J, Schlichtholz B, et al. Prognostic significance of
serum p53 antibodies in patients with limited-stage small cell lung cancer. Int
J Cancer (Pred Oncol) 2000;89:81–86.

60. Trivers GE, Cawley HL, DeBenedetti VM, et al. Anti-p53 antibodies in sera of
workers occupationally exposed to vinyl chloride. J Natl Cancer Inst
1995;87:1400–1407.

61. Trivers GE, De Benedetti VMG, Cawley H, et al. Anti-p53 antibodies in sera
from patients with chronic obstructive pulmonary disease can predate a diagnosis
of cancer. Clin Cancer Res 1996;2:1767–1775.

62. Metcalf RA, Welsh JA, Bennett WP, et al. p53 and Kirsten-ras mutations in
human mesothelioma cell lines. Cancer Res 1992;52:2610–2615.

63. Mor O, Yaron P, Huszar M, et al. Absence of p53 mutations in malignant
mesotheliomas. Am J Respir Cell Mol Biol 1997;16:9–13.

64. Kitamura F, Araki S, Suzuki Y, et al. Assessment of the mutations of p53
suppressor gene and Ha- and Ki-ras oncogenes in malignant mesothe-lioma in
relation to asbestos exposure: a study of 12 American patients. Ind Health
2002;40:175–181.

65. Ramael M, Lemmens G, Eerdekens C, et al. Immunoreactivity for p53 protein in
malignant mesothelioma and non-neoplastic mesothelium. J Pathol 1992;168:
371–375.

66. Esposito V, Baldi A, De LA, et al. p53 immunostaining in differential
diag¬nosis of pleural mesothelial proliferations. Anticancer Res 1997;17:733–
736.

67. Creaney J, McLaren BM, Stevenson S, et al. p53 autoantibodies in patients
with malignant mesothelioma: stability through disease progression. Br J Cancer
2001;84:52–56.

68. Isik R, Metintas M, Gibbs AR, et al. p53, p21 and metallothionein
immunoreactivities in patients with malignant pleural mesothelioma: correlations
with the epidemiological features and prognosis of mesothe-liomas with
environmental asbestos exposure. Respir Med 2001;95: 588–593.

69. Neri M, Betta P, Marroni P, et al. Serum anti-p53 autoantibodies in pleural
malignant mesothelioma, lung cancer and non-neoplastic lung diseases. Lung
Cancer 2003;39:165–172.

70. Amin KM, Litzky LA, Smythe WR, et al. Wilms’ tumor 1 susceptibility (WT1)
gene products are selectively expressed in malignant mesothe-lioma. Am J Pathol
1995;146:344–356.

71. Hecht JL, Lee BH, Pinkus JL, et al. The value of Wilms tumor susceptibil¬ity
gene 1 in cytologic preparations as a marker for malignant mesothe-lioma. Cancer
2002;96:105–109.

72. Cristaudo A, Vivaldi A, Sensales G, et al. Molecular biology studies on
mesothelioma tumor samples: preliminary data on H-ras, p21, and SV40. J Environ
Pathol Toxicol Oncol 1995;14:29–34.

73. Ni Z, Liu Y, Keshava N, et al. Analysis of K-ras and p53 mutations in
mesotheliomas from humans and rats exposed to asbestos. Mutat Res
2000;468:87–92.

74. Brandt-Rauf PW, Smith S, Hemminki K, et al. Serum oncoproteins and growth
factors in asbestosis and silicosis patients. Int J Cancer 1992;50: 881–885.

75. Baldi A, Groeger AM, Esposito V, et al. Expression of p21 in SV40 large T
antigen positive human pleural mesothelioma: relationship with survival. Thorax
2002;57:353–356.

76. Langerak AW, De Laat PA, Van der Linden-Van Beurden CA, et al. Expres¬sion
of platelet-derived growth factor (PDGF) and PDGF receptors in human malignant
mesothelioma in vitro and in vivo. J Pathol 1996;178: 151–160.

77. Ascoli V, Scalzo CC, Facciolo F, Nardi F. Platelet-derived growth factor
receptor immunoreactivity in mesothelioma and nonneo-plastic mesothelial cells
in serous effusions. Acta Cytol 1995;39:613– 622.

78. Metheny-Barlow LJ, Flynn B, van Gijssel HE, et al. Paradoxical effects of
platelet-derived growth factor-A overexpression in malignant mesothe-lioma.
Antiproliferative effects in vitro and tumorigenic stimulation in vivo. Am J
Respir Cell Mol Biol 2001;24:694–702.

79. Mossman BT, Gruenert DC. SV40, growth factors, and mesothelioma. Another
piece of the puzzle. Am J Respir Cell Mol Biol 2002;26:167– 170.

80. Ariad S, Seymour L, Bezwoda WR. Platelet-derived growth factor (PDGF) in
plasma of breast cancer patients: correlation with stage and rate of
pro¬gression. Breast Cancer Res Treat 1991;20:11–17.

