The effect of Chernobyl accident on the development of malignant diseases--situation after 20 years

The accident that occurred at the Chernobyl Nuclear Power Plant in 1986, released large quantities of radionuclides--among them radioiodine--into the atmosphere, thereby raising public concerns about its influence on thyroid structure and function, especially the development of malignancy. There were even reports about 700 deaths due to thyroid carcinoma in Russian Federation, Ukraine and Belarus, resulting from the accident. In this review we discussed the incidence of thyroid cancer in different parts of the world, especially in heavily contaminated countries, as Ukraine and Belarus, and the possible link between radioisotope activity in the thyroid and the development of malignancy. The study carried out in Minsk showed 40-fold increase of the incidence of thyroid cancer in the years 1986-1994, in comparison to the period 1977-1985. An increase of the incidence of thyroid cancer has generally been observed in many countries after the Chernobyl accident. We focused on the factors that may have an influence on this phenomenon, especially diagnostic tests, health care, social and environmental factors, like iodine level in water and soil. The results of molecular biology studies, e.g. RET translocation in carcinoma type RET/PTC1 in elderly and RET/PTC3 in children, and expression Ax1 and Gas6 in children were reviewed as well. We also mentioned other thyroid diseases, like nodular goitre, cysts, the disturbance of thyroid function and autoimmunity, possibly linked to the radiation after Chernobyl accident. Data obtained from the regions near Chernobyl showed no increased risk of other types of malignancy (leukaemia, Hodgkin's and non Hodgkin's lymphoma) in 1986-1996. In this article the epidemiology of thyroid diseases in Poland was also reviewed.     

Novel tumorigenic rearrangement,Delta rfp/ret,in a papillary thyroid carcinoma from externally irradiated patient

Molecular analysis of cDNA derived from a papillary thyroid carcinoma (PTC) (follicular variant of papillary thyroid carcinoma on histology) which developed in an externally irradiated patient 4 years after exposure identified a portion of the 5' region, exons 1-3, of the rfp gene juxtaposed upstream of the fragment encoding the tyrosine kinase (TK) domain of the ret gene. The fusion gene, termed Delta rfp/ret, was the result of a balanced chromosomal translocation t(6;10) (p21.3;q11.2) confirmed by interphase FISH painting, with breakpoints occurring in introns 3 and 11 of the rfp and ret genes, respectively. Both Delta rfp/ret and reciprocal ret/rfp chimeric introns had small deletions around breakpoints consistent with presumed misrepair of a radiation-induced double-strand DNA break underlying the rearrangement. No extensive sequence homology was found between the fragments flanking the breakpoints. The fusion protein retained the propensity to form oligomers likely to be mediated by a coiled-coil of the RFP polypeptide as assessed by a yeast two-hybrid system. NIH 3T3 fibroblasts stably transfected with a mammalian expression vector encoding full-length Delta RFP/RET readily gave rise to the tumors in athymic mice suggestive of high transforming potential of the fusion protein. Thus, the Delta rfp/ret rearrangement may be involved in a causative manner in cancerogenesis and provides additional evidence of the role of activated ret oncogene in the development of a subset of papillary thyroid carcinoma.     

siRNA nanoformulation against the ret/PTC1 junction oncogene is efficient in an in vivo model of papillary thyroid carcinoma

Delivery is a very important concern for therapeutic applications of siRNA. In this study, we have used chitosan-coated poly(isobutylcyanoacrylate) nanoparticles to deliver siRNA with a complementary sequence to the fusion oncogene ret/PTC1. By screening the mRNA junction we have selected a potent siRNA sequence able to inhibit this oncogene in a model of Papillary Thyroid Carcinoma cells. This siRNA sequence has then been validated by a shRNA approach using the same sequence. Furthermore, the high ret/PTC1 inhibition has triggered a phenotypic reversion of the transformed cells. We have designed well-defined chitosan decorated nanoparticles and succeeded to reduce their size. They have allowed to protect ret/PTC1 siRNA from in vivo degradation and leading to significant tumour growth inhibition after intratumoral administration.     

