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BRAF Mutations Are Not a Major Event in Post-Chernobyl Childhood Thyroid Carcinomas

Institute of Molecular Pathology and Immunology (J.L., V.T., P.S., V.M., J.M., A.A., M.S.-S.) and Medical Faculty (J.L., V.T., P.S., M.S.-S.), University of Porto, 4200-465 Porto, Portugal; Department of Pathology (J.M., M.S.-S.), Hospital S. João, 4200 Porto, Portugal; Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare (G.S., M.S.), Università di Napoli Federico II, 80137 Italia; Institute of Endocrinology and Metabolism (T.B., M.T.), 254114 Kiev, Ukraine; Medical Radiological Research Centre of Russian Academy of Medical Sciences (A.A.), 249020 Obninsk, Russian Federation; South West Wales Cancer Institute (S.J., G.T.), Swansea Clinical School, Singleton Hospital, SA2 8QA Swansea, United Kingdom; and Strangeways Research Laboratory (D.W.), University of Cambridge, CB1 8RN Cambridge, United Kingdom

Address all correspondence and requests for reprints to: Dr. Manuel Sobrinho-Simões, Institute of Molecular Pathology and Immunology, University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal. E-mail: ssimoes{at}ipatimup.pt.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
The BRAF gene has been shown to be a major target for mutations in papillary thyroid carcinoma (PTC) (36–69%), which forms almost all of the over 2000 cases of thyroid carcinoma that have occurred in Chernobyl. BRAF is activated by point mutation, and were it to occur at a high frequency in Chernobyl-related tumors, it would challenge the dominant role of double-strand breaks in radiation-induced PTC. In a previous study, we detected the BRAF V600E mutation in 46% (23 of 50) of sporadic adult PTC. Using the same methodology, we have analyzed 34 post-Chernobyl PTC and detected RET/PTC rearrangements in 14 (41%) and BRAF mutations (V600E) in four (12%). These two alterations did not coexist in any PTCs. The mean age at exposure of patients with PTC showing BRAF mutation was higher than that of patients with tumors without BRAF mutation irrespective of their RET status. We have also analyzed 17 sporadic cases of childhood PTC and found that only one (6%) harbored the BRAF V600E mutation. We conclude that the frequency of BRAF mutations is significantly lower (P = 0.0008) in post-Chernobyl PTC than in adult sporadic PTC, whereas no significant difference was found between post-Chernobyl and sporadic childhood PTCs.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
MOLECULAR BIOLOGICAL STUDIES of post-Chernobyl thyroid tumors revealed a high prevalence of rearrangements of the RET protooncogene (57–76%) in papillary thyroid carcinoma (PTC) (1, 2). RET/PTC3 was found to be the most frequent type of rearrangement and was also associated with more aggressive tumors (1, 2). Studies on the frequency of NTRK1 rearrangements, which are also associated with PTC, demonstrated that this type of rearrangement was present in a low percentage in post-Chernobyl PTC (3). Genes frequently activated by point mutation in human cancer (P53 and H-, N-, and K-RAS) were absent or only rarely involved in these radiation-induced tumors (4, 5, 6). Thus, RET/PTC rearrangements are, to date, the only genetic event consistently associated with post-Chernobyl PTC.

Recently, mutations in BRAF were found in various types of sporadic human cancers, including melanomas, thyroid cancer, colon cancer, lung cancer, and ovarian cancer (7, 8, 9, 10, 11, 12, 13, 14, 15), as well as benign nevi lesions (16). BRAF belongs to the RAF family of kinases, which includes two other isoforms: ARAF and CRAF (RAF-1). BRAF has a serine/threonine kinase activity and is located downstream of RAS and upstream of MEK in the classic MAPK cascade (17, 18, 19). Screening studies revealed a hotspot at position 1799, resulting in a substitution of a valine for a glutamate at residue 600 [previously position 1796 and residue 599, but according to recent data sequence, numbering of codons and nucleotides after exon 1 of BRAF were changed by +1 and +3, respectively (20)]. This mutation is located in the activating segment of BRAF and represents the vast majority of tumor-associated BRAF mutations (12). Functional studies led to the conclusion that this mutation resulted in a 70-to 138-fold increase of the activity of BRAF, independently of RAS stimulation, predicted to result in a proliferation stimulus to the cells and, ultimately, in cancer (12). Our group has shown that 46% (23 of 50) of sporadic adult PTCs harbored the BRAF V600E mutation (14) and, more recently, that BRAF V600E mutation appeared to be restricted to PTCs displaying a papillary or a mixed papillary/follicular growth pattern (21). The frequency of BRAF mutations in sporadic adult PTC is the second highest reported in human cancer, after melanoma (12, 16). These results fit with those published by other groups who found the V600E mutation in frequencies varying from 29–69% of PTCs (13, 15, 22, 23, 24, 25). In the series of sporadic PTCs we have previously published (14), RET/PTC rearrangements were present in 18% of cases and did not coexist with BRAF V600E mutation in any case. A similar finding was reported by Kimura et al. (13), who detected RET/PTC rearrangements in 16% of their cases. The same holds true regarding RAS mutations that did not coexist with BRAF mutations in our series nor in that of Kimura et al. (13, 14).

