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The Journal of Clinical Endocrinology & Metabolism 89(9):4280 – 4284 Copyright © 2004 by The Endocrine Society doi: 10.1210/jc.2004-0172
COMMENT Low Frequency of BRAFT1796A Mutations in Childhood Thyroid Carcinomas ATSUSHI KUMAGAI, HIROYUKI NAMBA, VLADIMIR A. SAENKO, KIYOTO ASHIZAWA, AKIRA OHTSURU, MASAHIRO ITO, NAOFUMI ISHIKAWA, KIMINORI SUGINO, KOICHI ITO, STEPHEN JEREMIAH, GERALDINE A. THOMAS, TATYANA I. BOGDANOVA, MYKOLA D. TRONKO, TAKESHI NAGAYASU, YOSHISADA SHIBATA, AND SHUNICHI YAMASHITA Department of Molecular Medicine (A.K., H.N., K.A., S.Y.), International Health and Radiation Research (V.A.S., S.Y.), Department of Radiation Epidemiology (Y.S.), Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, and Department of Surgery (A.K., T.N.), Takashi Nagai Memorial International Hibakusha Medical Center (A.O., S.Y.), Nagasaki University Hospital, Nagasaki 852-8523, Japan; Department of Pathology, National Nagasaki Medical Center (M.I.), Omura 856-8562, Japan; Ito Hospital (N.I., K.S., K.I.), Tokyo 150-8308, Japan; South West Wales Cancer Institute, Singleton Hospital (S.J., G.A.T.), Swansea, United Kingdom SA2 8QA; and Laboratory of Morphology of Endocrine System, Institute of Endocrinology and Metabolism, Academy of Medical Sciences of Ukraine (T.I.B., M.D.T.), Kiev 04114, Ukraine A high prevalence of the activating BRAF mutation, BRAFT1796A, is observed in adult papillary thyroid carcinomas (PTCs). The prognosis of childhood PTCs is generally fairly good despite the fact that distant metastases are often documented in these cases. To investigate the differences between the characteristics of childhood and adult PTCs, we analyzed both BRAFT1796A and RAS mutations in 31 Japanese and 48 post-Chernobyl Ukrainian thyroid carcinomas. In the 31 Japanese childhood cases, BRAFT1796A was found in only one instance (3.2%), and no RAS mutations were detected. In the Ukrainian subjects, of the 15 childhood and the 33 adolescent and young adult PTCs examined, the BRAFT1796A mutation was found in zero and eight cases, respectively, and RAS mu-
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tations were found in two of the young adult cases. In addition, 17 of the 48 Ukrainian cases showed expression of the RET tyrosine kinase region, indicating the existence of RET/PTC rearrangements. Unlike adult PTCs, we could detect no positive association between BRAFT1796A mutations and clinical parameters in the childhood carcinomas, suggesting that a low prevalence of BRAFT1796A is a common feature of PTCs in children regardless of radiation exposure levels. The differences in the prevalence of BRAFT1796A mutations between childhood and adult cases of PTC may well reflect inherent differences in the clinical features of these cancers between the two age groups. (J Clin Endocrinol Metab 89: 4280 – 4284, 2004)
RAF IS A RAF kinase isoform that functions as a component of the MAPK signaling cascade and is frequently mutated in adult papillary thyroid carcinoma (PTC) (1–7). Point mutations in the BRAF gene are almost exclusively found at nucleotide position 1796 (BRAFT1796A), as a thymine to adenine transversion resulting in a valine to glutamic acid substitution at codon 599. The prevalence of the BRAFT1796A mutation has been documented to be about 30 – 50% in PTCs. In our previous study we examined the correlation between BRAFT1796A and various clinical parameters in adult PTC patients and showed that this mutation significantly correlates with both distant metastases and an advanced clinical stage (4). Childhood thyroid carcinoma is often characterized by distant metastases, but is rarely diagnosed as immediately life threatening, and a relatively good prognosis is usually
recorded (8). The clinical and biological behavior of childhood thyroid carcinomas also seem to differ greatly from that of adult thyroid carcinoma, which may be due to either differences in the underlying genetic mutations or epigenetic alterations at a different age of onset. However, these differences are currently poorly understood. In this study we speculated that childhood thyroid carcinomas may be associated with a different prevalence of BRAFT1796A mutations compared with adult cases. Hence, BRAFT1796A mutations were examined in 31 cases of Japanese childhood thyroid carcinoma and in an additional 48 cases of PTC from the Ukraine, all of whom were less than 17 yr of age at the time of the Chernobyl accident. Our subsequent findings show a low frequency of BRAFT1796A mutations in childhood thyroid carcinomas.
