Molecular Epidemiologic Markers:
A New Concept in the Preventive Medicine with Special Attention to the Prevention of Cancer
István Ember1, Árpád Németh1, Csaba Varga1, Pál Perjési2, István Arany3, Katalin Fehér1, Katalin Németh1, Zsuzsanna Dombi1, and István Kiss1
1 Department of Public Health, Medical School, University of Pécs, Pécs, Hungary
2 Department of Biochemistry, Medical School, University of Pécs, Pécs, Hungary
3 Department of Internal Medicine, Division of Nephrology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
Corresponding author: Prof. István Ember
Department of Public Health
Medical School
University of Pécs
Szigeti u. 12
H-7643 Pécs, Hungary
Telephone: Phone: +36-72-536395
E-mail: istvan.ember@aok.pte.hu
CEJOEM 2005, Vol.11. No.1.: 3–15
Key words:
Early phase of carcinogenesis, gene alterations, biomarker, chemopreventive substances, risk assessment, primary prevention
Abstract:
In early diagnosis of cancer (secondary prevention), there was no real breakthrough, although amplification and/or overexpression
of onco-suppressor genes had been often observed in routine pathological studies. Numerous genetic alterations could be associated with
the early phase of carcinogenesis. Examination of these alterations, immediately after the exposure, could give us the opportunity to
use them as biomarkers. A relationship was proved between the early genetic alterations caused by the carcinogens and tumor development.
The effects were measured in “short-term” animal model in thymus, spleen, lymph nodes, bone marrow, liver, kidneys and lungs at 24, 48
and 72 hours after the treatment. Considering individual susceptibility and polymorphism of carcinogen metabolizing enzymes, we can
apply these biomarkers to identify the “high risk” individuals and groups of the population and this method could be suitable to
decrease cancer morbidity, follow up the effectiveness of therapy and chemopreventive agents.
INTRODUCTION
Despite the revolutionary discoveries in cancer research of the last decade, as new information about onco-suppressor gene cascades,
communication between cells, detection of apoptosis (Wild et al., 2002), there has been little success to find specific, diagnostic,
pathological and morphological alterations that would be characteristic of each tumor type (Balmain et al., 2003). The advances in
molecular genetics have led to the hypothesis that tumors are basically “genetic diseases”, mainly induced by environmental factors
(Minamoto et al., 1999). This is true primarily for sporadic tumors, constituting 90% of all tumors. Environmental chemical factors
are responsible for 90% of these tumors (Knudson, 2002).
It is known that ectopic activation of onco- and suppressor genes by environmental chemical factors
leads to development of tumors (Todd and Wong, 1999). However, this activation is complicated and interrelated that requires
multi-component systems. The detectable alterations in onco/suppressor genes and related pathways are not tumor specific and they are
often caused by various environmental carcinogens, i.e., chemical, physical, and biological factors. On the other hand, they can serve
as a basis to develop more effective therapy and achieve greater ability to differentiate tumor types in the diagnosis. In early
diagnosis (secondary prevention) as yet no real breakthrough occurred, even though amplification and/or overexpression of onco/suppressor
genes had been often observed in routine pathological studies. However, these findings have never been employed at the early diagnostic
period, they have been applied in advanced stages of tumorigenesis rather. From the point of view of prevention, a real achievement would
be to introduce the application of these early alterations to the premorbid diagnostics of tumorigenesis. Unfortunately, at this time it
seems impossible due to the lack of knowledge of tumor-specific changes. Consequently, the application of these genetic changes in tumor
pathology and early diagnostics faces limitations, not only in the secondary prevention of sporadic tumors but also in their early
diagnosis and screening. Nevertheless, this body of knowledge is effectively used in the field of tertiary prevention, i.e., in the
diagnosis of tumor relapses and follow-up of their possible remission, refinement of individually designed therapeutic regimens, and in
molecular and predictive clinical epidemiology (Hawk et al., 2000). On the other hand, in tertiary prevention the genetic approach has
seemed less effective to the early detection of metastases, despite the numerous promising signs. With familial tumors it is not the case,
because the expression of certain oncogenes is predictable, e.g., that of BRCA1 in breast cancer. The molecular method (assessment of
onco/suppressor genes), therefore, could be quite effective both in primary and secondary prevention of tumors (Owen, 2001; Garcea et al.,
2003), however, with methodological and ethical limitations.
