A MOLECULAR EPIDEMIOLOGY CASE CONTROL STUDY

Pleural malignant mesothelioma presents itself as a rare, highly aggressive neoplasm resulting mainly from the surface serosal cells found in the pleural cavity. A sharp rise in the incidence of mesothelioma has been recorded in the U.S. and Western Europe, and it has been predicted that around 250,000 deaths will occur as a result of malignant mesothelioma in the next 30 to 35 years. It can be difficult to diagnose diffuse pleural malignant mesothelioma. It is characterized by a poor survival rate, with fatalities generally occurring within 4-12 months after diagnosis has been made. While it is common knowledge that exposure to asbestos is the major causative agent responsible for the development of mesothelioma, associated with around 80% of cases, the number of cases involving individuals with a history of low level exposure to asbestos is increasing. The molecular changes that occur in the malignant mesothelioma carcinogenesis process remain unknown. In recent news, sequences belonging to SV40, which is a DNA tumor virus, have been linked with malignant mesothelioma, as a likely cofactor in the development of this malignancy.

A distinguishing characteristic of malignant mesothelioma is its long latency period, starting from the time of exposure to asbestos to eventual diagnosis. This suggests that several somatic genetic alterations may be needed for the tumorigenic changes occurring in a mesothelial cell. Initial evidence that supported this hypothesis was gathered via karyotypic analysis, which demonstrated multiple clonal cytogenetic changes in most cases of human malignant mesotheliomas. Many of the common cytogenetic aberrations occurring in malignant mesothelioma are characterized by deletions involving distinct regions in chromosome arms 1p, 3p, 4q, 6q, 9p, 13q, 14q, 15q, and 22q gains of chromosome 5, 7, and 20 or mutations of tumor-related genes, for instance neurofibromatosis type 2 (NF2) and p16 genes.

The high incidence of distinct chromosome region losses is concurrent with a latent mechanism of oncogenesis and it can be reckoned as a pointer to the locations of putative tumor suppressor genes that are responsible for the development and progression of malignant mesothelioma.

The existence of an intricate heterogeneity of the structural chromosomal anomalies in malignant mesothelioma appears to signify an inherent tendency of the cells to accrue genomic damage. Autosomal based spread of malignant mesothelioma, as evident in Turkey’s Cappadocian region, and the prevalence of malignant mesothelioma in families, provides support to the hypothesis that genetic vulnerabilities can act as a contributing factor in the etiology of this specific neoplasm. The function of genetic polymorphisms involving critical metabolic genes (for example GSTM1 and NAT2), as risk modifiers in malignant mesothelioma (asbestos-related), has recently been demonstrated in populations exposed to asbestos. Other types of heritable variations in hosts resistant to genetic alterations can be identified at distinct phases of the carcinogenic process, for instance chromosome stability and DNA repair competency. In such cases, a biomarker meant for genetic instability can be useful in identifying individuals who are at a greater risk for malignant mesothelioma.

Chromosomal changes have also been evident in various types of neoplastic diseases as risk predictors or as prognostic based factor. Human neoplasms demonstrate chromosomal anomalies in tumor tissue samples, and also in peripheral blood lymphocytes (PBL). The advantage of PBLs is that they allow noninvasive sample collection and also provide a considerable quantity of cells for analysis. Cytogenetic damage, which is measured as chromosomal anomalies in PBL, is an effective biomarker for human cancer risk, irrespective of the exposure to carcinogens.

The micronucleus examination in PBLs has been used as an easy and reliable technique for revealing cytogenetic damage. This analysis can be applied to measure chromosomal damage that can occur as chromosomal fragments or when entire chromosomes are excluded from the nucleus during mitosis. Micronucleus examination in PBLs can be deemed as an effective method for monitoring people with genetic instability and also as a screening test to identify carriers of specific mutations in the evaluation of cancer susceptibility.

Increased levels of micronuclei frequency in PBLs, as evident in cancer patients prior to the administration of chemotherapy and radiotherapy, have been reported in several papers. Ad hoc biomonitoring research studies involving specific types of cancer can help understand the significance of this biomarker in relation to individual genetic cancer risk.

Polyomaviruses, for instance JC virus (JCV) and SV40, are oncogenic in animal models and responsible for transformation of different types of animal and human cells. Also, JCV and SV40 have the ability to infect human PBLs, resulting in chromosomal instability.

A considerable increase in micronucleated binucleated lymphocytes was recorded in malignant mesothelioma patients in comparison to all other individuals. The most consistent demographic factors affecting the micronuclei frequency amongst human lymphocytes were evaluated. The effect that aging has on this specific biomarker is well-documented: age was linked with considerable increase in micronuclei levels in majority of the large-scale studies that specifically assessed this issue. These included two reanalysis of data collated from thousands of data.

