Reviewing Editor: Luca Persani

The authors have nothing to disclose

Correspondence to:
Vladimir Saenko, Department of International Health and Radiation Research, Atomic
Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto,
Nagasaki 852-8523, Japan; Email: saenko@net.nagasaki-u.ac.jp; Tel. +81-95-819-7122; Fax +81-95-918-7169

ABSTRACT
Chernobyl accident, the worst technogenic catastrophe involving massive radiation release into the environment, will soon reach the 25^th anniversary. Its major internationally recognized health consequence is thyroid cancer among the individuals affected by radioiodines at early ages. The largest in the world and unique series of radiation-induced thyroid malignancies has been a subject of investigations in many different aspects of sciences for decades. Here we review the results of investigations aimed at the elucidation of the “radiation signature”, a molecular classifier that could help discriminating between radiation-induced and sporadic tumors. The attempts to determine such employ a large variety of techniques, including measurements of DNA copy number variation on microarrays, differential gene expression profiling, proteomics, immunohistochemistry and genotyping of selected target genes or of the whole genome. From the point of view of study design and result interpretation, they could be broadly subdivided into those exploring molecular differences occurring after exposure to different etiological factors (i.e. radiation or other), thus looking for the damage pattern, and the ones seeking the markers of susceptibility to different etiological forms of thyroid cancer. There have been certain advances in both lines of investigations suggestive that establishment of the discriminative molecular signature is plausible. However, studies are far of being accomplished and require further efforts in following-up and investigating the Chernobyl cohort. Possible solutions to create comprehensive molecular concept will likely be integrative approaches combing clinico-pathological and extensive molecular data, and in-depth bioinformatic analyses.

Introduction
Ionizing radiation is a well known genotoxic agent that induces a variety of DNA lesions including nucleotide base modifications, abasic sites, strand cross-linking, DNA adducts, and singleand double-strand DNA breaks (DSBs) (1-3). Although all these types of lesions may potentially result in gene mutations, DSBs are considered to be the most significant for chromosomal aberrations, mutagenesis, genetic instability and carcinogenesis (2, 4-7). The multiplicity of DNA damages produced by radiation is thought to be one of the reasons for the diversity in biological consequences of exposure.
Human thyroid is an organ particularly vulnerable to ionizing radiation as was initially seen in the series of patients subjected to external beam therapy of the head and neck area for medical indications who then developed thyroid cancer (8). The Chernobyl accident, which occurred nearly 25 years ago on April 26, 1986, provided evidence of carcinogenic effect of environmental exposure to radioiodine isotopes, especially to ¹³¹I. A significant increase in thyroid cancer incidence was documented since early 1990-ies in Belarus, Ukraine and southwestern regions of Russia (9-12) (Fig. 1).
By 2002, the number of thyroid cancer cases registered in the individuals aged less than 18 years at the moment of exposure in the three most affected countries approached to 5,000 (13). Epidemiological studies have established qualitative and quantitative characteristics of causative association of thyroid cancer risk with internal exposure to radioiodine demonstrating that it is comparable to that after external irradiation (11, 14-20).
The outbreak of thyroid cancer in young patients suffered from the radioactive Chernobyl fallouts led to a great number of medical, epidemiological, dosimetric, sociological and laboratory investigations all aimed at evaluation of health impact, short and long-term consequences of the catastrophe for individuals, society and the environment as well as at elucidating the distinctive features of radiation-induced tumors. They resulted in important evidence-based conclusions which may be called lessons from Chernobyl; some of them could be drawn only after decades of observations. Applicably to the thyroid, the most important would be that ingestion of ¹³¹I at childhood may later cause thyroid cancer, that period of latency after exposure may be as short as only 4 years, that the use of stable iodine as a dietary supplement or as a thyroid-blocking agent may have a protective effect against cancer. From the molecular and pathological point of view, it has been recognized that radiation excess in thyroid cancer incidence is due to the papillary thyroid carcinoma (PTC) whose morphology and molecular characteristics, such as histological architecture and mutational pattern, appear to be changing with increasing latency or correlate with patient’s age (see reff. 20, 22, 23 for extensive reviews). The relative prevalence of RET/PTC3, RET/PTC1 and BRAF mutations implicated in molecular carcinogenesis of PTC has been proposed to tentatively parallel the dynamics of thyroid cancer incidence in children, adolescents and adults, respectively, shown in Fig. 1 (20).


