Radiation induced thyroid cancer: fundamental and applied aspects

Aim: To describe the epidemiology and pathology of thyroid cancer in Ukraine, and to perform the molecular analysis of genetic alterations more frequently found to be associated to papillary carcinomas (PTC) in a selected group of PTC. Materials and Methods: Relationship between the thyroid cancer incidence and gender, age, and place of residence of subjects aged 0–18 years at the time of the Chernobyl accident (5427 subjects of thyroid cancer, among which 3996 (73.6%) were children aged 0 to 14 years at the time of the accident, and 1431 (26.3%) were adolescents aged 15 to 18 years was studied. Pathologically analyzed thyroid carcinomas were obtained from 640 patients (20–40 years old at the time of surgery and born before the Chernobyl accident), and from 90 patients (11–22 years old at the time of surgery and born after the accident). All patients were operated during 2006–2008. RET/PTC rearrangements and BRAFV600E mutation were analyzed in 35 cases of PTC. Results: A comparison between the thyroid cancer incidence rates in the 6 highest contaminated regions of Ukraine and in the other 21 regions shows the most significant difference between the rates for the last three years of follow-up, which confirms that a direct relationship is still present between the rise in thyroid cancer incidence and the post Chernobyl radiation exposure. Much lower incidence of thyroid cancer in subjects, who were born after the accident, additionally confirmed a direct relationship between the Chernobyl accident and thyroid cancer development at least in those who were aged up to 18 years at the time of the nuclear accident. Pathological results showed that with increasing latency the decrease has been noted in the percentage of PTC with solid structure, a decrease in invasive properties of tumors, as well as an increase in the percentage of PTC with papillary-follicular structure, encapsulated forms, and «small» carcinomas measuring up to 1 cm. Molecular-biological studies of PTC revealed more common RET/PTC1 and RET/PTC3 rearrangements (34.3% of cases), than BRAFV600E mutation (24%cases). Conclusion: After 22 years from the Chernobyl nuclear accident the number and incidence of thyroid cancer cases in Ukraine was steadily increased in the cohort of those who were children and adolescents at the time of the accident. Most common thyroid tumors (PTC) were characterized by significant changes in histological structure with increasing latency. PTC with any RET/PTC rearrangements had more aggressive behavior than BRAFV600E-positive tumors or PTC without gene alterations

RET rearrangements in post-Chernobyl papillary thyroid carcinomas with a short latency analysed by interphase FISH.

Tissue samples from 13 post-Chernobyl childhood thyroid tumours that occurred within a short period of time (4-8 years) after the Chernobyl accident have been investigated by interphase FISH analysis for rearrangements of RET. In all, 77% of cases showed RET/PTC rearrangements and a distinct intratumoural genetic heterogeneity. The data were compared to findings on 32 post-Chernobyl PTCs that occurred after a longer period of time (9-12 years) after the accident. In none of the cases from either group were 100% of cells positive for RET rearrangement. In addition, the pattern of RET-positive cells was different in the two groups (short vs longer latency). A significant clustering of aberrant cells could be detected in the long-latency subgroup, whereas the aberrant cells were more homogeneously distributed among the short-latency tumours. The findings suggest that oligoclonal tumour development occurs in post-Chernobyl PTCs. This pattern of different clones within the tumour appears to become more discrete in cases with longer latencies, suggesting either outgrowth of individual clones or development of later subclones with time

Heterogeneity in the distribution of RET/PTC rearrangements within individual post-Chernobyl papillary thyroid carcinomas

The nuclear disaster that occurred in Chernobyl in 1986 offered the unique opportunity to study the molecular genetics of one human tumor type, papillary carcinoma of the thyroid gland, associated with a specific etiology. We have analyzed RET rearrangements in post-Chernobyl papillary thyroid carcinomas (n = 29), follicular thyroid adenomas (n = 2), and follicular thyroid carcinoma (n = 1) by interphase fluorescence in situ hybridization (FISH) analysis on paraffin-embedded tissue sections. Paraffin sections were microdissected before use to ensure that only tumor was present. Cell nuclei were scored for the presence of a split FISH signal (separated red and green signal) in addition to an overlapping signal. Only cells with either two overlapping signals or one split and one overlapping signal were counted to ensure that only complete cell nuclei had been scored. In total, 23 of 32 cases (72%) showed RET rearrangements diagnosed by FISH interphase analysis. In all cases, the tumors were composed of a mixture of cells with and without ret rearrangement on FISH. In some cases, this distribution was clearly nonrandom because clustering of rearranged cells was detected within the same tumor nodule. Accordingly, only 31% of the cases positive for rearrangement on FISH also scored positive using RT-PCR. These findings suggest that because RET/PTC rearrangements are not present in a majority of tumor cells, either a fraction of post-Chernobyl papillary thyroid tumors are of multiclonal origin, or ret rearrangement is a later, subclonal event

CLIP2 as radiation biomarker in papillary thyroid carcinoma.

A substantial increase in papillary thyroid carcinoma (PTC) among children exposed to the radioiodine fallout has been one of the main consequences of the Chernobyl reactor accident. Recently, the investigation of PTCs from a cohort of young patients exposed to the post-Chernobyl radioiodine fallout at very young age and a matched nonexposed control group revealed a radiation-specific DNA copy number gain on chromosomal band 7q11.23 and the radiation-associated mRNA overexpression of CLIP2. In this study, we investigated the potential role of CLIP2 as a radiation marker to be used for the individual classification of PTCs into CLIP2-positive and -negative cases-a prerequisite for the integration of CLIP2 into epidemiological modelling of the risk of radiation-induced PTC. We were able to validate the radiation-associated CLIP2 overexpression at the protein level by immunohistochemistry (IHC) followed by relative quantification using digital image analysis software (P=0.0149). Furthermore, we developed a standardized workflow for the determination of CLIP2-positive and -negative cases that combines visual CLIP2 IHC scoring and CLIP2 genomic copy number status. In addition to the discovery cohort (n=33), two independent validation cohorts of PTCs (n=115) were investigated. High sensitivity and specificity rates for all three investigated cohorts were obtained, demonstrating robustness of the developed workflow. To analyse the function of CLIP2 in radiation-associated PTC, the CLIP2 gene regulatory network was reconstructed using global mRNA expression data from PTC patient samples. The genes comprising the first neighbourhood of CLIP2 (BAG2, CHST3, KIF3C, NEURL1, PPIL3 and RGS4) suggest the involvement of CLIP2 in the fundamental carcinogenic processes including apoptosis, mitogen-activated protein kinase signalling and genomic instability. In our study, we successfully developed and independently validated a workflow for the typing of PTC clinical samples into CLIP2-positive and CLIP2-negative and provided first insights into the CLIP2 interactome in the context of radiation-associated PTC