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Molecular Biology and Genetics |
Departments of General Surgery [X. X., R. M. Q., R. A. P.] and Pathology [P. G.], Rush Presbyterian St. Luke’s Medical Center, Chicago, Illinois 60612; Thyroid Cancer Research Laboratory, Department of Internal Medicine, University of Kentucky Medical Center, Lexington, Kentucky 40536 [K. B. A.]; and the Veterans Affairs Medical Center, Lexington, Kentucky 40511 [K. B. A.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Davies et al. (31)
recently reported that somatic missense BRAF mutation occurs in 66% of malignant melanomas and at low frequencies in many human cancers, including 
11% of colon cancers, 14% of borderline ovarian cancers, and 0.5% of sarcomas. Mutations are restricted to the kinase domain of the BRAF gene at multiple sites located in exons 11 and 15. However, >80% of these mutations are attributable to a single-base thymidine substitution at the nucleotide position 1796 with adenosine, which leads to the conversion of valine at codon 599 to glutamic acid. These investigators did not find BRAF mutation in 33 breast, 23 prostate, 14 lung, and 19 head and neck squamous cell carcinomas, and 15 gliomas. It appears that BRAF mutations tend to occur in tumors that harbor RAS mutations at a higher frequency. Because RAS oncogenes are mutated in 30% of spontaneous thyroid tumors (6
, 32)
, it is possible that BRAF may be mutated in thyroid neoplasms at high frequency too. Here, we report that BRAF mutation at V599E occurred in 21 of 56 papillary thyroid carcinomas (38%). However, BRAF mutation at this site was not detected in any of 18 FTAs examined. These observations suggest that BRAF mutation may be involved in the oncogenesis of papillary thyroid carcinomas but not involved in the formation of benign FTAs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
Ten thyroid tumor cell lines were used in this study (Table 1)
. One follicular adenoma (KAK-1), 1 papillary carcinoma (KAT-10), and 3 anaplastic (KAT-4, KAT-18, and SW1736) carcinoma cell lines were originally established in Dr. Ain’s laboratory and have been described previously (33)
. The other 5 thyroid tumor cell lines, including 1 papillary (NPA87), 2 follicular (WRO82 and MRO87), and 2 anaplastic carcinomas (ARO81 and DRO90), were kindly provided by Dr. Guy J. F. Juillard at University of California at Los Angeles. All thyroid tumor cell lines were grown in complete RPMI 1640 containing 10% fetal bovine serum.
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MASA.
MASA-PCR was conducted in principle as previously described (34
, 35)
. A forward primer flanking the sequence of exon 15 (5'-TAGGTGATTTTGGTCTAGCTACAGT-3') was used as a positive control to amplify wild-type as well as mutant BRAF. A second primer with substitution of two bases at the 5'-end (GGTGATTTTGGTCTAGCTACAAA-3') was designed to amplify mutant BRAF gene only. The sequence of the reverse primer is 5'-GGCCAAAATTTAATCAGTGGA-3'. The PCR reaction was set with an initial denaturation of 2 min at 94°C and subsequent denaturation for 30 s at 94°C, annealing for 45 s at 52°C, and extension for 45 s at 72°C. Thirty-five cycles were used to amply the PCR product with the expected size of 129 bp. All negative samples were reexamined for BRAF mutation by PCR under the same condition but with 40 cycles. To confirm BRAF mutation detected by MASA, 9 BRAF-mutated DNA samples were randomly selected among 21 MASA-positive samples and used as the templates to amplify exon 15 of the BRAF gene. PCR products were extracted and sequenced.
RET/PTC Rearrangement Analyzed by IHC Staining.