Incidence of thyroid cancer in Belarus among the residents of radiocontaminated territories by age groups. This graph is inferred from the original one published earlier (21).
Molecular studies in Chernobyl thyroid cancer, depending on design, could be broadly classified into those attempting to determine a “damage signature” or “susceptibility signature” (24- 26). The first type of investigations explores frequencies and distribution of various mutations, in a comparative manner, between radiation-induced and sporadic thyroid cancers. Initial works on Chernobyl series were mostly mutational studies. As a whole, they demonstrated that none of oncogenes such as gene rearrangements (RET/PTC, NTRK, AKAP9-BRAF) or point mutations (BRAF, RAS family genes) could have been identified as radiation-specific.

Studies of the second type investigate if gene expression patterns or genetic factors may modify or serve as markers of inherited predisposition for developing cancer after radiation exposure. They generally require more advanced techniques because of the need to cover a large number of targets, ideally the whole genome. So far, several factors have been established to affect risk for developing thyroid cancer following internal exposure: radiation dose for the thyroid, younger age at exposure and iodine deficiency. Whether or not the genetics particularities of the individuals who developed thyroid cancer after Chernobyl remains largely unknown, but some facts, such as interpatient variations in the clinical course and latency as well as development of cancer only in a small proportion of the exposed victims, may be indicative of such a possibility. In this review we focus on the works performed to establish molecular classifiers capable of distinguishing radiation-induced Chernobyl cancers form sporadic PTCs. The importance and a need of a classifier is determined by the necessity to improve radiation risk assessment and risk communication, as well as to better manage and justify occupational and medical exposures tending to be expanding in the modern era of nuclear technologies.

Chromosomal imbalances
In an early study, chromosomal imbalances were examined using conventional comparative genomic hybridization (CGH) in a group of 60 Chernobyl childhood and adolescent PTCs (27). About 30% tumors were found to carry copy number variation (CNV). Both DNA gains (chromosomes 2, 7q11.2-21, 13q21-22, 21) and losses (16p/q, 20q, 22q) were found. Interestingly, deletions or loss of heterozygosity (LOH) on chromosomes 22q and 16p/q have been reported previously in PTC, FTC or ATC and associated with an aggressive tumor behavior (28-30). This study did not reveal correlations between the RET/PTC status of a tumor and specific DNA imbalance, yet the observation of a deletion at 22q in both RET/PTC-positive and RET/PTC-negative tumors was suggestive of the existence of alternate routes contributing to carcinogenesis, genetic heterogeneity or oligoclonal tumor development. The latter suggestion is supported by the observation of non-homogenous distribution of RET/PTC-harboring nuclei across tumor tissues (31). In a later work of the same group, employing an BAC-based array CGH, it was shown that RET/PTC-positive and RET/PTC-negative cases could be discriminated by the alteration pattern of chromosomes 1p, 3q, 4p, 7p, 9p/q, 10q, 12q, 13q and 21q (32). Furthermore, there was a significant difference between RET/PTC-positive childhood and adult PTCs: deletions on 1p35–36 were more frequent in adult cases. Regardless of RET/PTC rearrangement, chromosomal losses were more common than gains. In line with the previous study, the existence of additional, sometimes multiple, DNA alterations in both RET/PTCpositive and in RET/PTC-negative tumors could be interpreted as pointing at alternative paths of tumor development.
Another CGH study of 23 Chernobyl and 20 sporadic PTCs demonstrated that the overall prevalence of DNA gains was 2-4 higher in exposed patients as compared to non-exposed, and even more frequent (up to 10-fold) for recurrent gains (33). It was possible to determine the alteration pattern that discriminated radiation-related PTCs from sporadic (chromosomes 1p36.32-.33, 2p23.2- .3, 3p21.1-.31, 6p22.1-.2, 7q36.1, 8q24.3, 9q34.11, 9q34.3, 11p15.5, 11q13.2-12.3, 14q32.33, 16p13.3, 16p11.2, 16q21-q12.2, 17q25.1,19p13.31-qter, 22q11.21, 22q13.2) but because of limited sample size and non-uniform distribution of individual thyroid doses in the investigation the assessment of dose-response relationship has proved difficult. It was concluded that CNV, in addition to carcinogenesis-related alterations, also depend on radiation exposure and patient’s age at exposure.
Using a 50K Mapping array, 10 childhood Chernobyl PTCs were recently analyzed to demonstrate that DNA gains were more consistently observed at chromosome 1p, 5p, 9q, 12q, 13q, 16p, 21q, and 22q, while losses were found at 1q, 6q, 9q, 10q, 13q, 14q, 21q, and 22q (34). CNV amplifications were more frequent than deletions in line with the study by Kimmel et al. (33); no significant LOH was registered. This study is interesting because an overlay analysis was done to evaluate the concordance between CNV and gene expression. As a result, none of genes mapped to deleted regions was found to be downregulated. On the contrary, 87 genes that were amplified on CGH also displayed overexpression. After filtering gene expression profiles in Chernobyl PTCs against those reported previously for sporadic tumors and available from Gene Expression Omnibus, a radiation-related PTC identifier was established that included 113 messages among which 24 were downregulated and 41 were upregulated at least 3-fold. Six genes, CAMK2N1, AK1, DHRS3, FBXO2, ECE1 and PDE9A were unique to childhood radiation-induced PTC.
As a whole, the results of CGH analyses performed to date are not yet comprehensive enough to derive a CNV-based radiation signature. Usually the studies deal with small sample size, do not report validation experiments on independent specimens and employ platforms that are quite different in their resolution cumulatively making cross-analysis difficult. They, however, provide insights into the genomic regions, candidate genes and functional pathways involved in radiation-related thyroid carcinogenesis.