In the multi-step model of carcinogenesis, the chance to apply these markers is confined due to
their nonspecific nature (Perera, 1995), even though the general sequential steps of the tumorigenetic process are known (Balmain et al.,
2003). Thus, by applying step-by-step models we have the opportunity to identify high-risk groups but the early diagnosis of environmental
cancers is far away. A good example is the sequential predictive model (Fearon and Vogelstein, 1990; Fig. 1),
or other related models (Bellacosa, 2003).
Fig. 1. Molecular sequential model of cancer formation (Fearon and Vogelstein, 1990)
EVOLUTION OF MOLECULAR-EPIDEMIOLOGOCAL EVIDENCE
AND OUR RESEARCH DIRECTIONS
Numerous genetic alterations could be associated with the early phase of carcinogenesis. However, alterations in the incubation period
are describable only by means of molecular biological methods (Brambilla et al., 2003). It means opportunity to assess cancer risk both at
the individual and group levels (Ember et al. 2002). Examining these alterations immediately after exposure could give us the opportunity
to use them for risk assessment, as early biomarkers. It is worthwhile to emphasize that they are unrelated to the classical tumor markers
(Wild and Turner, 2001). Moreover, during the last 20 years, it has become clear that the models of multistep carcinogenesis (today
molecular carcinogenesis) might vary. Earlier they emphasized the importance of the sequential steps. Today it is believed that certain
somatic (oncogenic) alterations are interrelated and appear at a stage that depends on individual variability (Knudson, 2000). However,
sometimes the morphological counterparts of a given state could provide a basis to find the adequate biomarkers (Ember et al., 2002).
In this context, we cannot speak about diagnostic methods, but rather risk estimation and analysis.
Fig. 2 presents the pathogenesis taken from the first exposure to the actual disease. This scheme can also be
useful in evaluating other diseases beside tumors. It has become clear that before the morphological/clinical manifestations of tumors there
are gene alterations and the analysis and evaluation of these alterations would be highly informative on the actual condition or status of
tumor risk among patients or various groups. The validity of preventive markers implies the need of parameters that differ from those used for
diagnosis (Brambilla et al., 2003). The term prevention is defined in Fig. 3, which also includes the early
stages of carcinogenesis and molecular biological alterations (markers). At this point of the tumorigenetic process, i.e., before morphological
changes, the fields of prevention, epidemiology, and pathology (using similar molecular biological methods) have historically pursued
separate approaches.
Fig. 2. Cancer formation
Tumor research, the big expectation of the 80’s, prior to breakthroughs by molecular biology,
employed many nonspecific data. Today the use of DNA chip techniques also yields nonspecific data. The simultaneous measurement of thousands
of genes can be useful for clinicians to assess better the condition/status of the patient, its response to therapy, but it is less
informative for early and more specific diagnosis. It can be said that DNA chip microarray technology offers the possibility of broad
quantitative risk assessment, a concept to which less investment is required than to the present methods. The survey of somatic status is
not curative, but as a part of preventive/predictive medicine, the disease manifestation could be prevented by risk group survey. Still, it
is clear that preventive measures are now based on more solid scientific data than previously. Fig. 4 presents
the role of biological methods (i.e., onco/suppressor gene examination) that give the opportunity of prevention and quantitative risk estimation
as a part of molecular and predictive epidemiology. These measures are not diagnostic elements and naturally conform to different criteria
(they are nonspecific and approximate), therefore we interpret them as molecular epidemiological biomarkers.
Fig. 3. Preventive molecular epidemiology
Fig. 4. Expression of onco/suppressor genes (H-ras, p53, c-myc), as early biomarkers of chemical carcinogenesis
Before the discovery of onco- and suppressor genes, biotransformation by various adducts, carcinogens,
and events at the chromosomal level represented the markers that were used (Knudson, 2000; Wild and Turner, 2001; Ember et al., 2002). Now
when individual susceptibility, e.g., polymorphism of enzymes metabolizing various carcinogens is considered (Groopman et al., 1996; Vlastos
et al., 2003), we could apply molecular epidemiological biomarkers to identify individuals or groups at risk of carcinogenesis, to decrease
cancer morbidity and mortality, and to follow-up the effectiveness of the therapy. By means of these tools we have the possibility to
monitor early molecular events of carcinogenesis at the risk assessment level. At the same time we have a chance to assign the way of
intervention into these early events (primary prevention before the manifestation of tumors) and measure its effects with biomarkers.