Wild-type RET is not expressed in normal follicular thyroid epithelial cells nor in papillary thyroid carcinomas, whereas the rearranged RET/PTC driven by a promoter of its fusion partner is readily expressed and can be detected by IHC staining with the antibodies against the COOH terminus of RET. Several recent studies have shown that RET/PTC rearrangements detected by IHC staining are consistent with that identified by Southern blot or by reverse transcription-PCR analysis followed by Southern hybridization (36, 37, 38)
. To test whether RET/PTC rearrangements take place in BRAF-mutated PTC, we conducted IHC to analyze RET/PTC expression in 56 PTC specimens to determine whether BRAF mutation was overlapping with RET/PTC rearrangements in some PTC specimens. Tissue sections were dewaxed with xylene and rehydrated. Slides were then heat inactivated in 10 mM sodium citrate (pH 6.0) in a microwave for 3 min. Cooled slides were rinsed with PBS and then incubated with 1% H2O2 in methanol for 30 min at room temperature. Sections were then blocked with 5% normal goat serum in PBS for 30 min at room temperature followed by 1 h incubation with an anti-RET rabbit serum (Santa Cruz Biotechnology, Santa Cruz, CA) (1:300 dilution) in PBS. Slides were washed and then incubated with goat antirabbit antibody-biotin conjugate (PharMingen, San Diego, CA) diluted at 1:300 in PBS/5% normal goat serum. Strepavidin-horseradish peroxidase conjugate (Zymed, San Francisco, CA) diluted at 1:200 in PBS with 5% normal goat serum was added and incubated for 45 min at room temperature. Color development was done with 3,3'-diaminobenzidine substrate (Sigma, St. Louis, MO). Slides were counterstained with Mayer’s hemotoxylin for 2 min, dehydrated, and mounted. RET/PTC expression was graded by two investigators in a blinded fashion. The expression of RET/PTC in these specimens was additionally confirmed by IHC using a monoclonal antibody against the extreme COOH terminus of RET (1:40; Novocastra Laboratories Ltd., Burlingame, CA) with a Catalyzed Signal Amplification system (Dako Corp., Carpinteria, CA) following the manufacture’s protocol.
Statistical Analysis.
Correlation between HPR1 gene expression and the histological diagnosis or clinicopathological parameters of the thyroid neoplasms was determined by a 
2 test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Table 1
, 2
anaplastic thyroid carcinoma cell lines, ARO81 and DRO90, harbored a homozygous BRAF mutation at the nucleotide position 1796 where an adenosine was replaced by a thymidine, leading to the substitution of valine at 599 with glutamic acid. In the other 6 thyroid tumor cell lines (Table 1
; 1 follicular adenoma, 2 anaplastic, 2 papillary, and 1 follicular carcinoma cell lines), only one allele of the BRAF gene was mutated at this site (Fig. 1
and Table 1
). No mutation at T1796 was detected in an anaplastic cancer cell line (KAT18) and a follicular carcinoma cell line (WRO82; Fig. 1
and Table 1
). No other point mutation within exon 15 was detected. Overall, our results show that 8 of 10 thyroid tumor cell lines originally established in two laboratories (Dr. Guy J. F. Juillard, University of California at Los Angeles; Dr. Kenneth B. Ain, University of Kentucky Medical Center) contained BRAF gene mutation at the T1796 nucleotide position. This suggests that BRAF may be mutated at high frequency in thyroid neoplasms.
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, the BRAF exon 15 was amplified by using the mismatched primer in 4 BRAF-mutant thyroid tumor cell lines (DRO90 and ARO81 with homozygous mutation; NPA87 and MRO87 with heterozygous mutation) but not in 2 wild-type cell lines (WRO82 and KAT18). A PCR reaction that amplifies BRAF exon 15 was included as a positive control. BRAF exon 15 was amplified in all samples. These results suggest that screening of BRAF mutation using this MASA-PCR is specific, and this approach can be used to rapidly screen BRAF mutation at codon 599 in a large number of tumor samples.
We next analyzed BRAF mutation in microdissected thyroid tumor tissues by using MASA. As shown in Fig. 2A
, BRAF mutation at T1796 was detected in 21 of 56 papillary carcinomas but not detected in 18 benign follicular adenomas and 6 nodular goiters. All negative DNA samples were reanalyzed for BRAF mutation by PCR reaction with 40 cycles, no BRAF mutation was found. Nine of 21 positive samples were randomly selected for DNA sequencing to confirm BRAF mutation. All 9 samples were positive with BRAF mutation. Shown in Fig. 2B
are three representative DNA sequences of BRAF-mutated samples, two of which had almost equal peak areas of A and T nucleotides, indicating a high homogeneity of tumor cells in these two microdissected sections. A third sample had a smaller A peak than T peak, reflecting heterogeneity of tumor cells and nontumor cells in the microdissected section. Nevertheless, these sequencing data additionally suggest that BRAF mutation detected by MASA is reliable and specific.
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Higher Frequency of BRAF Mutation in Male Patients than in Female Patients with PTC.