The results we and other groups obtained in sporadic thyroid cancer support the claim that BRAF mutations are a major event in sporadic thyroid papillary carcinogenesis (13, 14, 15, 21, 22, 23, 24, 25). However, to date, only one study has analyzed specifically radiation-induced PTC that occurred as a consequence of the Chernobyl accident. In this study, the authors found a similar frequency of BRAF mutations in these radiation-induced PTCs (three of 10, 30%) when compared with sporadic PTCs (29–69%), even though a lower frequency (one of five, 20%) was found when analyzing only radiation-induced PTCs in children (26). We undertook the present study to determine the frequency of such mutations in a series of post-Chernobyl childhood thyroid tumors, to find out whether or not the inverse relationship between BRAF mutations and RET/PTC rearrangements observed in sporadic thyroid tumors is also present in cases occurring in a radiation setting and to increase our understanding of the mechanisms involved in radiation carcinogenesis.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
We analyzed 34 cases of PTC (nine males and 25 females) and 11 follicular adenomas (FAs) (two males and nine females) that occurred in contaminated regions in Ukraine. For each case of PTC and each FA, we studied DNA and RNA extracted from frozen samples of tumor tissue and adjacent normal thyroid tissue. The pathology of the samples was confirmed by frozen section before homogenization and nucleic acid extraction. DNA from the peripheral blood of 15 PTCs and four FAs was also available and studied. The material we studied (DNA and RNA from tumor and normal thyroid tissue and blood samples) was obtained from the Chernobyl Tissue Bank (http://www.chernobyltissuebank.com). We have also studied DNA from 17 paraffin-embedded cases of sporadic PTC that occurred in nonexposed children under the age of 18 [12 cases from the files of Hospital S. João in Porto and five cases from a survey of childhood thyroid cancer in England and Wales (27)].

The diagnosis of all the Chernobyl cases was confirmed by the members of the Pathology Panel of the Chernobyl Tumor Bank. The diagnosis of the nonexposed cases and the subclassification of all the cases were carried out by at least two of four experienced thyroid pathologists among the authors (T.B., J.M., D.W., and M.S.-S.).

BRAF mutation analysis

To screen for BRAF mutations, we analyzed DNA from tumor tissue and, when available, adjacent thyroid tissue and peripheral blood of the patients. We studied the two regions of BRAF where mutations have been identified, G loop region and activation segment, which are encoded by exons 11 and 15, respectively. Both exons were amplified by PCR using conditions described elsewhere (12). The amplicons were then subjected to single strand conformation polymorphism (SSCP) analysis: PCR products of BRAF exons 11 and 15 were diluted 1:1 with loading buffer (95% formamide, 0.05% bromophenol blue, and 0.05% xylene cyanol), denatured at 98 C for 10 min, and cooled on ice for 5 min. Electrophoresis of the denatured PCR products was carried out in nondenaturing 0.8x mutation detection enhancement gels (BMA, Rockland, ME) at 180 V and 8 C for 15 h. PCR/SSCP products were visualized by standard DNA silver staining.