Chemopreventive materials are synthetic or naturally occurring substances – mainly of plant and rarely of
animal origin – which exert their effect before the morphological detection of the tumor is possible (Bartsch, 1999; Fenech, 2002; Visakorpi,
2003). Based on their chemical, physical, or biological characteristics, their application is collectively termed chemoprophylaxis in analogy
with the successful prevention of pox and other infective diseases. Their mechanism of action is different and covers a broad range, including
inhibition of chemical binding to macromolecules, modulation of biotransformation, and altering the somatic mutation and repair system, etc.
Their main action is inhibition of carcinogenesis (anti-initiators, antioxidants, anti-promoters, etc). We presented an animal model that not
only meets the criteria of biomarkers, detection of chemical carcinogen exposure, and early biological effects (Fig. 5),
but also other preconditions. This system would satisfy many other functional aspects as well. Essentially, this approach applies a mouse strain,
which is especially sensitive to the effect of chemical carcinogens and which responds to their effects by overexpressing certain key onco- and
suppressor genes (Ha-ras, c-myc, and p53) soon after exposure (within 24–72 hours) (Hong and Lippman, 1995; Warren and Shields, 1997; Howell
at al., 2002). The model signals carcinogenic effects at an early phase, which otherwise could only be detected in long-term studies (Shureiqi
et al., 2000; Turini and Dubois, 2002). On the one hand, it is capable of signaling exposure, the early effect of the examined chemical,
physical, or biological carcinogens as a part of primary prevention. On the other hand, it gives an opportunity to quantitatively assess the
risk of carcinogenesis in human populations. By this approach, we can develop a complex molecular epidemiological marker system (Fig. 6),
which could provide information on tumorigenic factors endangering humans with potential biological effects which could give the opportunity
of primary prevention (chemoprophylaxis, hygienic actions, immunological and genetic interventions) and thereby to formulate an integral part
of primary preventive systems in molecular and preventive epidemiology (Ember et al., 2002). In addition, this method is capable to measure and
follow-up the effectiveness of applied chemoprophylactic interventions at the level of genes (Gyöngyi et al., 2001b). The model is a “short-term”
sensitive animal strain (CbA/Ca/H-2k) model for determining carcinogenicíty by monitoring gene alterations (Ember et al., 1999; Ember et al.,
2000). This model proved a relationship between the early genetic alterations (gene amplification, gene expression and mutation) induced by
carcinogens and tumor development in the thymus, spleen, lymph nodes, bone marrow, liver, kidneys, and lungs at 24, 48, and 72 hours after the
treatment with the carcinogens. From these organs and the white blood cells of the peripheral blood RNAs were isolated. The expression of three
genes (H-ras, c-myc, and p53) was investigated. H-ras, with its key role in chemical carcinogenesis, is known to play a significant role in
signal transduction, while p53 is a tumor suppressor gene involved in the regulation of apoptosis, DNA repair, and cell-cycle. The gene c-myc
exerts its effect in the proliferation and immortalization processes. The early alterations of these three genes detected at three time points
predict the biological effects of carcinogens (Gyöngyi et al., 2001a). The model signalizes if any effect of a chemopreventive agent applied and
the stage of tumorigenesis in which the intervention has occurred. The investigation of various chemical carcinogens in this model is not only
an important primary preventive tool, but due to consistent early biological effects it can also be used to monitor exposure. It is necessary to
note that as a biomarker it can be used for risk estimation but not to establish diagnosis.
Fig. 5. A biomarker test system in an animal model
Fig. 6. Complex risk assessment model
The above mentioned method has predictive value, since it indicates the fate of affected animals or
individuals by examining gene expression and amplification. By means of the Kaplan-Mayer survival curves, the model offers the opportunity to
measure the effectiveness of individually designed chemotherapy protocols (chemoprevention) together with the follow-up stages of the disease
and metastasis. The significance of the animal model experiment allows one to test chemopreventive agents. However, early tests of carcinogenesis
before the manifestation of tumors are not able to test chemotherapeutical effectiveness. For the latter, long-term experiments are necessary
along with in vitro models. To our knowledge, this is the first in vivo short-term model particularly utilizing predictive molecular biomarkers.