We analyzed whether BRAF mutation correlated with patient age, gender, and tumor stage. Interestingly, we found that BRAF mutation occurred in 9 of 14 male PTC patients and in 12 of 42 female patients (Table 2)
. Statistical analysis revealed that BRAF mutation occurred at a significantly higher frequency in male patients than in female patients (P < 0.05). In addition, we found that BRAF mutation occurred in 15 of 33 PTC patients older than 40 years old and in only 6 of 23 PTC patients younger than 40 years old. BRAF mutation in patients with PTC tended to occur at a higher frequency in older patients than in younger patients, but this was not a significant difference (P > 0.05). We did not find that BRAF mutation was associated with tumor volume (Table 2)
. BRAF was mutated at a higher rate in PTC with invasive and metastatic potential (Table 2)
than in the noninvasive tumors, but again, this was not statistically significant (P > 0.05).
Overlapping of BRAF Mutation with other Genetic Alterations in PTC.
RET/PTC rearrangements frequently occur in papillary thyroid carcinomas. To test whether BRAF mutation concurs with RET/PTC rearrangements, we conducted IHC to determine the status of RET/PTC rearrangement. IHC analysis using an anti-RET rabbit serum revealed that RET/PTC expression was abundantly present in the cytoplasm of the tumor cells in a BRAF-mutated specimen (Fig. 3A
, inset) and a PTC with wild-type BRAF but not in the neighboring normal thyroid follicular cells (Fig. 3A)
. Similar results were obtained when an anti-RET mAb was used (Fig. 3, C and D)
. Normal mouse IgG and normal rabbit serum included as negative controls did not show any positive signal (data not shown). All samples analyzed for RET/PTC expression using anti-RET rabbit serum were additionally tested for RET/PTC expression by IHC with an anti-RET mAb. We found that RET/PTC expression was consistent in 85% of the specimens. The samples graded as having RET/PTC expression with either method were considered as RET/PTC positive.
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, we found that 21 of 56 PTCs (38%) were RET/PTC positive. Among them, 13 of 35 PTCs (37%) with wild-type BRAF were RET/PTC positive. Unexpectedly, we found that 8 of 21 BRAF-mutated samples (38%) were also RET/PTC positive, suggesting that BRAF mutation and RET/PTC rearrangements are overlapping in a large number of PTC.
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, we did not find any NRAS mutations in all 21 BRAF-mutated specimens, indicating that there is no overlapping in NRAS and BRAF mutations in these PTC. Because KRAS and HRAS mutations were not analyzed in this study, we could not exclude the possibility that KRAS and HRAS mutation might be overlapping with BRAF mutation. However, a recent study by Kimura et al. (39)
demonstrated that 28 BRAF-mutated PTCs did not contain any form of mutated RAS genes. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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80–90% of all mutations detected in several types of tumors, in particular in melanomas and colon cancers (31
, 40)
. Several other sites in the kinase domain of BRAF in exons 11 and 15 can also be mutated but with much lower frequency (31
, 40)
. Our present study using MASA demonstrated that BRAF mutation at the hot-spot codon 599 occurred in 21 of 56 papillary carcinomas (38%). While our manuscript was being revised, Kimura et al. (39)
reported that BRAF mutation occurs in 28 of 78 PTCs (35.8%) and that all mutations are located at codon 599. Thus, these observations collectively suggest that the rate of BRAF mutation in PTC is the second highest to that in melanomas (>60%; Refs. 31
, 41
, 42
) and is much higher than other cancers such as colorectal adenocarcinomas (5–10%; Refs. 31
, 40
, 43
) and lung cancers (1.8%; Ref. 44
).
Although our present study revealed that BRAF was mutated in 38% of PTC at codon 599, we did not find BRAF mutation at this site in any of 18 follicular adenomas studied. Statistical analysis revealed that BRAF was differentially mutated in these two different types of thyroid neoplasms (Table 1
; P = 0.006). Consistent with this observation, Kimura et al. (39)
reported that no BRAF mutation was detected in 14 FTAs, as well as in 10 follicular thyroid carcinoma. These observations are in sharp contrast to previous studies showing that the upstream activators of BRAF, the RAS genes (HRAS, KRAS, and NRAS), are mutated in both benign FTAs and papillary thyroid carcinomas (10
, 45
, 46)
. It is not clear why BRAF is only mutated in papillary thyroid carcinomas but not in FTAs, whereas RAS is mutated in both benign and malignant thyroid neoplasms. Nevertheless, a higher BRAF mutation rate in carcinomas than in adenomas was also observed in another type of tumor: Rajagopalan et al. (40)
reported that BRAF is mutated in 30 of 276 clinical colon cancers (11%) but is mutated in only 2 of 54 colon adenomas (3.8%); Similarly, Yuen et al. (43)
reported that BRAF is mutated in 11 of 215 colorectal adenocarcinomas (5.1%) and in 3 of 108 (2.8%) sporadic adenomas.