Abbreviations: CTB, Chernobyl Tissue Bank; EC, extracellular; PTC, papillary thyroid carcinoma; TK, tyrosine kinase. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.
Materials
Subjects and Methods We analyzed surgically removed thyroid tumors from 31 Japanese patients who were younger than 15 yr of age when treated for thyroid carcinoma between 1962 and 1995 at Ito Hospital (Tokyo, Japan). The clinical details of these patients were retrieved from the hospital files and
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revealed that none of these individuals had any prior history of internal or external radiation exposure. Each of the thyroid tumors was independently reclassified by two pathologists based on the histopathological typing of the World Health Organization (9). DNA samples from 50 Chernobyl childhood thyroid carcinomas were also obtained from the Chernobyl Tissue Bank (CTB) (10) (www.chernobyltissuebank.com). Two of these subjects were eliminated from the experimental analysis because we were unable to PCR-amplify either the BRAF or RAS gene from the corresponding DNA sample. Hence, 48 Chernobyl samples were further analyzed in this study. Our study protocol was approved by the human ethics review committee of Nagasaki University.
DNA extraction and molecular analysis DNA was isolated from formalin-fixed, paraffin-embedded tumor specimens from 31 Japanese thyroid carcinoma patients. Extractions were performed on 10-m-thick sections, following mechanical microdissection, with a DXPAT kit (Takara Co., Kyoto, Japan), resulting in successful isolation from greater than 90% of the tumor cells. DNA samples obtained from the CTB had already been extracted from surgically removed thyroid carcinoma tissues. Tumor DNA was amplified by PCR to generate products from common hot spots in thyroid tumors, including a portion of BRAF exon 15, codons 12 and 13 of K-RAS, and codon 61 of both H- and N-RAS (11). Exon 15 of the BRAF gene was amplified with the forward primer 5⬘-TAAAAAGGTGATTTTGGTCTAGCTCTAG-3⬘ and the reverse primer 5⬘-ACTATGAAAATACTATAGTTGAGA-3⬘; exon 2 of the N-RAS was amplified with the forward primer 5⬘-TTGAAACTTCCCTCCCTCCCTGC-3⬘ and the reverse primer 5⬘-AGCTCTATCTTCCCTAGTGTGGTAA-3⬘; exon 2 of H-RAS was amplified with the forward primer 5⬘-CAGGGAGAGGCTGGCTGTGTG-3⬘ and the reverse primer 5⬘-CCACCTGTGCGGCGTGGGCT3⬘; exon 1 of K-RAS was amplified with the forward primer 5⬘-GGTACTGGTGGAGTATTTGATAGT-3⬘ and the reverse primer 5⬘CTCATGAAAATGGTCAGAGAAACCT-3⬘. PCRs were performed under standard conditions (95 C for 2 min, 94 C for 30 sec, 58 C for 30 sec, 72 C for 30 sec, for 38 cycles; then 72 C for 10 min) in a final volume of 50 l, using Ex Taq polymerase (Takara Co.). The amplified products were purified with a MinElute PCR Purification Kit (Qiagen, Chatsworth, CA) and sequenced on an ABI PRISM 3100 automated capillary DNA sequencer using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA). It was confirmed that the quality of the DNA extracted from paraffin sections was comparable to that of fresh tissue DNA and could be successfully used to amplify fragments less than 400 bp in size.