Gene expression profiles
Several studies have been undertaken to elucidate characteristic expressiosome features of Chernobyl thyroid cancers. The earliest one analyzed 12 Ukrainian and 8 sporadic PTCs from French patients, and 13 thyroid adenomas using Micromax microarrays with a set of 2400 known human cDNA probes (24). Neither unsupervised nor supervised classification algorithms could distinguish radiation-related from sporadic PTCs, perhaps in part due to the relatively small number of tested genes. However, separation from benign thyroid neoplasia was effective: based on a 36-gene signature a 3% misclassification rate was achieved. The importance of this investigation was in obtaining molecular evidence of similarity between PTCs of different etiology which confirmed previous observations of their morphological resemblance once again proving that radiation-induced and sporadic PTCs are closely related diseases presumably having much in common pathogenetically.
The whole genome study used Human Genome Survey Microarray V2.0 platform that combines >29000 genes (35). Screening was done on pooled RNA samples from 11 Chernobyl patients aged 15-22 years at diagnosis and 41 patients from southeastern Germany aged 15-83 years and the results were confirmed on an RTQ-PCR low-density array for selected genes.
Microarray analysis detected 646 differentially upregulated and 677 downregulated genes (>5-fold difference) between the groups. Interestingly, the genes predominantly overexpressed in Chernobyl tumors included G-proteins (RAS family genes), growth factors and receptors (VEGFA, EGFL9, PDGFC, PDGFRB, IGF1R, IGBP1) and some of oxidoreductases (cyclooxygenase 2 (PTGS2), superoxide dismutase (SOD1)) which were associated with tumor aggressiveness and poorer prognosis in previous studies (36-42). Such overexpression was interpreted as supportive to the notion that Chernobyl PTC manifested particularly high aggressiveness with frequent lymph node metastases and extrathyroidal invasion. This work also identified a molecular classifier consisting of 7 genes (SFRP1, MMP1, ESM1, KRTAP2-1, COL13A1, BAALC and PAGE1) that enabled a confident classification into radiation-related and sporadic PTCs.
One more investigation explored transcriptomes in 12 Chernobyl and 14 French patients using Human 1 cDNA Microarray slides covering 8000 genes (43). Similarly to the previous report from this group (24), unsupervised classification did not provide distinction between the two groups of cancers on a global scale. A supervised analysis, however, using four different algorithms, succeeded to determine classifiers that included from one to several thousands genes (median 256) with overall error rates ranging 12-27%. This study is noteworthy because the effects of possible etiological agents, which are presumably gamma radiation in Chernobyl tumors and hydrogen peroxide in sporadic tumors, were taken into account. Hydrogen peroxide is produced during thyroid hormone synthesis (44) and may play a role in thyroid tumorigenesis (45). Furthermore, it is a potent DNAdamaging substance which produces not only single-strand DNA breaks and base modifications but also double-strand breaks and, as recently shown, is capable to generate RET/PTC1 rearrangement in a human thyroid cell line (46). Using previously available data (47), the authors found that in a Blymphocyte cell line treated with 10 different genotoxic agents, in vitro gene expression responses to 200 µM of hydrogen peroxide and 2.5 Gy of gamma-rays were the most resembling. There were however 293 genes whose expression levels differed >1.5-fold between the two types of treatment of which, after removing genes related to immune reactions, 118 were present on the arrays used to profile PTCs. These genes were tested as a molecular classifier and, as a result, led to the separation of Chernobyl and sporadic PTC with the error rates 15-27%. In addition, whether the genes whose products are involved in five major DNA repair mechanisms, i.e. base-excision repair, mismatchexcision repair, nucleotide-excision repair, homologous recombination and nonhomologous end joining, may constitute a classifier was explored. Thirteen genes of homologous recombination pathway were found to make a classifier that distinguished radiation-induced and sporadic PTCs with error rates of 15-31%. It was proposed that, given DNA repair is largely accomplished within hours after damage while differential gene expression in the tumors persisted for many years, such profile may be a signature of susceptibility to different etiological forms of thyroid cancer. If these results find further support in independent PTC series, they may well be considered as a piece of evidence suggesting the existence of inherited predisposition to radiation-induced PTC.
Similarly to the results obtained in CGH studies, gene expression data provide valuable information for the attempts of elucidating molecular radiation signature, but they are not completed yet. So far reported works, being generally encouraging, have been done using relatively small series of cancers and produce the results that do not converge to yield a reliable set of markers. This points at the need to expand the number of analyzed cancers of both etiologies with better matching in terms of clinico-pathological and molecular characteristics to achieve the desired reproducibility and avoid biases.