The RET proto-oncogene is rearranged in 40% of spontaneous PTC when analyzed by IHC staining and/or reverse transcription-PCR followed by Southern hybridization (36, 37, 38)
and with a much higher rate in radiation-associated PTC (56–84%) as well as in pediatric PTCs (70%; Ref. 47
). A few prior studies demonstrated that combined RET/PTC rearrangements or TSHRand RAS mutation can occur in papillary thyroid carcinomas (18
, 46 , 48, 49, 50)
. For example, Bounacer et al. (48)
reported that combined RAS mutation and RET/PTC rearrangements are detected in 2 of 28 radiation-associated thyroid tumors. In another study, Sugg et al. (50)
analyzed genetic alterations in 20 PTCs and found one of them harboring both NRAS mutation and a RET/PTC3 rearrangement. In this study, we tested whether BRAF mutation would occur together with RET/PTC rearrangements in PTC. To our surprise, IHC analysis revealed that a large number (8 of 21) of BRAF-mutated PTC-expressed RET, indicating that the RET proto-oncogene is rearranged in these tumors. This observation is totally unexpected not only because it contradicts recent studies showing that BRAF mutation at codon 599 does not overlap with RET/PTC rearrangements nor with RAS mutation (39)
in PTC, as well as in other malignancies such as melanomas and colorectal adenocarcinomas (31
, 40
, 43)
but also because two combined genetic alterations in the same signaling pathway appear to be redundant and therefore to be unnecessary. Nevertheless, based on our observation that 38% of the 21 BRAF-mutated PTCs also harbored RET/PTC rearrangements, we speculate that RET/PTC may cooperate with mutated BRAF to induce clinically overt PTC (24)
. In fact, both mutant BRAF gene and RET/PTC have a relatively low oncogenic potential. For example, the oncogenic potential of mutant BRAF gene is 50-fold lower than V12 HRAS when tested in NIH3T3 cells (31)
, whereas RET/PTC1 is unable to fully transform the rat thyroid PC Cl 3 cells (e.g., unable to grow in soft agar and to develop tumorigenicity in athymic mice) but can cooperate with RAS oncogenes to fully transform PC Cl3 cells (24)
. It is possible that BRAF may cooperate with RET/PTC to fully transform thyroid epithelial cells, leading to the progression of occult microcarcinomas, which have a very high rate of RET/PTC rearrangements (>70%), into clinically overt papillary thyroid carcinomas. Bearing in mind that concomitant BRAF mutation and RET/PTC rearrangements were not confirmed in a similar study recently published by Kimura et al. (39)
, this double-hit model should be taken with caution.
Detailed mutation analyses in a variety of tumors by Davies et al. (31) demonstrated that BRAF is generally mutated at a higher frequency in tumor cell lines than that in their corresponding tumor samples. For example, these investigators reported that BRAF is mutated in 7 of 40 colon cancer cell lines (18%), 5 of 59 sarcoma cell lines (9%), 4 of 38 glioma cell lines, and 4 of 131 lung cancer cell lines. In contrast, BRAF mutation is only detected in 4 of 33 colon cancers (12%), 1 of 182 sarcomas (0.5%), and none of the 15 gliomas and 14 lung cancers (31) . Our present study demonstrated that BRAF was mutated overall in 8 of 10 thyroid tumor cell lines (80%), including one follicular adenoma cell line, whereas BRAF was only mutated in 38% of papillary thyroid carcinomas. The higher BRAF mutation rate in tumor cell lines than in their corresponding tumor specimens may be attributable to: (a) the cell lines from the original tumors with BRAF mutations may be selectively established because of an advantage in cell growth (31) and an antiapoptotic effect (51) ; (b) established cell lines are homogenous, whereas microdissected tumor sections contain many normal cells such as stromal cells, endothelial cells, and infiltrating immune cells. Therefore, a low percentage of tumor cells in a specimen may lower the sensitivity of the methods used to detect BRAF mutation, resulting in a false negative result; and (c) BRAF mutation in cell lines may be gained during long-term in vitro cell culture.