RT-PCR analysis of expression of the RET tyrosine kinase (TK) and extracellular (EC) domains RNA that had been extracted from frozen sections of control, paired samples of papillary carcinoma and normal thyroid tissues was obtained from the CTB. RT was performed on 1 g RNA in a final volume of 25 l. Two microliters of each reaction mixture were used in subsequent PCRs with the primers 5⬘-CACCGGATGGAGAGGCCAGACAACTGCAGC-3⬘ and 5⬘-ACCGGCCTTTTGTCCGGCTC-3⬘ for TK and 5⬘-GTGCAGTTCTTGTGCCCCAACATCAG-3⬘ and 5⬘-CCCAGCGCGTGCTCACCT-3⬘ for EC in a final volume of 50 l. Primers used to amplify the 3⬘ end of the ret oncogene (TK) correspond to exon 16 (5⬘ upstream) and exon 17 (3⬘ downstream), spanning a 1150-bp intron. The primers used to amplify regions of the EC domain were designed to span intron 4, yielding a fragment of 106 bp. PCRs consisted of 30 cycles of 95 C for 30 sec, 50 C for 30 sec, and 72 C for 1 min. Fifteen microliters of each PCR were separated on 2% agarose gels. The specificity of each PCR product was confirmed by Southern blotting using a probe specific for the 3⬘ region of the ret gene and also by direct sequencing.
Immunohistochemistry Paraffin-embedded tissue was deparaffinized in xylene and rehydrated in PBS. Antigen retrieval was performed by heating each slide three times in 200 ml PBS in a microwave oven at 50% power for 4 min, followed by washing with 0.01 m PBS for 10 min. Endogenous peroxidase activity was blocked with 0.3% H2O2/methanol for 10 min. After washing three times with 0.01 m PBS for 5 min each time, the section was
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blocked with 1% BSA for 15 min, followed by rinsing with PBS. A primary mouse monoclonal antibody against human CD15 (diluted 1:50; DAKO, Glostrup, Denmark) was applied overnight at 4 C, and bound antibody was subsequently visualized with biotinylated horse antimouse IgG, followed by avidin-peroxidase and diaminobenzidine treatment.
Results
Both the clinical parameters and oncogene alterations, including BRAFT1796A; H-, N-, and K-RAS; and RET/PTC genes, for each of the Japanese and Ukrainian cases examined in this study are summarized in Table 1. All of the Japanese patients were less than 15 yr of age at the time of tumor diagnosis (identified as 29 PTCs, one follicular carcinoma, and one poorly differentiated carcinoma). Distinct histological variants were classified according to the following criteria: follicular variant, more than 50% of the tumor contained follicular components and no well formed papillae were evident; or solid variant, more than 50% of the tumor has solid and/or trabecular components and, again, no well formed papillae were found. In total, 29 PTCs were subclassified as 27 classical papillary type and two follicular variant tumors. Seven of the 29 PTCs had distant metastases, and 21 cases had neck lymph node metastases. In the 48 cases of Ukrainian thyroid carcinoma that we analyzed, each of the patients was less than 17 yr of age at the time of Chernobyl accident. However, both the age at tumor diagnosis and the age at surgical treatment in each case varied significantly from 11–31 yr. Thus, we divided the Ukrainian subjects into two groups, one consisting of 15 