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The frequency of micronuclei MN in peripheral blood lymphocytes PBL is extensively used as a biomarker of chromosomal damage and genome stability in human populations. Much theoretical evidence has been accumulated supporting the causal role of MN induction in cancer development, although prospective cohort studies are needed to validate MN as a cancer risk biomarker.
A total of subjects from of 10 countries, screened in 20 laboratories for MN frequency between and in ad hoc studies or routine cytogenetic surveillance, were selected from the database of the HUman MicroNucleus HUMN international collaborative project and followed up for cancer incidence or mortality. To standardize for the inter-laboratory variability subjects were classified according to the percentiles of MN distribution within each laboratory as low, medium or high frequency.
The same groups also showed a decreased cancer-free survival, i. The results from the present study provide preliminary evidence that MN frequency in PBL is a predictive biomarker of cancer risk within a population of healthy subjects.
The current wide-spread use of the MN assay provides a valuable opportunity to apply this assay in the planning and validation of cancer surveillance and prevention programs. Measurement of micronucleus MN frequency in peripheral blood lymphocytes PBL is extensively used in molecular epidemiology and cytogenetics to evaluate the presence and the extent of chromosomal damage in human populations exposed to genotoxic agents or bearing a susceptible genetic profile 1.
This assay has been also successfully applied to identify dietary and genetic factors that have a significant impact on genome stability 2.
The high reliability and low cost of the MN technique, has contributed to the worldwide success and adoption of this biomarker for in vitro and in vivo studies of genome damage 3. MN originate from chromosome fragments or whole chromosomes that are not included in the main daughter nuclei during nuclear division Figure 1. The formation of MN in dividing cells is the result of chromosome breakage due to unrepaired or mis-repaired DNA lesions, or chromosome malsegregation due to mitotic malfunction.
All these events can cause the formation of MN through chromosomal rearrangements, altered gene expression or aneuploidy, effects associated with the chromosome instability phenotype often seen in cancer 5 , 9— A Schematic diagram showing the origin of MN from either a lagging chromosome fragment or a whole chromosome.
B A photomicrograph of a mitogen-stimulated, cytokinesis-blocked lymphocyte containing one MN. The presence of an association between MN induction and cancer development is supported by a number of observations.
The most substantiated include: i the high frequency of this biomarker in untreated cancer patients and in subjects affected by cancer-prone congenital diseases, e. Bloom syndrome or ataxia telangiectasia 1 , 9 ; ii the presence of elevated MN frequencies in oral mucosa, used as a surrogate biomarker of cancer in clinical chemoprevention trials 12 ; iii the correlation existing between genotoxic MN-inducing agents and carcinogenicity, e. Further evidence—based on the mechanistic and experimental correlation existing between chromosomal aberrations CA and MN 1 , 9 —comes from the results of recent cohort studies, which in most cases demonstrated that the frequency of CA in PBL of healthy subjects is a predictor of cancer risk 15— The possible association of lymphocyte MN frequency with cancer risk has earlier been examined in Swedish and Italian cohorts 15 , 16 , although no conclusions could be drawn from those studies because of the young age of the cohorts and the small number of events 18 cancer cases and 9 cancer deaths.
To test the hypothesis that PBL MN are predictive of increased cancer risk, we assembled a large international cohort of subjects whose lymphocytes had been screened for MN frequency between and and who were free of cancer at the time of testing. The study was conducted within the framework of the HUman MicroNucleus project HUMN , an international collaborative project, which allowed gathering of data on individuals studied in 20 cytogenetics laboratories from 10 countries 1.
The international collaborative HUMN project was developed to improve knowledge of the biology and relevance of MN induction and its application to human population studies 1. Details about this initiative and the list of publications produced under the HUMN project can be found on the project website, Author Webpage. About 50 laboratories actively involved in the HUMN project or included in the mailing list were invited to contribute their databases to the cohort study, assuming that personal identification was available for all subjects and a link with local or national cancer registry was possible.
An arbitrarily chosen minimum size of subjects analysed in the same laboratory even in different studies was required to be eligibile for inclusion in the cohort. A detailed description of the protocol used for measurement of MN frequency in PBL was collected from each laboratory submitting a database and evaluated by the HUMN steering committee for compliance to acceptable standard methodology Altogether, 20 laboratories from 10 countries, that were measuring MN frequency in human populations as a routine procedure, submitted databases that fulfilled the inclusion criteria, and all were included in the study.
Only individual MN frequencies based on the scoring of at least interphase cells were considered for statistical analysis. The large majority of laboratories adopted the cytokinesis-block assay 20 , scoring MN frequency in binucleated lymphocytes cells. The dataset from Sweden was produced using a different protocol, and MN were scored in mononuclear lymphocytes 21 , but this difference did not influence the statistical analysis of data. The subjects were originally selected by the testing laboratories for cytogenetic ad hoc studies, or routine biological dosimetry because of their exposures to mutagens or carcinogens, or as unexposed referents.