The molecular mechanisms by which BRAF is mutated at high frequency in melanoma and papillary thyroid carcinomas are not known. Davies et al. (31)
proposed that BRAF mutation in melanoma is somehow related to features of melanocyte biology. This supposition is based on the observations that besides the RAS signaling pathway, BRAF in melanocytes can be directly activated by cyclic AMP, a secondary messenger regulated by binding of 
-melanocyte stimulating-hormone, and other proopiomelanocortin-derived peptides to their melanocortin receptor I (52
, 53)
. This alternative BRAF activation pathway plays a critical role in controlling melanocyte proliferation and differentiation, particularly in response to UVB radiation (52
, 53)
. Interestingly, in thyrocytes, thyroid-stimulating hormone strongly activates cyclic AMP and promotes thyrocyte cell proliferation (54, 55, 56, 57)
. Therefore, it is likely that mutation of BRAF in melanocytes and thyrocytes may be related to cyclic AMP, although how exactly cyclic AMP results in BRAF gene mutation remains elusive. In colon cancer, Rajagopalan et al. (40)
reported that BRAF mutation is associated with the deficiency of mismatch repair. Although it is not clear whether mismatch repair activity is also compromised or deficient in papillary thyroid carcinomas, ß-catenin, another gene frequently mutated in colon cancer because of the deficiency of mismatch-repair, is also mutated in papillary thyroid carcinomas (58)
. Therefore, it is possible that BRAF mutation in papillary thyroid carcinomas may, in part, result from the defective DNA mismatch repair.
Another interesting observation in this study is that among 8 mutant thyroid tumor cell lines, 6 were heterozygous (Table 2)
. The other two cell lines (ARO81 and DRO90) were either homozygous because of gene conversion or hemizygous because of the loss of a second allele of the BRAF gene. Similar to this observation, Rajagopalan et al. (40)
found that 2 of 28 colon adenocarcinomas with BRAF mutation at residue 599 were not heterozygous. The underlying mechanism for this nonheterozygous BRAF gene mutation and its role in trigging cell transformation and tumorigenesis remain to be defined.
In summary, our present study demonstrates that BRAF was mutated at a high frequency in papillary thyroid carcinomas but not in follicular adenomas and that a large number of BRAF-mutated PTC also harbored RET/PTC rearrangements. These observations provide a molecular basis for the oncogenesis of PTC and suggest that PTC and FTA may be developed through the mutation of different oncogenes within the same signaling pathway. It will be interesting to find out whether quantitative and/or qualitative differences in the RAS/BRAF-signaling pathway determine the type of thyroid neoplasm.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported, in part, by a grant from Thyroid Research Advisory Council, NIH R29 Grant CA76407 (to X. X.) and the Department of General Surgery at Rush Presbyterian St. Luke’s Medical Center. 
2 To whom requests for reprints should be addressed, at Department of General Surgery, Rush Presbyterian St. Luke’s Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. Phone: (312) 942-6623; Fax: (312) 942-2867; E-mail: xxu{at}rush.edu
3 The abbreviations used are: PTC, papillary thyroid carcinoma; FTA, follicular thyroid adenoma; IHC, immunohistochemistry; MASA, mutant-allele-specific amplification; mAb, monoclonal antibody.
Received 1/ 6/03. Accepted 5/22/03.