Proteomic investigation
To date only one proteomic study involving Chernobyl thyroid cancers has been reported to the best of our knowledge. Boltze et al. analyzed protein extracts from 86 Chernobyl and 91 sporadic PTCs from patients of southeastern Germany (48). On 2-D electrophoresis, around 2000 spots were identified on the reference gels and among them 18 candidates upregulated in radiation-induced PTCs were determined. Immunohistochemistry was performed for all these candidates and in addition for two other proteins, potential markers for PTC. The results were evaluated semiquantitatively eventually leaving 6 proteins (NTRK1, MMP-1, MMP-13, MMP-9, Cathepsin W and Cathepsin X) that allowed most efficient separation between the groups. When adjusted for patients’ age, NTRK1, MMP-1 and MMP-13 staining resulted in a complete separation of the two etiological groups. Without age adjustment, NRTK1 alone and a combination of either two MMPs or of two Cathepsins also worked well with no false positive and false negative test results. Note that MMP1 gene upregulation in Chernobyl PTCs was reported previously (35). Interestingly, NTRK1 overexpression in radiationinduced PTCs may indicate structural mutation-independent role of this receptor tyrosine kinase as chromosomal rearrangements involving the NTRK1 gene are observed in less than 10% of Chernobyl cancers (49).
Whether a relatively simple immunostaining approach can be universally used to discriminate radiation-induced from sporadic PTCs remains to be established. Concerns are related first of all to patients’ age (and/or duration of latent period) and associated changes in tumor morphology as well as underlying mutational events all potentially leading to the shifts in the spectrum of expressed proteins. This direction certainly needs further investigation.

Genetic association studies
The purpose of this type of investigations is to determine genetic factors associated with disease thus addressing issue of inherited susceptibility. In general, there are two methodologies of selecting gene polymorphisms, usually SNPs, to be analyzed. The first one, termed candidate gene approach, is based on a hypothesis that genetic variations in one or in a limited number of genes may affect risk for or the phenotype of a given disease. A more comprehensive way is initially hypothesisfree and employs analysis throughout the genome; it is termed genome-wide association study (GWAS). While a substantial number of studies has been done in sporadic thyroid cancers, only few explored radiation-induced thyroid malignancies.