81. Nowak AK, Lake RA, Kindler HL, et al. New approaches for mesothe-lioma:
biologics, vaccines, gene therapy, and other novel agents. Semin Oncol
2002;29:82–96.

82. Thirkettle I, Harvey P, Hasleton PS, et al. Immunoreactivity for cadherins,
HGF/SF, met, and erbB-2 in pleural malignant mesotheliomas. Histopathology
2000;36:522–528.

83. Brandt-Rauf PW, Luo JC, Carney WP, et al. Detection of increased amounts of
the extracellular domain of the c-erbB-2 oncoprotein in serum during pulmonary
carcinogenesis in humans. Int J Cancer 1994;56:383– 386.

84. Lahat N, Froom P, Kristal-Boneh E, et al. Increased serum concentration of
growth factor receptors and Neu in workers previously exposed to asbestos. Occup
Environ Med 1999;56:114–117.

85. Krajewska B, Lutz W, Pilacik B. Determination of blood serum oncopro-tein
NEU and antioncoprotein p-53—molecular biomarkers in various types of
occupational exposure. Int J Occup Med Environ Health 1998;11: 343–348.

86. Filiberti R, Marroni P, Paganuzzi M, et al. c-erbB-2 protein in serum of
primary lung cancer patients. Cancer Detect Prev 2002;26:64–68.

87. Ardizzoni A, Cafferata MA, Paganuzzi M, et al. Study of pretreatment serum
levels of HER-2/neu oncoprotein as a prognostic and predictive factor in
patients with advanced nonsmall cell lung carcinoma. Cancer 2001;82:1896–1904.

88. Morocz IA, Schmitter D, Lauber B, et al. Autocrine stimulation of a human
lung mesothelioma cell line is mediated through the transforming growth factor
alpha/epidermal growth factor receptor mitogenic pathway. Br J Cancer
1994;70:850–856.

89. Vogelzang NJ. Emerging insights into the biology and therapy of malig¬nant
mesothelioma. Semin Oncol 2002;29:35–42.

90. Pache JC, Janssen YM, Walsh ES, et al. Increased epidermal growth
factor-receptor protein in a human mesothelial cell line in response to long
asbestos fibers. Am J Pathol 1998;152:333–340.

91. Manning CB, Cummins AB, Jung MW, et al. A mutant epidermal growth factor
receptor targeted to lung epithelium inhibits asbestos-induced pro¬liferation
and proto-oncogene expression. Cancer Res 2002;62:4169–4175.

92. Janne PA, Taffaro ML, Salgia R, et al. Inhibition of epidermal growth factor
receptor signaling in malignant pleural mesothelioma. Cancer Res 15:5242–5247.

93. Tolnay E, Kuhnen C, Wiethege T, et al. Hepatocyte growth factor/scatter
factor and its receptor c-Met are overexpressed and associated with an
in¬creased microvessel density in malignant pleural mesothelioma. J Cancer Res
Clin Oncol 1998;124:291–296.

94. Harvey P, Warn A, Newman P, et al. Immunoreactivity for hepatocyte growth
factor/scatter factor and its receptor, met, in human lung carci¬nomas and
malignant mesotheliomas. J Pathol 1996;180:389–394.

95. Maulik G, Shrikhande A, Kijima T, et al. Role of the hepatocyte growth
factor receptor, c-Met, in oncogenesis and potential for therapeutic
inhi¬bition. Cytokine Growth Factor Rev 2002;13:41–59.

96. Siegfried JM, Weissfeld LA, Luketich JD, et al. The clinical significance of
hepatocyte growth factor for non-small cell lung cancer. Ann Thorac Surg
1998;66:1915–1918.

97. Cacciotti P, Strizzi L, Vianale G, et al. The presence of simian-virus 40
sequences in mesothelioma and mesothelial cells is associated with high levels
of vascular endothelial growth factor. Am J Respir Cell Mol Biol
2002;26:189–193.

98. Catalano A, Romano M, Martinotti S, et al. Enhanced expression of vas¬cular
endothelial growth factor (VEGF) plays a critical role in the tumor progression
potential induced by simian virus 40 large T antigen. Onco-gene
2002;25:2896–2900.

99. Masood R, Kundra A, Zhu S, et al. Malignant mesothelioma growth inhi¬bition
by agents that target the VEGF and VEGF-C autocrine loops. Int J Cancer
2003;104:603–610.

100. Strizzi L, Catalano A, Vianale G, et al. Vascular endothelial growth factor
is an autocrine growth factor in human malignant mesothelioma. J Pathol
2001;193:468–475.

101. Strizzi L, Vianale G, Catalano A, et al. Basic fibroblast growth factor in
mesothelioma pleural effusions: correlation with patient survival and
angiogenesis. Int J Oncol 2001;18:1093–1098.