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K. Mercer, S. Giblett, S. Green, D. Lloyd, S. DaRocha Dias, M. Plumb, R. Marais, and C. Pritchard Expression of Endogenous Oncogenic V600EB-raf Induces Proliferation and Developmental Defects in Mice and Transformation of Primary Fibroblasts Cancer Res., December 15, 2005; 65(24): 11493 - 11500. [Abstract] [Full Text] [PDF]
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M. Xing, W. H. Westra, R. P. Tufano, Y. Cohen, E. Rosenbaum, K. J. Rhoden, K. A. Carson, V. Vasko, A. Larin, G. Tallini, et al. BRAF Mutation Predicts a Poorer Clinical Prognosis for Papillary Thyroid Cancer J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6373 - 6379. [Abstract] [Full Text] [PDF]
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V. Guarino, P. Faviana, G. Salvatore, M. D. Castellone, A. M. Cirafici, V. De Falco, A. Celetti, R. Giannini, F. Basolo, R. M. Melillo, et al. Osteopontin Is Overexpressed in Human Papillary Thyroid Carcinomas and Enhances Thyroid Carcinoma Cell Invasiveness J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5270 - 5278. [Abstract] [Full Text] [PDF]
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V. Vasko, S. Hu, G. Wu, J. C. Xing, A. Larin, V. Savchenko, B. Trink, and M. Xing High Prevalence and Possible de Novo Formation of BRAF Mutation in Metastasized Papillary Thyroid Cancer in Lymph Nodes J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5265 - 5269. [Abstract] [Full Text] [PDF]
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M. O. Hoque, E. Rosenbaum, W. H. Westra, M. Xing, P. Ladenson, M. A. Zeiger, D. Sidransky, and C. B. Umbricht Quantitative Assessment of Promoter Methylation Profiles in Thyroid Neoplasms J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 4011 - 4018. [Abstract] [Full Text] [PDF]
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M Xing BRAF mutation in thyroid cancer Endocr. Relat. Cancer, June 1, 2005; 12(2): 245 - 262. [Abstract] [Full Text] [PDF]
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S Rossi, L Fugazzola, L De Pasquale, P Braidotti, V Cirello, P Beck-Peccoz, S Bosari, and A Bastagli Medullary and papillary carcinoma of the thyroid gland occurring as a collision tumour: report of three cases with molecular analysis and review of the literature Endocr. Relat. Cancer, June 1, 2005; 12(2): 281 - 289. [Abstract] [Full Text] [PDF]
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J. A. Knauf, X. Ma, E. P. Smith, L. Zhang, N. Mitsutake, X.-H. Liao, S. Refetoff, Y. E. Nikiforov, and J. A. Fagin Targeted Expression of BRAFV600E in Thyroid Cells of Transgenic Mice Results in Papillary Thyroid Cancers that Undergo Dedifferentiation Cancer Res., May 15, 2005; 65(10): 4238 - 4245. [Abstract] [Full Text] [PDF]
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V. Porra, C. Ferraro-Peyret, C. Durand, S. Selmi-Ruby, H. Giroud, N. Berger-Dutrieux, M. Decaussin, J.-L. Peix, C. Bournaud, J. Orgiazzi, et al. Silencing of the Tumor Suppressor Gene SLC5A8 Is Associated with BRAF Mutations in Classical Papillary Thyroid Carcinomas J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 3028 - 3035. [Abstract] [Full Text] [PDF]
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 N. H. Tran, X. Wu, and J. A. Frost B-Raf and Raf-1 Are Regulated by Distinct Autoregulatory Mechanisms J. Biol. Chem., April 22, 2005; 280(16): 16244 - 16253. [Abstract] [Full Text] [PDF]
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N. Mitsutake, J. A. Knauf, S. Mitsutake, C. Mesa Jr., L. Zhang, and J. A. Fagin Conditional BRAFV600E Expression Induces DNA Synthesis, Apoptosis, Dedifferentiation, and Chromosomal Instability in Thyroid PCCL3 Cells Cancer Res., March 15, 2005; 65(6): 2465 - 2473. [Abstract] [Full Text] [PDF]
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R. Sorrentino, S. Libertini, P. L. Pallante, G. Troncone, L. Palombini, V. Bavetsias, D. Spalletti-Cernia, P. Laccetti, S. Linardopoulos, P. Chieffi, et al. Aurora B Overexpression Associates with the Thyroid Carcinoma Undifferentiated Phenotype and Is Required for Thyroid Carcinoma Cell Proliferation J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 928 - 935. [Abstract] [Full Text] [PDF]
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 P. Smyth, S. Finn, S. Cahill, E. O'Regan, R. Flavin, J. J. O'Leary, and O. Sheils ret/PTC and BRAF Act as Distinct Molecular, Time-Dependant Triggers in a Sporadic Irish Cohort of Papillary Thyroid Carcinoma International Journal of Surgical Pathology, January 1, 2005; 13(1): 1 - 8. [Abstract] [PDF]