The original cytogenetic studies were performed between and , and most of them have been published in the peer-reviewed scientific literature. The most commonly studied exposures were to ionizing radiation subjects , pesticides subjects , polycyclic aromatic hydrocarbons subjects organic solvents subjects and cytostatic drugs subjects.
The cohorts investigated in this study were different from those that were previously studied to determine the relationship between CA in PBL and cancer risk 15— All subjects included in the HUMN cohort had a valid personal identification code, were at least 15 years old, and free of cancer at the time of cytogenetic testing. The study protocol was approved by the ethics committee of the coordinating center at the National Cancer Research Institute of Genoa, Italy.
The list of national cohorts with selected characteristics is reported in Table I. Seventy subjects included in the cohort were tested more than once. The follow-up period began with the date of MN testing and ended with death, cancer diagnosis, emigration, 85th birthday or end of follow-up —, depending on the country , whichever occurred first. The median duration of follow-up was 8.
Information on cancer incidence was obtained by linking the cohorts with national or regional cancer registries. In Poland, an active system of follow-up was set up via contacts with local cancer registries, municipalities of residence, employers, pension funds and general practitioners. In Italy and Belgium, mortality data were available and information on causes of death was collected via postal follow-up from the Municipality of residence. It was possible to follow the incidence for a subset of Italian subjects 7 cancer cases; person-years , and these data were included in the statistics of cancer incidence.
Twenty-one subjects diagnosed with a non-melanoma skin cancer ICD-IX were excluded from the analyses. In order to standardize for the marked inter-laboratory variability see Table I MN frequency was categorized by tertiles of the laboratory-specific distributions, i. Information about occupational exposure to mutagens or carcinogens was collected as reported in the original studies, and re-classified according to the job-exposure matrix described by the ESCH group Data on smoking status at the moment of cytogenetic testing were available in all cohorts, and subjects were classified as current, former or never smoker without consideration of the level of smoking.
The effect of MN frequency on cancer incidence was evaluated by comparing cancer incidence rates for the medium and high levels versus the low level, after adjusting for the confounding effects of age, gender, smoking status and occupational exposure to mutagens or carcinogens. A random effect term was then included in the models to adjust for the differences in cancer rates occurring among countries.
The presence of effect modification was tested by computing the log-likelihood ratio test for two hierarchical models, the first with, and the second without interaction terms involving MN. To verify if the presence of pre-clinical stages of cancer might have influenced MN frequency at test we repeated the analyses excluding the first 2 years of follow-up.
Furthermore, we stratified the entire cohort according to the median follow-up time among the cancer cases, i. Given the homogeneous pattern of cancer incidence and cancer-free survival in the medium and high tertiles, the analyses by country and cancer site, which generally had a number of events too low to allow the analysis on three strata levels, were performed combining these two tertiles.
These additional analyses were performed in groups with at least 10 observed cases. The effect of MN frequency on the probability to be cancer free at the end of the follow-up was estimated by means of Cox's proportional hazard model 24 , using time since test as the time variable and adjusting for age, gender, smoking status and occupational exposure.
STATA software was used for all statistical analyses An overall number of incident cancers including 7 cases from Italy and 56 cancer deaths were registered at the end of the follow-up periods. The most frequent cancer sites were colon and rectum 37 , stomach 35 , lung 28 and breast 20 , mostly contributed by Japan, Italy and Sweden where the person-years were the largest and the mean ages of the cohorts were the highest.
Removing the first 2 years of follow-up which most likely are affected by undiagnosed cancers from the statistical analysis, did not change the risk estimates 13 cancer cases occurred within this time frame. The limited impact of confounding due to variables that affected MN frequency i.
However, after stratifying by gender, females showed a linear trend of RRs by MN frequency level, i. Occupational exposure to mutagens or smoking status did not significantly modify the relationship between MN frequency and cancer risk. Relative risk of cancer incidence by MN frequency, gender, occupational exposure to carcinogens and smoking status. RR estimated by negative binomial regression analysis subjects.
Cancer free survival refers to time from MN test to the first cancer diagnosis. The association between MN and survival was estimated using the multivariate Cox proportional hazard model.
Only findings from Japan, which is the oldest national cohort and has the largest number of events, reached statistical significance, i. No statistics could be provided for breast cancer because none of the 14 incident cases fell in the reference tertile 7 in the medium and 7 in the high, respectively.
Relative risk of cancer incidence and mortality by MN frequency and cancer site. The results from the present study support the hypothesis that MN frequency in PBL is a predictive biomarker of cancer risk.
These data strengthen and extend the evidence accumulated by experimental, mechanistic and association studies 1 , that the extent of genetic damage measured in lymphocytes reflects the occurrence of early carcinogenic events in the target tissues. The strengths of this study include the relatively large size of the study group, the same direction of risk estimates in all countries, and the independence of results from the time elapsed between MN testing and cancer diagnosis.
However there are some limitations, which are discussed below. A specific association was found in a number of cancer sites, although statistical significance was reached only in the groups of urogenital and gastro-intestinal cancers. In particular, the higher risks for stomach 1. Nevertheless, we acknowledge that the number of cancers per organ site is relatively small, and that the statistical estimates, which are suggestive of an association with MN, are likely to become more stable as further cancers accumulate with increasing age of the cohort.