RET rearrangements in radiation-induced papillary thyroid carcinomas: high prevalence of topoisomerase I sites at breakpoints and microhomology-mediated end joining in ELE1 and RET chimeric genes

Children exposed to radioactive iodine after the Chernobyl reactor accident frequently developed papillary thyroid carcinomas (PTC). The predominant molecular lesions in these tumors are rearrangements of the RET receptor tyrosine kinase gene. Various types of RET rearrangements have been described. More than 90% of PTC with RET rearrangement exhibit a PTC1 or PTC3 type of rearrangement with an inversion of the H4 or ELE1 gene, respectively, on chromosome 10. To obtain closer insight into the mechanisms underlying PTC3 inversions, we analyzed the genomic breakpoints of 22 reciprocal and 4 nonreciprocal ELE1 and RET rearrangements in 26 post-Chernobyl tumor samples. In contrast to previous assumptions, an accumulation of breakpoints at the two Alu elements in the ELE1 sequence was not observed. Instead, breakpoints are distributed in the affected introns of both genes without significant clustering. When compared to the corresponding wildtype sequences, the majority of breakpoints (92%) do not contain larger deletions or insertions. Most remarkably, at least one topoisomerase I site was found exactly at or in close vicinity to all breakpoints, indicating a potential role for this enzyme in the formation of DNA strand breaks and/or ELE1 and RET inversions. The presence of short regions of sequence homology (microhomologies) and short direct and inverted repeats at the majority of breakpoints furthermore indicates a nonhomologous DNA end-joining mechanism in the formation of chimeric ELE1/Ret and Ret/ELE1 genes.     

Analysis of the expression of RET/PTC oncogenes in post-chernobyl papillary thyroid carcinomas of patients from different age groups

A comparative analysis of the expression of both, RET/PTC1 and RET/PTC3 oncogenes in papillary thyroid carcinomas (PTC) of patients from different age groups was carried out. Those were the following groups: children (mean age - 13 years, mean latency period - 13 years), young adults (mean age - 24 years, mean latency period - 14 years), adults (mean age - 38 years, mean latency period - 22 years). The presence of RET/PTC oncogenes was detected using polymerase chain reaction. In all cases the samples of both tumor and normal thyroid tissue were studied. It was established that induction of both, RET/PTC1 and RET/PTC3 rearrangements was present only in carcinoma samples. In PTCs the percentage of RET/PTC-positive tumors with increasing the age of patients has been decreasing. It should be noted that the part of carcinomas with induction of RET/PTC1 did not change with increasing the age of patients. At the same time the frequency of RET/PTC3 rearrangements with the increasing both the latency period and age of patients, significantly decreased. In conclusion, our data can evidence for the presence of correlation between the age of patients, latency period and induction of RET/PTC3 oncogenes.     

Molecular cytogenetic characterization of chromosome 9-derived material in a human thyroid cancer cell line

The incidence of papillary thyroid carcinoma (PTC) increases significantly after exposure of the head and neck region to ionizing radiation, yet we know neither the steps involved in malignant transformation of thyroid epithelium nor the specific carcinogenic mode of action of radiation. Such increased tumor frequency became most evident in children after the 1986 nuclear accident in Chernobyl, Ukraine. In the eight years following the accident, the average incidence of childhood PTCs (chPTC) increased 70-fold in Belarus, 200-fold in Gomel, 10-fold in the Ukraine and 50-fold in Tschnigov, Kiev, Rovno, Shitomyr and Tscherkassy compared to the rate of about 1 tumor incidence per 106 children per year prior to 1986 (Likhtarev et al., 1995; Sobolev et al., 1997; Jacob et al., 1998). To study the etiology of radiation-induced thyroid cancer, we formed an international consortium to investigate chromosomal changes and altered gene expression in cases of post-Chernobyl chPTC. Our approach is based on karyotyping of primary cultures established from chPTC specimens, establishment of cell lines and studies of genotype-phenotype relationships through high resolution chromosome analysis, DNA/cDNA micro-array studies, and mouse xenografts that test for tumorigenicity. Here, we report the application of fluorescence in situ hybridization (FISH)-based techniques for the molecular cytogenetic characterization of a highly tumorigenic chPTC cell line, S48TK, and its subclones. Using chromosome 9 rearrangements as an example, we describe a new approach termed 'BAC-FISH' to rapidly delineate chromosomal breakpoints, an important step towards a better understanding of the formation of translocations and their functional consequences.     