With the application of chemopreventive agents, we realized that a significant decrease in oncogene
expression could be reached by treatment with these chemicals. Examining many potential chemopreventive agents, we concluded that the model is
capable of testing the effects of potential chemopreventive agents, too, even those of different mechanisms of action. Our investigations showed
that the model was able to detect chemopreventive effects as efficiently as they had been detected in long-term models. In other words, this
model can be considered a chemopreventive testing model that enables us to investigate the initiation and all steps of the carcinogenic effect as
shown in the case of the carcinogen (7,12-dimethylbenz[a]anthracene) (DMBA) (Ember et al., 1998b).
First we wanted to rely on the early diagnosis and the screening pathway. However, as tumor-specific
genetic alterations were lacking, the animal experiments suggested us to direct our efforts to identify susceptible and exposed individuals by
applying early biological markers and quantifying risk assessment by means of their molecular-predictive character. Thus, a way was found from
secondary to primary prevention, with due attention to the ethical and technical limitations and the fact that the orthodox therapeutic medicine
cannot give proper answers to molecular medicine in terms of pharmacological development or in effectiveness of therapeutic interventions.
For this reason, in the Western European countries ethical limitations to genetically based (somatic)
diagnostic measures have been raised (Ember et al., 2000; Vahakangas, 2004). For quantitative risk assessment, molecular epidemiological
developments on animal-based experiments and further human epidemiological studies are equally required. In the new animal model, we found gene
alterations (biomarkers) that can also be used as early approximative biomarkers not only in epidemiology but also in chemopreventive models as a
part of the primary prevention complex extrapolated to humans (Ember et al, 1998a). This so far has been achieved in relation to risk assessment.
Its capability to analyze the effectiveness of interventions is, however, a task yet to be achieved. In spite all of these considerations, a high
number of statistical studies are required, mainly due to the individual variability of onco-suppressor genes, to enable us to use the model at a
population level. At the same time, we should not forget to apply individual gene polymorphism (enzyme polymorphism) to risk assessment.
CONCLUSIONS
In summary, our suggested model aptly applies to epidemiology, prevention, and prediction and can be used as an integral part of primary tumor
prevention. Our mouse model can also be used as a complex model (exposure, early risk assessment) and it can play a role in testing the
effectiveness of chemoprophylaxis. However, in the absence of better therapies, chemoprevention is a natural, effective tool. In accordance with
the preventive (chemopreventive) methods, this model provides information in the “silent” period of chemical carcinogenesis, i.e., when no
morphological and biochemical signs are present. At this stage, chemopreventives cannot be investigated. Therefore, the ability to test
chemopreventives would help in formulating interventional and preventive strategies with an estimated effectivity of 30–35%, which has not been met
by other known means to decrease morbidity and mortality. For this purpose, we developed our molecular biological, genetic, and mathematical model
of high scientific and experimental background. In order to measure their effects, we investigated numerous potential chemopreventive substances,
e.g., we studied the changes in oncogene expression caused by the known carcinogenic DMBA, as a positive control. We did not expect to measure the
therapeutic effects in this early induction model, since we were far from the point of tumor development, but we examined substances that inhibited
overexpression of certain oncogenes. We concluded that for measuring the effects of substances in this period, we do not have any other possibility
but using molecular epidemiological markers in in vivo “short-term” or classical “long-term” induction models.
Our model seems to be suitable for testing preventive substances because it utilizes biomarkers, which are
more potent and useful, at this early stage, than the other late markers. Therefore this animal model is applicable as a molecular epidemiological
predictive test for pre-signaling the effects of a chemical carcinogen and protection against it as well. It is fast, cheap, and interpretable.
According to the literature, no model like this – capable of monitoring both the individual steps of tumorigenesis and the effects of interventions
undertaken at those stages – is available. Therefore, our model is suitable to test the strategy of potential preventive substances, which exert
their effect in the early stage of tumorigenesis while controlling the effectiveness of interventions.
In summary, by establishing this animal model we have a molecular, epidemiological and preventive marker
system capable of indicating the risk on the one hand, and serving as a model for testing substances with preventive characters, on the other. The
next step would be extrapolation of the model to humans.
ACKNOWLEDGEMENT
The present work was supported by grants of the National Fund of Scientific Research (OTKA No.: T037279), and the Council of Health Sciences
(ETT No.: 323/2003) of Hungary.
We are grateful to Ms. Mardelle Susman (Department of Microbiology and Immunology, UTMB, Galveston, TX) for
reviewing this manuscript.
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Received: 2 February 2005
Accepted: 20 May 2005
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