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A Perren, S Schmid, T Locher, P Saremaslani, C Bonvin, P U Heitz, and P Komminoth BRAF and endocrine tumors: mutations are frequent in papillary thyroid carcinomas, rare in endocrine tumors of the gastrointestinal tract and not detected in other endocrine tumors Endocr. Relat. Cancer, December 1, 2004; 11(4): 855 - 860. [Abstract] [Full Text] [PDF]
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 J A Fagin How thyroid tumors start and why it matters: kinase mutants as targets for solid cancer pharmacotherapy J. Endocrinol., November 1, 2004; 183(2): 249 - 256. [Abstract] [Full Text] [PDF]
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G. Salvatore, R. Giannini, P. Faviana, A. Caleo, I. Migliaccio, J. A. Fagin, Y. E. Nikiforov, G. Troncone, L. Palombini, F. Basolo, et al. Analysis of BRAF Point Mutation and RET/PTC Rearrangement Refines the Fine-Needle Aspiration Diagnosis of Papillary Thyroid Carcinoma J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5175 - 5180. [Abstract] [Full Text] [PDF]
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J. A. Fagin Challenging Dogma in Thyroid Cancer Molecular Genetics--Role of RET/PTC and BRAF in Tumor Initiation J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4264 - 4266. [Full Text] [PDF]
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J. Lima, V. Trovisco, P. Soares, V. Maximo, J. Magalhaes, G. Salvatore, M. Santoro, T. Bogdanova, M. Tronko, A. Abrosimov, et al. BRAF Mutations Are Not a Major Event in Post-Chernobyl Childhood Thyroid Carcinomas J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4267 - 4271. [Abstract] [Full Text] [PDF]
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A. Kumagai, H. Namba, V. A. Saenko, K. Ashizawa, A. Ohtsuru, M. Ito, N. Ishikawa, K. Sugino, K. Ito, S. Jeremiah, et al. Low Frequency of BRAFT1796A Mutations in Childhood Thyroid Carcinomas J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4280 - 4284. [Abstract] [Full Text] [PDF]
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M. Xing, R. P. Tufano, A. P. Tufaro, S. Basaria, M. Ewertz, E. Rosenbaum, P. J. Byrne, J. Wang, D. Sidransky, and P. W. Ladenson Detection of BRAF Mutation on Fine Needle Aspiration Biopsy Specimens: A New Diagnostic Tool for Papillary Thyroid Cancer J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2867 - 2872. [Abstract] [Full Text] [PDF]
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T. Ikenoue, Y. Hikiba, F. Kanai, J. Aragaki, Y. Tanaka, J. Imamura, T. Imamura, M. Ohta, H. Ijichi, K. Tateishi, et al. Different Effects of Point Mutations within the B-Raf Glycine-Rich Loop in Colorectal Tumors on Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase/Extracellular Signal-Regulated Kinase and Nuclear Factor {kappa}B Pathway and Cellular Transformation Cancer Res., May 15, 2004; 64(10): 3428 - 3435. [Abstract] [Full Text] [PDF]
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E. Puxeddu, S. Moretti, R. Elisei, C. Romei, R. Pascucci, M. Martinelli, C. Marino, N. Avenia, E. D. Rossi, G. Fadda, et al. BRAFV599E Mutation Is the Leading Genetic Event in Adult Sporadic Papillary Thyroid Carcinomas J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2414 - 2420. [Abstract] [Full Text] [PDF]
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 R H Edwards, M R Ward, H Wu, C A Medina, M S Brose, P Volpe, S Nussen-Lee, H M Haupt, A M Martin, M Herlyn, et al. Absence of BRAF mutations in UV-protected mucosal melanomas J. Med. Genet., April 1, 2004; 41(4): 270 - 272. [Abstract] [Full Text] [PDF]
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M. Xing, V. Vasko, G. Tallini, A. Larin, G. Wu, R. Udelsman, M. D. Ringel, P. W. Ladenson, and D. Sidransky BRAF T1796A Transversion Mutation in Various Thyroid Neoplasms J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1365 - 1368. [Abstract] [Full Text] [PDF]
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M. Xing, Y. Cohen, E. Mambo, G. Tallini, R. Udelsman, P. W. Ladenson, and D. Sidransky Early Occurrence of RASSF1A Hypermethylation and Its Mutual Exclusion with BRAF Mutation in Thyroid Tumorigenesis Cancer Res., March 1, 2004; 64(5): 1664 - 1668. [Abstract] [Full Text] [PDF]
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