Thyroid cancer risk in Belarus after the Chernobyl accident: Comparison with external exposures

Within the time period 1990–1993, childhood thyroid cancer incidence due to the Chernobyl accident increased dramatically in Belarus, especially with regard to the birth cohort January 1, 1971, to May 31, 1986. This rise subsequently slowed down, i.e. during the period 1994–1996. The respective data were analysed and compared with the results of an analysis on the time dependence of thyroid cancer incidence in a pooled cohort of persons who had been exposed during childhood to external radiation with high dose rates. Concerning the period of 5–10 years following exposure, the excess absolute cancer risk per unit thyroid dose in the latter (external) exposure group was found to exceed the one in the Belarus group by a factor of two. This difference, however, is not statistically significant. The age-adjusted average excess absolute risk per unit thyroid dose for the period of 5–50 years following external childhood exposure was found to be 8 female and 14 male cases per 10⁴ person-year · Gy, which is a factor about 2.5 times higher than for the non-adjusted risk in the pooled cohort, as reported by Ron et al. in 1995. Assessments of future excess thyroid cancer cases due to the Chernobyl accident were done on the basis of the time dependence of thyroid cancer risk following external exposure. The thyroid cancer incidence among the birth cohort considered in Belarus and for a period starting from the cessation of the available observation data (1 January 1997) and extending to 50 years after the Chernobyl accident has been estimated to be about 15,000 cases, with an uncertainty range of 5000–45,000 cases. According to our calculations, 80% of these cases exceed the baseline risk under enhanced thyroid surveillance. Received: 8 June 1999 / Accepted in revised form: 20 November 1999     

Mechanisms of mutagenesis in mammalian cells. Application to human thyroid tumours