BRAF sequencing analysis

Whenever a sample presented aberrant bands in the SSCP analysis, these bands were excised from the mutation detection enhancement gel, and the PCR products were eluted and used as template for a second PCR using the aforementioned PCR conditions. PCR products from this second PCR were subjected to a purifying treatment using Exonuclease I (New England Biolabs, Beverly, MA) and Shrimp Alkaline Phosphatase (Amersham Biosciences, Piscataway, NJ) and subjected to automatic sequencing, using ABI Prism dGTP BigDye Terminator Ready Reaction Kit (Perkin-Elmer, Foster City, CA) and an ABI prism 3100 Genetic Analyzer (Perkin-Elmer). Sequencing was performed on both strands using the aforementioned primers. Whenever an alteration was identified by sequencing, the DNA sample was subjected to a second mutation analysis, including PCR amplification from genomic DNA of the exon containing the alteration, SSCP analysis, and sequencing of the amplified fragment, to confirm the existence of the alteration.

In both SSCP and sequencing analysis, we used the TPC-1 cell line as a negative control for BRAF mutations and the HT-29 cell line as a positive control for BRAF V600E mutation.

Expression of RET/PTC1 and RET/PTC3

The prevalence of RET/PTC1 and RET/PTC3 rearrangements were analyzed by using the RT-PCR assay. A common antisense and different forward oligodeoxynucleotides, specific for the H4 and RFG genes, were used as before (6). The sequence of the forward primers used were: RET/PTC1, 5'-ATTGTCATCTCGCCGTTC-3' (nucleotides 196–214 of RET/PTC1); and RET/PTC3, 5'-AAGCAAACCTGCCAGTGG-3' (nucleotides 697–714 of RET/PTC3). The sequence of the reverse primer was: 5'-TGCTTCAGGACGTTGAAC-3' (nucleotides 543–561 of RET/PTC1). One microgram of RNA was reverse transcribed and subsequently subjected to 35 cycles of PCR with a thermal cycler (Perkin-Elmer-Cetus). The product of the reaction was analyzed on a 2% agarose gel and hybridized with a RET probe covering the TK domain. The quality of the RNA samples was assayed by amplifying the HPRT mRNA. The HPRT specific primers were: forward primer, 5'-CCTGCTGGATTACATCAAAGCACTG-3', corresponding to nucleotides 316–340 of the third exon of the human gene; and reverse primer, 5'-CCTGAAGTATTCATTATAGTCTCAAGG-3', corresponding to nucleotides 685–661 of the eighth exon of the human gene.

Statistical analysis

The statistical analysis of the results was performed using the ² test with the Yates correction and Fisher’s exact test. A P value < 0.05 was considered statistically significant.

Informed consent

Informed consent was obtained in accordance with National Institutes of Health guidelines for all the tissues from the Chernobyl-related tumors. The use of spare sections from the paraffin blocks of the sporadic tumors was in accordance with the guidelines of the ethical committee of the University Hospital of S. João (Porto, Portugal).

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
We detected the presence of a BRAF mutation in four of 34 post-Chernobyl PTCs (12%); all four cases showed the 1799T-A mutation in exon 15 of BRAF, leading to substitution of a valine for glutamate at position 600 (Table 1). The four post-Chernobyl PTCs with BRAF mutation displayed signs of local invasiveness; vascular invasion or distant metastasis were not detected, and only one showed cervical node metastasis. The BRAF V600E mutation was also detected in one of 17 PTCs (6%) that occurred in nonexposed children (Table 1). The mutation was detected in the tumor tissue and was not present in normal thyroid samples or peripheral blood.

Three of the four post-Chernobyl cases presenting the V600E mutation were conventional forms of PTC with a predominantly papillary growth pattern, and the remaining case was a follicular variant of PTC (Table 2). No solid variant of PTC displayed the BRAF V600E mutation, in contrast to the occurrence of RET/PTC rearrangements in four of the eight cases of this variant (Table 2).

Among the five cases who were oldest at time of exposure, three showed a BRAF mutation, whereas only one showed a RET/PTC rearrangement. Patients with RET/PTC rearrangements had the lowest mean age at exposure of any group (Table 3).

No mutations in exon 11 of BRAF were found in any of 45 post-Chernobyl tumors (Table 1).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
The Chernobyl accident resulted in the highest number of a specific type of human cancer (PTC) that occurred as a consequence of a single event in a single place, at a specific time, ever recorded (28). This dramatic increase in the number of thyroid cancers in Belarus, Ukraine, and parts of the Russian Federation, which began just 4 years after the Chernobyl accident, has provided a unique chance to study the molecular pathology of thyroid cancer, specifically radiation-induced thyroid cancer.