Candidate gene approach
In a study by Stephens et al. (50) no evidence for LOH in the RET gene was found in 28 of 46 PTCs from Ukraine heterozygous for at least one of three SNPs of interest (G691S, S904S and L769L); this observation is in line with the later microarray findings (33). Investigation of the additional 68 cases demonstrated that the rare S allele of G691S was significantly overrepresented in patients aged more than 30 years (30-72 years old, range and exposed 10-14 years before operation) as compared to the younger ones. Since excess radiation risks for PTC in the individuals exposed at the age older than 20 years old is very low and further declines with age at exposure, it was proposed that RET polymorphisms may influence carcinogenesis in sporadic but not in radiation-induced PTCs. The Arg72Pro polymorphism of the TP53 gene (encodes tumor suppressor protein p53) was assessed in 48 pediatric/adolescent and 68 adult Ukrainian and Russian patients with PTC, residents of radiocontaminated territories in Chernobyl areas (51), and 53 adult patients with sporadic PTC and 313 healthy controls from Russia. The Arg/Arg homozyotes were found to be significantly underrepresented in adult patients, but not in children and adolescents. In tumor tissues, no LOH or imbalanced TP53 allele expression in heterozygous individuals was found. These findings suggested that germline TP53 allele combinations other than Arg/Arg may contribute to the risk of development of PTC in individuals exposed to radiation during their late childhood, adolescence or in young adulthood, particularly females aged between 18 and 30. Of note, elevated risk for thyroid cancer was reported in females exposed to Chernobyl radiation at the age below 30 years in an epidemiological investigation (52).
A recent study of 9 SNPs in 5 genes (ATM, XRCC1, TP53, XRCC3 and MTF1) involved in DNA damage response in 255 PTC patients (123 from Chernobyl areas and 132 sporadic) and 596 healthy controls (198 residents of Chernobyl areas and 398 subjects without history of radiation exposure) showed that the ATM G5557A and XRCC1 Arg399Gln polymorphisms, regardless of radiation exposure, were associated with a decreased risk of cancer (53). Interestingly, the ATM IVS22-77 T>C and TP53 Arg72Pro SNPs interacted with radiation exposure: the ATM IVS22-77 associated with the increased risk of sporadic PTC whereas TP53 Arg72Pro correlated with the higher risk of radiation-induced PTC in adult patients, in support to the previous report (51). A possibility of gene-gene and gene-environment interactions was demonstrated. Some particular ATM/TP53 genotypes strongly associated with either sporadic or radiation-induced cancer indicating that variability of these genes may be potential risk modifiers for developing PTC of different etiology.