cases who were operated on before the age of 15 yr (childhood group) and another containing the 33 cases who were surgically treated at an age above 15 yr (adolescent and young adult group). All of the tumors in both groups were pathologically diagnosed as a PTC by the CTB pathological panel. The 15 PTCs in the childhood group were subclassified into two cases of a classical papillary type (13.3%), four cases of solid variant (26.7%), two cases of follicular variant (13.3%), and seven cases of mixed variant tumors (46.7%; which are defined as a tumor having a mixture of papillary, solid, and/or follicular components) by the CTB pathological panel. The 33 PTCs in the young adult group were subclassified into seven classical papillary types (21.2%), one solid variant (3.0%), nine follicular variants (27.3%), and 16 mixed variant tumors (48.5%). Distant metastases were observed in three of the 15 childhood group patients (20.0%) and four of the 33 adolescent and young adult group cases (12.1%). Regional lymph node metastases were observed in nine of the 15 childhood group cases (60.0%) and 13 of 33 young adult cases (39.4%). Extrathyroidal invasions were also found in three of the childhood cases (20.0%) and 11 of the young adult cases (33.3%). DNA sequence analysis of BRAF exon 15 in the Japanese patients found only one case of a BRAFT1796A mutation, which was pathologically diagnosed as a poorly differentiated carcinoma. To confirm whether this tumor originated from a papillary or a follicular carcinoma, we performed immunohistochemical analysis of CD15, which has been reported to be specifically expressed in PTCs (12). Tumor cells in small foci of papilloid growths were found to be positively stained with anti-CD15 antibody (data not shown), suggest-
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TABLE 1. Clinical parameters and oncogenic mutations of Japanese childhood and post-Chernobyl Ukrainian thyroid carcinomas Japanese (n ⫽ 31)
Mean age at operation (yr) Female/male ratio Mean tumor size (cm) Micropapillary carcinoma Histological type Papillary carcinoma (PTC) Follicular carcinoma Poorly differentiated carcinoma Histological subtype of PTCs Classical type Solid variant Follicular variant Mixed variant Extrathyroidal invasion Regional lymph node metastasis Distant metastasis Gene mutation BRAF V599E (T1796A) K-RAS codon 12/13 H-RAS codon 61 N-RAS codon 61 Ret TK expression Negative Moderately positive Strongly positive
Ukrainian ⬍ or ⫽15 yr old (n ⫽ 15)
Ukrainian ⬎15 yr old (n ⫽ 33)
11.3 24/7 4.1 4/29 (13.8)
14.3 11/4 2.3 0/15 (0.0)
23.5 24/9 2.4 3/33 (9.1)
29/31 (87.9) 1/31 (3.33) 1/31 (3.33)
15/15 (100) 0/15 (0.0) 0/15 (0.0)
33/33 (100) 0/33 (0.0) 0/33 (0.0)
27/29 (93.1) 0/29 (0.0) 2/29 (6.9) — 7/31 (22.6) 21/31 (67.7) 7/31 (22.6)
2/15 (13.3) 4/15 (26.7) 2/15 (13.3) 7/15 (46.7) 3/15 (20.0) 9/15 (60.0) 3/15 (20.0)
7/33 (21.2) 1/33 (3.0) 9/33 (27.3) 16/33 (48.5) 11/33 (33.3) 13/33 (39.4) 4/33 (12.1)
1/31 (3.2) 0/22 (0.0) 0/22 (0.0) 0/16 (0.0)
0/15 (0.0) 0/14 (0.0) 0/14 (0.0) 0/14 (0.0)
8/33 (24.2) 0/33 (0.0) 1/32 (3.1) 1/33 (3.0)
NE NE NE
10/15 (66.7) 4/15 (26.7) 1/15 (6.7)
21/33 (63.6) 7/33 (21.2) 5/33 (15.2)
NE, No examination. Percentages are given in parentheses.