Mutations are defined as stable and irreversible modifications of the normal genetic message due to small changes in the number or type of bases, or to large modifications of the genome such as deletions, insertions or chromosome rearrangements. These lesions are due to either polymerase errors during normal DNA replication or unrepaired DNA lesions, which will give rise to mutations through a mutagenic pathway. The molecular process leading to mutagenesis depends largely on the type of DNA lesions. Base modifications, such as 8-oxo-guanine or thymine glycol, both induced by ionizing radiations (IR), are readily replicated leading to direct mutations, usually base-pair substitutions. The 8-oxo-G gives rise predominantly to G to T transversions, the type of mutations found in ras or p53 gene from IR-induced tumors. Bulky adducts produced by chemical carcinogens or UV-irradiation are usually repaired by the nucleotide excision repair (NER) pathway which is able to detect structural distortion in the normal double-strand DNA backbone. These lesions represent a blockage to DNA and RNA polymerases as well as some signal for p53 accumulation in the damaged cell. In the absence of repair, these lesions could be eventually replicated owing to the induction of specific proteins at least in bacteria during the SOS process. The precise nature of the error-prone replication across an unexcised DNA lesion in the template is not fully understood in detailed biochemical terms, in mammalian cells. IR basically produce a very large number of DNA lesions from unique base modifications to single- or double-strand breaks and even complex DNA lesions due to the passage of very high energy particles or to a local re-emission of numerous radicals. The breakage of the double-helix is a difficult lesion to repair. Either it will result in cell death or, after an incorrect recombinational pathway, it will induce frameshifts, large deletions or chromosomal rearrangements. Most of the IR-induced mutations are recessive ones, requiring therefore a second genetic event in order to exhibit any harmful effect and a long latency period before the development of a radiation-induced tumor. The fact that IR essentially induced deletions and chromosomal translocations renders very difficult the use of the p53 gene as a marker for mutation analysis. In agreement with the type of lesions induced by IR, it is interesting to point out that the presence has been observed, in a vast majority of radiation-induced papillary thyroid carcinomas (PTC), of an activated ret proto-oncogene originated by the fusion of the tyrosine kinase 3' domain of this gene with the 5' domain of four different genes. These ret chimeric genes which are due to intra- or inter-chromosomal translocations, were called RET/PTC1 to PTC5. The RET/PTC rearrangements were found in PTC from children contaminated by the Chernobyl fall-out as well as in tumours from patients with a history of therapeutic external radiation, with a frequency of 60-84%. This frequency was only 15% in 'spontaneous' PTC. The type of ret chimeric gene predominantly originated by the accidental or therapeutic IR was different. Indeed, PTC1 was present in 75% of the tumours linked to a therapeutic radiation and PTC3 in 75% of the Chernobyl ones. The other forms of RET/PTC were observed in only a minority of the post-Chernobyl PTC (< 20%). The difference in the frequency of PTC1 and PTC3 in both types of PTC, is statistically significant (P < 10(-5), Fischer's exact test). In two of the post-therapeutic radiation PTC, RET/PTC1 and PTC3 were simultaneously present. A PTC1 gene was also observed in 45% of the adenomas appearing after therapeutic radiation. The long-period of latency between exposure to IR and the appearance of thyroid tumours is probably due to the conversion of a heterozygote genotype of IR-induced mutations to a homozygote one. It will be interesting to use this time lag in accidental or therapeutic-irradiated p     

Thyroid cancer risk in areas of Ukraine and Belarus affected by the Chernobyl accident

The purpose of the present study was to analyze the thyroid cancer incidence risk after the Chernobyl accident and its degree of dependence on time and age. Data were analyzed for 1034 settlements in Ukraine and Belarus, in which more than 10 measurements of the (131)I content in human thyroids had been performed in May/June 1986. Thyroid doses due to the Chernobyl accident were assessed for the birth years 1968-1985 and related to thyroid cancers that were surgically removed during the period 1990-2001. The central estimate for the linear coefficient of the EAR dose response was 2.66 (95% CI: 2.19; 3.13) cases per 10(4) PY-Gy; for the quadratic coefficient, it was -0.145 (95% CI: -0.171; -0.119) cases per 10(4) PY-Gy(2). The EAR was found to be higher for females than for males by a factor of 1.4. It decreased with age at exposure and increased with age attained. The central estimate for the linear coefficient of the ERR dose response was 18.9 (95% CI: 11.1; 26.7) Gy(-1); for the quadratic coefficient, it was -1.03 (95% CI: -1.46; -0.60) Gy(-2). The ERR was found to be smaller for females than for males by a factor of 3.8 and decreased strongly with age at exposure. Both EAR and ERR were higher in the Belarusian settlements than in the Ukrainian settlements. In contrast to ERR, EAR increases with time after exposure. At the end of the observation period, excess risk estimates were found to be close to those observed in a major pooled analysis of seven studies of childhood thyroid cancer after external exposures.     

