THYROID Volume 15, Number 4, 2005 © Mary Ann Liebert, Inc.
BRAF Mutations are Uncommon in Papillary Thyroid Cancer of Young Patients Karen Penko,1 Jeffrey Livezey,1 Cydney Fenton,1 Aneeta Patel,1 Diarmuid Nicholson,2 Michael Flora,3 Kevin Oakley,1 R. Michael Tuttle,4 and Gary Francis1
Mortality is low for young patients (younger than 21 years) with papillary thyroid cancer (PTC), and different mutations might contribute to this. Previous studies detected ret/PTC rearrangements more frequently in PTC from children than adults, and recent reports describe a high incidence of BRAF T1796A transversion in adult PTC. However, BRAF mutations have not been adequately studied in PTC from young patients. We amplified and sequenced segments of the BRAF gene spanning the T1796A transversion site in 14 PTC from patients 10–21 years of age (mean, 17.5 3.5 years). The PTC (7 class 1; 5 class 2; 1 class 3) ranged from 0.7–2.9 cm in diameter (mean, 1.4 0.75 cm). None of them (0/14) contained BRAF T1796A and none recurred (mean follow-up, 66 40 months). This incidence of BRAF T1796A is significantly less than that reported for adult PTC (270/699, 38.6%, p 0.0015) in several series. None of our PTC (0/10) contained ras mutations, but 7/12 (58%) contained ret/PTC rearrangements. We conclude that BRAF mutations are less common in PTC from young patients, and ret/PTC rearrangements were the most common mutation found in these childhood PTC.
Introduction
S
suggest that papillary thyroid carcinomas (PTC) follow a more favorable course in young patients (younger than 21 years of age) (1–3). They have lower mortality rates than older patients when matched for similar stage disease, and in most series, disease-specific mortality is only 1%–2% (1–3). The prognosis remains favorable despite a high incidence of cervical lymph node metastasis and even for those with diffuse pulmonary metastases (3). The reasons for this variation between children and adults are unknown but are generally thought to arise from differences in mutations and oncogene expression in the thyroid (4–9). Several studies have shown that ret/PTC mutations are more common in young patients (47%–65%) compared to older adults (3%–34%) (7,10,11). We also found that ras mutations are less common (6.5%) than in older adults with PTC (12%) (4). Despite these differences, recent studies show that mutations in the BRAF gene are seen in a wide variety of cancers and are very common in PTC from older adults (12–22). The EVERAL OBSERVATIONS
vast majority of these mutations (92%) consist of a T to A switch at nucleotide 1796 in the coding region of exon 15 in the BRAF gene (T1796A), and generate unregulated B-Raf activity (13). Constitutively activated B-raf leads to increased activity of extracellular signal-regulated kinase (ERK) through the ret/PTC-SHC-RAS-RAF-MEK-MAPK ERK pathway, resulting in increased cellular proliferation (23). Multiple groups have investigated this BRAF T1796A mutation in adult PTC and have reported frequencies ranging from 29%–69% (14–22). Together, these studies have identified a total of 270 positive mutations in 699 adult PTC (38.6%), making the BRAF T1796A mutation the most common genetic alteration so far identified in adult PTC. Furthermore, this BRAF mutation has not been found in follicular thyroid carcinomas (FTC), medullary thyroid carcinomas (MTC), Hürthle cell carcinomas, or benign adenomas (14–20). Several studies have also found no overlap among mutations in BRAF, ret/PTC, and ras (15,17,22). This finding supports the concept of alternative mutations in that any activating mutation along the ret/PTC-SHC-RASRAF-MEK-MAPK ERK pathway plays a similar role in thyroid cell dedifferentiation and cancer pathogenesis (23).
Departments of 1Pediatrics and 3Biomedical Instrumentation Center, Uniformed Services University of the Health Sciences, Bethesda, Maryland. 2Department of Clinical Investigation, Walter Reed Army Medical Center, Washington, D.C. 4Department of Endocrinology, Memorial Sloan Kettering Cancer Center, New York, NY. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or to reflect the opinions of the Uniformed Services University of the Health Sciences, Walter Reed Army Medical Center, the Department of the Army, or the Department of Defense.
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BRAF IN PTC OF YOUNG PATIENTS
321
In this study, we determined the incidence of the most common BRAF mutation (T1796A) in archived thyroid tissue specimens from patients 21 years of age or younger, compared this to the reported incidence in PTC from older adults, and investigated the presence or absence of mutations in ras and ret/PTC.
tient who has been free of disease for at least 4 months (no palpable disease and negative 131Iodine scan) (3). Patients were classified as having persistent disease if they were at least 4 months beyond initial treatment and had persistent 131I uptake, magnetic resonance imaging (MRI) evidence of disease, or elevated serum thyroglobulin despite prior surgery and radioiodine (3).