Molecular epidemiology based on whole genome association data
To date only one investigation of Chernobyl PTCs employing GWAS has been published (54). A total of 667 patients from Belarus diagnosed for PTC in 1989–2009 and 1275 controls from Belarus and Russia were studied, of which 408 cases and 627 controls were genotyped using Illumina Human610-Quad BeadChips (>500,000 SNPs) and the remaining samples were used for validation study. Statistical meta-analysis identified 4 SNPs at chromosome 9q22.33 showing significant association with disease. For one of them, rs965513, used for validation, a P-value of 4.8x10-12 was obtained which far surpasses the threshold of genome-wide significance of 5x10-8 (55). This SNP is located within a linkage disequilibrium (LD) block centromeric to the FOXE1 gene which encodes a thyroid-specific transcription factor TTF2 playing pivotal roles in thyroid morphogenesis. In addition, two candidates SNPs on chromosomes 9p and 12p that strongly tended to associate with disease risk were identified but genotyping of additional samples would be necessary to validate the significance of those.
To better understand the importance of this finding, it is necessary to mention two studies of genetic predisposition to sporadic differentiated thyroid cancer published last year just before the study by Takahashi et al. The first one reported rs965513, the same polymorphism described in the Chernobyl series, as the strongest genetic marker associating with thyroid malignancy in individuals of European descent. This study also claimed another SNP, rs944289 on chromosome 14q13.3 in the proximity of the NKX2-1 gene that encodes the TTF1 transcription factor, to be a marker for thyroid cancer (56) but it was not confirmed in the Chernobyl series. The second study, employing candidate gene approach, initially genotyped 768 SNPs in 97 genes in 615 cases and 525 controls from Spain and used 482 patients and 532 controls from Italy for validation (57). The target genes were selected based on their differential expression in primary thyroid tumours or the involvement in thyrocyte biology, metabolism and/or carcinogenesis such as the MAP kinase, JAK/STAT and TGF-beta pathways. An SNP, rs1867277, within the LD block spanning FOXE1 and located at the 5’UTR of the gene was identified as associating with PTC. Functional study demonstrated that this SNP affects FOXE1 expression by recruiting the USF1/USF2 transcription factors. Since forkhead transcription factors have been implicated in several human cancers (58-61) including epithelial-mesenchymal transition in colon cancer (62), it was proposed that FOXE1 may influence thyroid tumor call migration and invasion. While its precise role remains to be elucidated, this was an important clue to the understanding the molecular pathogenesis of PTC.
Thus, the three studies, two of sporadic thyroid cancers and one of radiation-induced tumors, have concordantly identified the FOXE1 (TTF2) locus as a marker of inherited susceptibility for PTC of different etiology. This leads to an important corollary that among the genetic factors affecting risk for radiation-induced Chernobyl PTC the strongest one is the same that confers predisposition to the sporadic form of this type of malignancy. Therefore, it is likely that “radiation-sensitive genotype”, whose existence may be expected given the possible existence of putative radiation-associated markers on chromosomes 9p and 12p and the absence of sporadic PTC marker on 14q13.3 (i.e. NKX2-1 or TTF1), comes next to and after, in terms of the effect strength, the general susceptibility to thyroid cancer. As outlined in Fig. 2, the results of genetic association studies allow to add genetic predisposition to the list of risk factors for radiation-induced thyroid carcinogenesis known from the earlier experience. Further investigation of etiology-specific marker(s) will probably refine our understanding of radiation-induced carcinogenesis by addressing issues of gene-gene and geneenvironment interactions.

Figure 2. Genetic predisposition as an emerging risk factor for both sporadic and radiation-induced papillary thyroid carcinoma. Sporadic and radiation-induced PTC share the major genetic determinant of inherited susceptibility to thyroid cancer, FOXE1 at chromosome 9q22.33, which appears to be stronger than possible etiology-specific genetic markers: on chromosome 14q13.3 (NKX2-1 or TTF1) for sporadic PTC and putative markers on chromosomes 9p and 12q for radiation-induced PTC.

Conclusion
A rapidly growing body of evidence suggests that the identification of molecular “radiation signature” in thyroid cancer is likely to become possible, with certain degree of certainty, in the coming years. The advances in exploring both the damage pattern by genomic microarrays, differential gene expression or immunohistochemically and inherited susceptibility by GWAS and expression arrays keep on bringing encouraging results yet they are far of being finalized. At present they rather contribute to work out a proof of principle that radiation-induced and sporadic thyroid cancers could be distinguished using a definite set of validated markers. Perhaps this set will include not only the above-mentioned markers as well as essential clinico-pathological information but also other, such as e.g. miRNA and proteomics, whose integration into the spectrum of potential targets and in-depth analyses may enable better insights into the possible classifiers. Its availability will likely allow future personalized cancer risk prediction which is of a significant importance in view of the growing thyroid cancer incidence in the world and also because of the relevance to occupational and expanding medicinal exposures, and radiation emergency medicine issues.
Undoubtedly, Chernobyl cohort is an inestimable source of knowledge in the area. Continuous observation, follow-up and thorough studies are warranted to yield the higher level of understanding. In this regard, international initiatives, such as the Chernobyl Tissue Bank (http://www.chernobyltissuebank.com/) or EC-coordinated GENRISK-T consortium (http://www.helmholtz-muenchen.de/isb/genrisk-t/index.html), Nagasaki University GCOE Program Global Strategic Center for Radiation Health Risk Control (http://www-sdc.med.nagasakiu. ac.jp/gcoe/projects/index_e.html) and other cooperative efforts would be the principal roadways to solving the problem.

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