RET rearrangements in papillary thyroid carcinomas and adenomas detected by interphase FISH

Activation of the RET protooncogene through somatic rearrangements represents the most common genetic alteration in papillary thyroid carcinoma (PTC). Three main rearranged forms of RET have been described: RET/PTC1 and RET/PTC3, which arise from a paracentric inversion of the long arm of chromosome 10, and RET/PTC2, which originates from a 10;17 translocation. We have developed a dual-color FISH approach to detect RET/PTC rearrangements in interphase nuclei of thyroid lesions. By using a pool of three cosmids encompassing the RET chromosome region and a chromosome 10 centromeric probe, we could discriminate between the presence of an inversion (RET/PTC1 and RET/PTC3) or a translocation (RET/PTC2). We have investigated a series of thyroid tissue samples from Italian and French patients corresponding to a total of 69 PTCs and 22 benign lesions. Among PTCs, 13 (18.8%) showed a RET rearrangement, and 11 (15.9%) of these carried an inversion (RET/PTC1 or RET/PTC3) in more than 10% of the nuclei examined. Activated forms of RET were also observed in three adenomas. RT-PCR analysis on the same samples confirmed the presence and the type of rearrangement predicted using FISH analysis. An interesting difference in the frequency and type of RET rearrangements was detected between the Italian and the French patients. Furthermore, we identified a putative novel type of rearrangement in at least one PTC sample. Several PTCs carried a significant number of cells characterized by a trisomy or a tetrasomy of chromosome 10. Overall, the FISH approach in interphase nuclei represents a powerful tool for detecting, at the single cell level, RET/PTC rearrangements and other anomalies involving the RET chromosome region.     

Rearrangements of NTRK1 oncogene in papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) represents an example of tumour with high incidence of oncogenic sequences, such as RET/PTC and Trk. Both of them arise from the fusion of 3' terminal sequences of TK domain of RET or NTRK1 gene, respectively, with 5' terminal sequences of their activating genes. In case of NTRK1 oncogene, several rearrangement types are observed, characteristic for PTC: Trk (TMP3), Trk-T1, Trk-T2, Trk-T3 and Trk-2h, observed in human breast cancer cell line. Studies from different geographical regions, revealed significant population differences in the incidence of Trk rearrangements (0-50%), while within the same population, the frequency of Trk in spontaneous and radiation-associated PTCs is similar. The results of studies, focused on the correlation between tumour genotype and the histopathological type of tumour, involving cases of both RET/PTC and Trk rearrangements in PTC, are not unequivocal. In many studies, no correlation was observed between the presence of RET and/or NTRK1 rearrangement and such parameters, as patient's age at diagnosis, gender, histopathological type of tumour or clinical stage (TNM stage grouping), although the earliest clinical symptoms and the worst disease outcomes were observed for RET/NTRK1 rearrangement positive tumours. Differences in the rearrangement incidence between male and female patients were associated with the latency period of radiation-associated tumours, being significantly lower in women. In general, it is assumed that oncogenic Trk sequences are typical for the spontaneous type of PTC.     

Irradiation and second cancers. The thyroid as a case in point

The thyroid gland is highly sensitive to radiation during childhood: the risk of thyroid tumours is increased for mean doses as low as 100 mGy and for higher doses, the risk increases linearly with the dose. Excess relative risk is important, being 7.7 for 1 Gy delivered to the thyroid gland during childhood. The risk of thyroid tumours is modified by several factors: a) age at exposure: in childhood, the risk decreases with increasing age at exposure and is not significant after 20 years; b) gender: females are two times more likely than males to develop thyroid tumours; c) genetic predisposition due to a defect in DNA repair mechanisms, and dietary and hormonal factors may modify the risk; d) the influence of fractionation and dose rate is not well established. Radioiodine 131 (1311) used for medical purposes has almost no tumourigenic effect on the adult thyroid gland. The consequences of the Chernobyl accident have clearly shown that the risk of thyroid cancer after exposure to 1311 in childhood is important, and that such exposure should be prevented by potassium iodine prophylaxis. RET/PTC rearrangements are found in 60-80% of papillary carcinomas and in 45% of adenomas occurring after radiation exposure. They are found in 5-15% of papillary carcinoma and in no follicular adenomas that occurred in the absence of radiation exposure.     