Materials and Methods DNA extraction
Thyroid tissue specimens After approval by the Institutional Review Boards at Walter Reed Army Medical Center, Washington, D.C.; and the Uniformed Services University of the Health Sciences, Bethesda, Maryland; 40 specimens were obtained from an archival bank of formalin-fixed thyroid tissue previously removed from military dependents 21 years of age or younger (3). The extent of disease at diagnosis was defined according to DeGroot et al.: class 1 disease was confined to the thyroid gland, class 2 involved regional lymph nodes, class 3 extended beyond the thyroid capsule or was inadequately removed, and class 4 showed distant metastasis (1). MACIS scores were calculated according to Hay et al. (24). Recurrence was defined as the appearance of new disease (new radioactive iodine uptake or biopsy proven disease) in any pa-
TABLE 1. DEMOGRAPHIC
AND
Paraffin-embedded tissue sections (5 m) were deparaffinized using sequential washes with xylene and absolute ethanol. DNA was extracted using the Qiagen DNeasy (Valencia, CA) extraction kit. The integrity of the extracted DNA was confirmed by polymerase chain reaction (PCR) amplification of the internal housekeeping gene, glyceraldehyde-3phosphate dehydrogenase (GAPDH). BRAF gene amplification by polymerase chain reaction (PCR) Extracted DNA (100 ng) was amplified to generate a 224bp sequence of BRAF exon 15 that spans the 1796 coding region using polymerase chain reaction (PCR). PCR conditions were as follows: an initial denaturation step at 96°C for 5
MUTATIONAL FEATURES
OF
THYROID TUMORS
Focality
Classa
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Unifocal Unifocal Unifocal Unifocal Unifocal Unifocal Unifocal MultiUnifocal Unifocal MultiMultiMultiUnk
Papillary Thyroid Carcinoma 1 3.31 T Yes 1 3.43 T Unkf 1 3.46 T No 1 3.55 T No 1 3.64 T Unk 1 3.82 T Yes 1 3.97 T Yes 2 3.31 T Yes 2 3.34 T Yes 2 3.40 T Yes 2 3.40 Unk Unk 2 3.40 T Unk 3 4.88 T Yes Unk Unk Unk Unk
No Yes No No No Unkf Unk No No No Unk No No Unk
0.7 1.1 1.2 1.5 1.8 2.4 2.9 0.7 0.8 1.0 1.0 1.0 2.6 Unk
YOUNG PATIENTS
MACISb Followscore Surgeryc RAId Recurrence up (mo)
Case Age (yrs) Radiation Size # /gender exposure (cm) 19 F 19 M 20 M 11 F 21 F 17 F 18 F 15 F 10 F 20 F 19 M 21 F 17 M Unkf
IN
ret/PTC
Ras
BRAFe
No No No No No No No No No No No No No Unk
106 77 118 18 68 50 100 85 15 101 104 22 3 Unk
PTC2 PTC1&3 Unk Neg PTC1&2 PTC3 Neg Neg PTC1 PTC1 Neg PTC1 Neg Unk
Neg Neg Neg Neg Neg Neg Neg Neg Unk Neg Unk Neg Neg Unk
WT WT WT WT WT WT WT WT WT WT WT WT WT WT
Yesg No No Unk
33 43 18 Unk
Neg Neg Neg Unk
Unk Neg Neg Unk
WT WT WT WT
Unk
No
59
Neg
Neg
WT
Unk
Unk
Unk
Unk
Unk
WT
Follicular Thyroid Carcinoma 15 16 17 18
17 F 17 F 19 F Unk
Unk No No Unk
2.2 Multi2.5 Unifocal 2.8 Unifocal Unk Unk
Unk Unk Unk Unk
Unk Unk Unk Unk
Unk T T T
Yes Yes Unk Unk
Medullary Thyroid Carcinoma 19
21 M
Unk
0.7
Multi-
Unk
Unk
T
Benign Adenoma 20
Unk
aClass
Unk
Unk
according to DeGroot, et al. (1). according to Hay, et al. (24). cSurgery: T total thyroidectomy. dRAI radioactive iodine ablation. eBRAF WT wild type sequence. fUnk unknown. gTime to recurrence: 9 months. bMACIS
Unk
N/A
Unk
322
PENKO ET AL.