It is well known that sporadic PTCs are commonly associated with rearrangements of the RET protooncogene (3–60%) (29). In the post-Chernobyl irradiation setting, this association is even more evident; 57–76% of cases have been reported to show RET/PTC rearrangements, with RET/PTC3 and RET/PTC1 being the most prevalent (1, 2). RET/PTC3 was found in an unusually high frequency in the earlier tumors and was associated with a solid pattern of growth and clinical aggressiveness (1). It is still unclear whether this apparent high frequency of RET/PTC rearrangements is related to radiation or to age; a similar frequency of RET/PTC rearrangement was found in unexposed children with papillary carcinoma in England and Wales (30). Nevertheless, there is still a high percentage of post-Chernobyl PTCs with no genetic alterations yet disclosed.

After the discovery of BRAF mutations as a major event in sporadic PTC (13, 14, 15), we were interested both to assess the prevalence of BRAF mutations in a cohort of post-Chernobyl tumors and sporadic childhood PTCs and to ascertain whether BRAF mutations and RET/PTC rearrangements were mutually exclusive. Our results revealed a significantly lower percentage of BRAF mutations in post-Chernobyl PTC (12%) than in our own series of sporadic adult PTC studied with the same techniques (46%) (P = 0.0009) (14), whereas the opposite occurs (as one would expect) regarding the prevalence of RET/PTC rearrangement (14). The frequency of BRAF mutations in sporadic adult PTC in our series (14) is similar to most of those reported in other studies (Table 4).

However, there are other interrelated possibilities that must be considered. The earlier BRAF studies have been carried out in adult patients, whereas our Chernobyl-related studies have included only children and adolescents. To investigate possible age-related effects, we have compared the results in post-Chernobyl PTC with the findings in 17 sporadic PTCs that occurred in children under 18 yr. In this setting, we observed that only one PTC of 17 (6%) harbored the BRAF V600E mutation. This frequency is similar to that observed in post-Chernobyl PTC but significantly lower than that of sporadic PTC occurring in adults (P = 0.0016) (14).

Our findings revealed that BRAF mutations are usually rare in sporadic childhood thyroid carcinoma, both in an irradiation setting and in sporadic tumors. Therefore, it is possible that the route to thyroid carcinogenesis that involves a BRAF mutation may have a longer latent period than the route involving a rearrangement. This explanation can only be tested by waiting another few years and then reassessing the frequency of BRAF mutations and RET/PTC rearrangements in adult Chernobyl-related thyroid carcinomas and in the more readily available age-matched adult patients.

In addition, our study has confirmed codon 600 (nucleotide 1799) as a mutational hotspot in BRAF coding sequence and the V600E mutation as specific to PTC. As has been observed in other studies (13, 14, 24), no mutation was found in BRAF exon 11 in PTC, nor in FA. Interestingly, we observed in one FA a mutation in codon 601 (K601E); an identical mutation had been previously detected in one sporadic FA (14) as well as in 9% of cases of follicular variant of PTC (21). Further studies are needed to assess the frequency and the putative clinical significance of this mutation in both benign and malignant thyroid tumors.

Our findings show that BRAF mutations are much less common in PTC in children and adolescents, both sporadic and radiation related, than in any of the reported series of sporadic PTC. They emphasize the importance of taking age into account when comparing mutation frequency in different series. Continuing study of the Chernobyl-related tumors and of the relationship between the frequency of BRAF mutation and age in sporadic tumors is needed to ascertain whether the etiological agent, latent period, and/or age is the cause of this marked difference in mutation frequency.

	Acknowledgments

 
We thank the Chernobyl Tissue Bank (http://www.chernobyltissuebank.com) for providing the material used in this study.

	Footnotes

 
This work was partially supported by the Portuguese Science and Technology Foundation (Ph.D. Grants SFRH/BD/8425/2002 to J.L. and SFRH/BD/13055/2003 to V.T. and Postdoctoral Grant SFRH/BPD/14594/2003 to V.M.) and by further funding from the same source (Project Programa Operacional Ciência Tecnologia e Inovação/Ciências Biomédicas e Oncológicas/47477/2002).

Abbreviations: FA, Follicular adenoma; PTC, papillary thyroid carcinoma; SSCP, single strand conformation polymorphism.

Received December 30, 2003.

Accepted April 16, 2004.

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