A radioecological model for thyroid dose reconstruction of the Belarus population following the Chernobyl accident

A radioecological model was developed to estimate thyroid exposures of the Belarus population following the Chernobyl accident. The input of the model includes an extensive data set of the ¹³⁷Cs activity per unit area deposited during the Chernobyl accident, the rainfall data for different regions of Belarus, the ¹³¹I/¹³⁷Cs ratio in the deposit and the start of the grazing period in Belarus in April/May 1986. The output of the model is the age-dependent thyroid exposure due to the intake of ¹³¹I with fresh milk. Age-dependent average thyroid doses were assessed for selected regions of Belarus. The maximum thyroid doses were estimated for the inhabitants of Gomel oblast where the highest deposition was observed among the regions considered here. The lowest doses were estimated for Vitebsk oblast with the lowest level of depositions. The mean exposures for the oblasts of Grodno, Minsk, Mogilev and Brest were very similar. The results were compared with estimations of thyroid exposure that were based on ¹³¹I measurements in human thyroids, and they are in good agreement. The model may be used for the assessment of thyroid doses in Belarus for areas where no ¹³¹I measurements are available.     

Thyroid cancer incidence in Ukraine: trends with reference to the Chernobyl accident

For the first time, a comparative analysis of thyroid cancer incidence in Ukraine after the Chernobyl accident was done in a cohort that is almost as large as the general population. On the basis of thyroid doses from radioactive iodine in individuals aged 1-18 years at the time of accident, geographic regions of Ukraine with low and high average accumulated thyroid doses were established and designated "low-exposure" and "high-exposure" territories, respectively. A significant difference of thyroid cancer incidence rates as a function of time between the two territories was found. That is, the increase in the incidence was higher in high-exposure regions than in low-exposure regions. The incidence rates varied substantially among the different attained age-groups, especially in the youngest one (up to 19 years old). The analysis that was adjusted for screening and technological effects also indicated that in the high-exposure regions, thyroid cancer incidence rates at the age of diagnosis of 5-9, 10-14 and 15-19 years were significantly higher in those born in 1982-1986 compared to those born in 1987-1991, while in the low-exposure regions, no significant difference was observed. The observed probable excess of radiation-induced thyroid cancer cases in adults exposed to radioactive iodine from the Chernobyl accident, especially in females, may be due to the high power of the present study. However, it should be noted that our investigation was not essentially free from ecological biases.     

Strontium fallout from Chernobyl and perinatal mortality in Ukraine and Belarus

Perinatal mortality rates in the regions of Ukraine and Belarus surrounding the Chernobyl site increased in 1987, the year following the Chernobyl accident. The same year, increases of perinatal mortality were also observed in Germany and Poland, and the effect can be associated with the caesium burden in pregnant women. After 1989, there is an unexpected second rise of perinatal mortality in Belarus and Ukraine. This increase is shown to correlate with the strontium content in pregnant women. The findings parallel an increase of perinatal mortality in Germany following the atmospheric bomb tests in the 1950's and 1960's. While the effect from caesium is essentially limited to 1987, the effect from strontium persists until the end of the study period in 1998. The cumulative effect from strontium around Chernobyl outweighs the effect from caesium by at least a factor of 10. This is contrary to the assertion that the caesium content in the Chernobyl fallout was more than 10-times greater than the strontium content. Thus, the dose factor presently used seems to severely underestimate the effect of strontium on perinatal mortality.     

Nuclear-power-plant accidents: thyroid cancer incidence and radiation-related health effects from the Chernobyl accident

Following the Chernobyl accident, enormous amounts of radioisotopes were released in the atmosphere and have contaminated surrounding populations in the absence of rapid protective countermeasures. The highest radiation doses were delivered to the thyroid gland, and the only direct consequence of radiation exposure observed among contaminated population is the increased incidence of thyroid cancers among subjects who were children in 1986 and who lived at that time in Belarus, Ukraine or Russia.