A feature of this study is the non-linearity of the dose—response relationship between MN frequency and overall cancer incidence, showing that subjects in the medium and high tertile have a higher risk of cancer relative to the low tertile, but there was no significant difference between medium and high tertiles.
A non-linear relationship between MN frequency and the risk of cancer seems the most likely explanation, assuming that there is a value of MN frequency beyond which no further increase in cancer risk occurs, e.
Alternative explanations include selective loss of genetic material, i. Furthermore, genome damage-induced cell death has been found in many other diseases, such as neurodegenerative disease 35 , which could also explain the plateau effect given that association with other degenerative disease was not explored.
The presence of a linear trend of RRs in the subgroup of women, who were much less occupationally exposed relative to males data not shown , suggests that the observed non-linearity may have, to some extent, been caused by incomplete adjustment for occupational exposure. While a residual confounding due to occupational exposure to mutagens or smoking status is still possible as shown by the small difference between adjusted and unadjusted relative risk estimates , statistical analysis showed a lack of effect modification, i.
Formation of nuclear anomalies such as MN, chromosomal rearrangements and anaphase bridges leading to breakage-fusion-bridge cycles and generation of more MN are events commonly seen in the early stages of carcinogenesis 4 , 26 , Elevated levels of MN are indicative of defects in DNA repair and chromosome segregation which could result in generation of daughter cells with altered gene dosage, or deregulation of gene expression that could lead to the evolution of the chromosome instability phenotype often seen in cancer 1 , 2 , 9 , 10 , 13 , These considerations give mechanistic support to a possible causal association between MN frequency and the risk of cancer.
The observed association between MN frequency and cancer risk in non-haematological malignancies in our study suggests that genome damage events in lymphocytes may be correlated with cancer initiating events in other tissues via a common genetic, dietary or environmental factor.
This is also supported by a recent paper indicating that specific chromosomal rearrangements play a role not only in haematological malignancies but also in non-haematological malignancies, and that there may be no fundamental tissue-specific differences in the genetic mechanisms by which neoplasia is initiated A major challenge in the mechanistic interpretation of our findings is the fact that MN can be generated through different processes, i.
In contrast to chromosome breakage, whose role in early stages of carcinogenesis has extensively been studied, the significance of aneuploidy is still poorly understood, although it is well known that aneuploidy is a hallmark of the majority of human tumours, and is associated with high grade invasiveness and poor prognosis Studies of the cytogenetic evolution in breast cancer have suggested that a highly aneuploid state could originate from a polyploidization event concurrent with a gradual loss of individual chromosome copies Similar findings have also been reported for preneoplastic lesions of the colon 41 , oesophagus 42 and cervix Cancer risk estimates associated with MN frequencies in the present study are roughly similar to the estimates reported for CA.
Our findings confirm the predictive role of chromosome anomalies but they do not provide definitive evidence concerning the role of aneuploidy. A resolution of this issue may result from future studies designed to distinguish between these mechanisms using centromere and telomere detection in MN, and including measurement of nucleoplasmic bridges which are a marker of misrepair of DNA lesions and telomere end-fusion 9 , 28 , The international multi-centre nature of this study may be considered among its strengths because this design allowed us to explore various environmental exposures in diverse genetic backgrounds using the country as a proxy of genetic features.
On the other hand, this approach has also introduced a number of limitations. These include the large inter-laboratory variability of MN frequency which is most likely due to technical differences in slide preparation and scoring, the heterogeneous quality of data on genotoxic exposures such as cigarette smoking and occupational carcinogens or the availability of a single measure of MN per individual which may have resulted in misclassification among MN frequency levels.
THz Exposure of Whole Blood for the Study of Biological Effects on Human Lymphocytes
In the present study, we investigated the induction of genotoxic effects in human peripheral blood lymphocytes after exposure to electromagnetic fields used in mobile communication systems frequency MHz. For this purpose, the incidence of micronuclei was evaluated by applying the cytokinesis-block micronucleus assay. Cytotoxicity was also investigated using the cytokinesis-block proliferation index. The experiments were performed on peripheral blood from 20 healthy donors, and several conditions were tested by varying the duration of exposure, the specific absorption rate SAR , and the signal [continuous-wave CW or GSM Global System of Mobile Communication modulated signal]. The SARs were estimated numerically. No statistically significant differences were detected in any case in terms of either micronucleus frequency or cell cycle kinetics.
Institute for Electromagnetic Sensing of Environment Naples, Italy
The exposures were carried out in wire patch cells under strictly controlled conditions of both temperature and dosimetry, and the induction of genotoxic effects was evaluated in lymphocyte cultures from 10 healthy donors by applying the cytokinesis-block micronucleus assay. Positive controls were provided by using mitomycin C. Each laboratory tested five donors, and the resulting slides were scored by both laboratories. Following this experimental scheme, it was also possible to compare the results obtained by cross-scoring of slides.
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