As far as the current study is concerned, a considerable age-related increase was not recorded in the micronuclei frequencies, as this outcome is fully evident only when wide age ranges are considered. A significant percentage of the subjects enrolled for this study were instead found to belong to a comparatively narrow age interval.

A gender-based outcome was clearly visible in concurrence with earlier reports: a higher micronuclei frequency was recorded amongst females in comparison to males. However, no prominent variation can be documented, apparently due to the fewer number of women enrolled.

In context of cigarette consumption, the micronuclei frequency was found slightly higher in non-smokers in comparison to smokers. However, the effect of specific types of cigarette smoking indices such as duration, pack/years, intensity, and time from cessation, could not be ascertained. These results are in concurrence with a recently conducted reanalysis of collated databases. The authors noted that in comparison to non-smokers, current as well as former smokers tend to register slightly lower micronuclei frequencies. Only very heavy smokers (who did not face occupational exposure to genotoxic agents) registered significantly higher micronuclei frequencies. However, this was a subgroup that represented very poorly in our population.

The study also analyzed data based on exposure to asbestos. A marginal increase in micronuclei frequency was recorded amongst asbestos-exposed individuals in comparison to unexposed subjects. However, this observation was recorded only in mean values and not in median values. Also, 4 of the 21 subjects (individuals diagnosed with mesothelioma) in the study did not report a history of exposure to asbestos, although it may be possible that 2 of them were exposed to very low levels of asbestos as housewives of dockers.

Since long, asbestos has been identified as the primary cause of mesothelioma and lung cancer. Even though the association between asbestos and mesothelioma was established, 20% of the cases involved individuals who did not have a known history of exposure to asbestos. The mechanism that can help explain the carcinogenicity and cytotoxicity of asbestos has not yet been identified: asbestos fibers were classified as nongenotoxic carcinogens because they failed to induce gene mutation in majority of short-term assays. However, recent revisions carried out on scientific literature have clearly revealed that asbestos fibers can result in DNA damage and structural and numerical chromosomal anomalies in various types of mammalian cell systems.

Different types of asbestos fibers demonstrate their ability to stimulate micronuclei using different alterations of the micronucleus test. Results obtained through kinetochore analysis have shown evidence that clastogenic events as well as loss of entire chromosomes were linked with the initiation of micronuclei by asbestos fibers. Recently, two key mechanisms have been proposed for genotoxicity induced by asbestos: the first relates to physical effects that asbestos fibers have on spindle apparatus and chromosome; the second relates to the production of reactive nitrogen species or reactive oxygen species, directly or indirectly induced by the fibers.

Oxidative DNA damage and oxidative stress have been recorded in workers who were exposed to high levels of asbestos fibers in the past, which confirms the existence of an oxidative mechanism. A probable explanation for the difference in personal risks faced by people exposed to asbestos may be the individual antioxidant defense capacity. Evidence related to cytogenetic damage, as presented through micronuclei frequency in people diagnosed with mesothelioma, can be associated with endogenous and exogenous cofactors in addition to asbestos exposure.

The effectiveness of the DNA repair enzymes and genetic metabolic polymorphisms have been identified as risk factors responsible for the high prevalence of cytogenetic damage in controlled groups of subjects. The identification of the cofactors that may render certain people more susceptible to asbestos or those that can induce mesothelioma in individuals not exposed to asbestos has been a major area of study in many laboratories worldwide.

Virus expressions affect the protective cellular mechanisms with a considerable increase of micronuclei in cells, and recently these viruses have been identified as a probable cause of mesothelioma. The focus is particularly on investigating the role played by SV40 Tag sequences that have been usually found present and overexpressed in mesothelioma tissues. Researchers have noted that SV40 large T antigen presentation in mesothelial cells can weaken the control of DNA integrity and augment apoptosis. It can work as a carcinogen in conjunction with asbestos exposure, thereby playing a significant role in the development of mesothelioma. Data available on the incidence of SV40 Tag sequences in cancer cases reveal that, in specific setting of PCR assay and DNA extraction, samples of human T lymphocyte carry SV40 DNA at low prevalence (10%).

The conclusion based on the findings of the study is that malignant mesothelioma is linked with a considerable increase of micronuclei frequency. The findings show no relationship between micronuclei and other types of explanatory variables, for instance severity of asbestos exposure, polyoma virus and smoking habits. The quantum of cytogenetic damage evaluated using micronuclei frequency may be associated with individual susceptibility. The above results have to be verified in a larger population of individuals diagnosed with cancer by assessing other biomarkers and essaying the presence of SV40 virus.

 

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