ing that these poorly differentiated tumor cells may have arisen from a papillary carcinoma. The samples that were included in this study were also analyzed for mutations in codons 12 and 13 of K-RAS and in codons 61 of H- and N-RAS by DNA sequencing, but in each case none was found. Neither RET rearrangements nor expression levels could be analyzed in the Japanese cases, because RNA could not be extracted from the paraffin blocks to perform the RT-PCR analysis. We also analyzed BRAFT1796A and RAS mutations in the post-Chernobyl thyroid carcinomas obtained from the CTB (Table 1). We did not find any BRAFT1796A mutations in our childhood group, but this mutation was found in eight of the 33 cases (24.2%) in the adolescent and young adult groups. Similarly, although no RAS mutations were observed in the childhood group, one CAA3 CGA mutation in N-RAS codon 61 and one CAG3 CGG mutation in H-RAS codon 61 were observed in the adolescent and young adult groups. Additionally, the presence of rearrangements in the RET oncogene was examined by analyzing the expression of two different areas, the EC and intracellular tyrosine kinase regions (13). Samples were considered positive upon detectable expression of RET TK in the absence of RET EC expression. The validity of this method was confirmed by subsequent subcloning and sequencing of RET rearrangements in some of the post-Chernobyl PTCs (13a). We observed strong and moderate expression of RET TK in one subject (6.7%) and in four other cases (26.7%), respectively, from the childhood group and in seven cases (21.2%) and five cases (15.2%), respectively, from the adolescent and young adult groups. Of the tumors that were positive for BRAFT1796A, none was positive for RAS mutations, and all were negative for RET expression, except one case that showed weak RET expression. Furthermore, BRAFT1796A-
positive cases did not show any significant association with large tumor size, regional lymph node invasion, extrathyroidal invasion, or distant metastases (Table 2). Discussion T1796A
In this study BRAF mutations, RAS hotspot mutations, and RET TK expression levels were examined in childhood thyroid carcinomas. Previous studies showed that BRAFT1796A can be detected in 30 –50% of adult PTCs. In this study, however, this mutation was found in only one of 31 Japanese cases (3.4%) and in zero of 15 Ukrainian childhood cases, although we found the mutation in eight of 33 Ukrainian young adult cases (24.2%). These findings indicate that a low incidence of BRAFT1796A mutations is a common feature of childhood thyroid carcinomas. The differences in the biological behavior of childhood and adult thyroid carcinomas may therefore be at least partly dependent on the frequency of the BRAFT1796A mutation. We observed that distant metastasis occurred in seven of 31 Japanese cases (22.6%) and three of 15 Ukrainian childhood cases (20.0%). This high frequency of distant metastases is consistent with the previously characterized features of childhood thyroid carcinomas (14). Extrathyroidal invasions were also observed at a high frequency. However, the BRAFT1796A mutation was not found in any case displaying metastases and in only one case with an extrathyroidal invasion. In addition to the low prevalence of BRAFT1796A in childhood thyroid carcinoma, it is noteworthy that in childhood cases we examined that are BRAFT1796A positive, no association was found with either distant metastases or extrathyroidal invasion. There is no relationship between the behavior of PTC in childhood and the presence of BRAFT1796A mutation.
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TABLE 2. Pathohistological subtypes and clinical data of BRAFT1796A mutation cases in the patients with thyroid carcinomas observed in our Japanese (JP) and Ukrainian (UA) series Patient
BRAF mutation Case 1 (UA) Case 2 (UA) Case 3 (UA) Case 4 (UA) Case 5 (UA) Case 6 (UA) Case 7 (UA) Case 8 (UA) Case 9 (JP) Subtotal or mean value BRAF wild cases (JP) ⱕ15 yr old BRAF wild cases (UA) ⱕ15 yr old BRAF wild cases (UA) ⬎15 yr old
Age (yr)
16 31 28 25 17 26 24 22 12 22.3
Diagnosisa
Tumor size (mm)
Subtypeb
PTC PTC PTC PTC PTC PTC PTC PTC PTCc
P P P SP F F FP FSP F
23 22 60 21 18 22 9 18 6 21.6d 43.0d 22.9d 18.3d
Extend beyond gland
None None Positive None None None None None None 1/9 (1.1)e 7/30 (23) 3/15 (20) 11/25 (44)
Regional LN metastasis
Distant metastasis
None None Positive None None None Positive None Positive 3/9 (3.3) 20/30 (67) 9/15 (60) 11/25 (44)
None None None None None None None None None 0/9 (0.0) 7/30 (23) 3/15 (20) 4/25 (16)
a
Diagnosed by the CTB Pathology Panel. P, Papillary variant; F, follicular variant; S, solid variant; SP, FP, or FSP, mixed variant. c Diagnosed by Japanese pathologists. d Mean value. e Percentage is given in parentheses. b
In this study we analyzed two distinct childhood thyroid carcinoma groups living in regions with or without radiation exposure. The pathological and genetic features of childhood thyroid carcinoma have been more extensively studied in the Ukraine after the sharp increase in the incidence of this disease after the Chernobyl accident. The resulting pathological examinations have shown that the frequency of solid tumor growth patterns was significantly higher in post-Chernobyl childhood thyroid carcinomas than in Japanese cases, suggesting that there are differences in the process of carcinogenesis between these two groups (15). Previous molecular studies have demonstrated that RET/PTC rearrangements occur in some 50 –70% of the childhood PTCs that have arisen in the Chernobyl region (16 –18), whereas RAS mutations are uncommon (19). In addition, the solid follicular variant of post-Chernobyl PTCs has been frequently associated with RET/PTC3 rearrangements (16). In contrast, a previous study from our laboratory has shown that there is a low incidence of RET/PTC rearrangements in Japanese childhood thyroid carcinomas (20). It is therefore generally believed that RET/PTC rearrangements often arise due to exposure of the thyroid gland to both internal and external radiation, although several studies have showed that RET/PTC rearrangements, especially RET/PTC1, are frequently observed in childhood and adolescent papillary thyroid carcinomas without any history of radiation exposure (21, 22). We observed RET TK expression in 17 of 48 Ukrainian cases (35.4%). The reason for the relatively low frequency of RET alterations in this group compared with previous reports is unknown, but may be due to the difference in methodologies. We also assessed whether radiation exposure affects the induction of BRAFT1796A in thyroid carcinoma, but found no BRAFT1796A mutations in Ukrainian childhood PTCs and a very low frequency of this mutation (3.2%) in Japanese childhood PTCs. Therefore, radiation exposure seems not to influence the induction of a BRAFT1796A mutation, unlike RET/PTC rearrangements. There is also almost no overlap between PTCs harboring BRAFT1796A and con-
taining RAS and RET/PTC rearrangements, except for one case that has both a BRAFT1796A mutation and weak RET TK expression. In that case, the low level RET expression observed in this tumor would be consistent with either a small number of cells expressing the rearrangement or with a low level of expression in many cells. We need further studies to clarify the biological and clinical characters of this case. A common feature of BRAFT1796A, RAS mutations, and RET/ PTC rearrangement is the constitutive activation of the MAPK pathway, suggesting that this signal transduction pathway is likely to contribute to papillary thyroid tumorigenesis. It is therefore quite plausible that other oncogenes, which activate the MAPK pathway, may play a role in the carcinogenesis of Japanese childhood PTCs that have very low incidences of BRAF, RAS, and RET/PTC mutations (20). The pathological diagnosis of the single BRAFT1796A-positive Japanese case in our study was quite difficult. As this case presented with prominent solid cellular proliferation, occasional microfollicle foci, and no typical nuclear features of papillary carcinoma, the pathologists involved with the analysis initially diagnosed it as a poorly differentiated follicular carcinoma. However, judging retrospectively from the results of the BRAFT1796A mutation screening and antiCD15 positive staining, which are both thought to be specific for papillary carcinomas (12, 23), this case is now more likely to be a progression of a papillary carcinoma to a more dedifferentiated state in which it has, therefore, lost its well differentiated papillary characteristics. Consistent with this result, Nikiforova et al. (5), by examination of well differentiated papillary areas of undifferentiated carcinomas, demonstrated that poorly differentiated and anaplastic carcinomas with the BRAFT1796A mutation can arise from papillary carcinomas. Thus, analysis of the BRAFT1796A mutation may also be useful in assisting with the final diagnosis of cases that cannot easily be distinguished as either papillary or follicular carcinomas. In conclusion, this study provides the first evidence that the BRAFT1796A mutation is uncommon in childhood PTCs
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despite its high incidence in adult PTCs. The BRAFT1796A mutation might therefore be one of the factors that contributes to the differing biological behaviors observed between childhood and adult PTCs. Acknowledgments Received February 3, 2004. Accepted April 26, 2004. Address all correspondence and requests for reprints to: Dr. Hiroyuki Namba, Department of Molecular Medicine, Atomic Bomb Disease Institute, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. E-mail:
[email protected]. This work was supported by Grants-in-Aid for Scientific Research 13671158, 14380256, 14406017, and 15590981 and the 21st Century COE Program “International Consortium for Medical Care of Hibakusha and Radiation Life Science” from the Ministry of Education, Culture, Sports, Science, and Technology.
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JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.