minutes; followed by 35–40 amplification cycles of: denaturation at 96°C for 30 seconds, annealing at 55°C for 1 minute, and primer extension at 72°C for 30 seconds; followed by a final run-off extension at 72°C for 10 minutes. The following previously published primers were used (19): Forward (sense):
5-TCATAATGCTTGCTCTGATAGGA-3
Reverse (antisense): 5-GGCCAAAAATTTAATCAGTGGA-3 This method was successful in amplifying BRAF from 9 samples. However, due to DNA degradation from prolonged storage of formalin fixed tissues, amplification was not successful in the remaining samples. New primers spanning a smaller (124 bp) sequence of the BRAF gene, to include the T1796A transversion site were used to amplify BRAF from all 20 samples that were ultimately successfully sequenced: Forward:
5-CCATCCACAAAATGGATCCAGA-3
Reverse:
5-TCAGATATATTTCTTCATGAAG-3
PCR conditions were identical to those outlined above except an annealing temperature of 52°C was used. All amplified PCR products were resolved on a 2% agarose gel and examined for the presence of the appropriate sized product. In 12 samples, nested PCR was required to amplify sufficient product for sequence analysis. Nested PCR was performed using 1 L of the initial product in a new PCR reaction using the above conditions and 20–40 amplification cycles. DNA sequencing of amplified products The PCR products were sequenced using the Applied Biosystems ABI 1377 instrument. The presence or absence of the T to A transversion at position 1796 in the coding region of exon 15 in the BRAF gene was determined by direct comparison to the published wild-type BRAF gene sequence. DNA extracted from NPA (papillary thyroid cancer) cell cultures was used as a positive control and DNA extracted from WRO (follicular thyroid cancer) cell cultures was used as a negative control (15,19). Statistical analyses Statistical analyses were performed using SPSS software for Windows 95 (SPSS Inc., Chicago, IL). Nonparametric analyses were performed using the Fisher’s exact test. Results We successfully amplified the pertinent portion of the BRAF gene and sequenced the PCR product in 20 samples. Table 1 shows the clinical and pathologic data associated with each sample. The patients ranged in age from 10 to 21 years (mean, 17.7 3.2 years). Twelve (60%) were females, 5 (25%) were males, and 3 (15%) were of unspecified gender. There were 14 PTC, 4 follicular thyroid carcinomas (FTC), 1 medullary thyroid carcinoma (MTC), and 1 benign adenoma. One patient with PTC was known to have a history of radiation exposure. Table 2 shows the characteristics of the 14 PTC. Their histologic subtypes included 5 (36%) typical PTC, 5 (36%) follicular variants, 2 (14%) invasive solid
TABLE 2. CHARACTERISTICS OF THE FOURTEEN PATIENTS PAPILLARY THYROID CANCER Number Gender (F/M/Unknown) Mean age (yr) Radiation exposure (Y/N/Unknown) Mean tumor size (cm) Average MACISa score Multi-/Unifocal/Unknown Histologic type (TcFVd/Ie/TCf/DSg Follow-up (months) Classb 1/2/3/4/Unknown Surgery (Total thyroidectomy/Subtotal thyroidectomy/Lobectomy/ Unknown) Radioactive iodine ablation (Yes/No/Unknown) Recur (Yes/No/Unknown) Ras mutation (Neg/H-ras/N-ras/Unknown) ret/PTC mutation (Positive/negative/Unknown)
WITH
n 14 9/4/1 15.6 0.65 1/9/4 1.4 0.21 3.6 0.11 4/9/1 5/5/2/1/1 67 11 7/5/1/0/1 12/0/0/2
7/2/5 0/13/1 11/0/0/3 7/5/2
aMACIS
according to Hay, et al. (24). according to DeGroot, et al. (1). cT typical. dFV follicular variant. eI invasive solid. fTC tall cell variant. gDS diffuse sclerosing variant. bClass
variants, 1 (7%) tall cell variant, and 1 (7%) diffuse sclerosing variant. They ranged in size from 0.7 to 2.9 cm (mean, 1.4 0.21 cm). Nine (64%) were unifocal and 4 (29%) were multifocal. Seven (50%) were confined to the gland (DeGroot class 1), 5 (36%) had regional node involvement (DeGroot class 2), 1 (7%) had direct invasion beyond the thyroid capsule (DeGroot class 3), and none recurred (1). One of the FTC recurred after 9 months. All of the tumors (n 20), including the PTC (n 14) contained the wild type BRAF gene sequence and none contained the BRAF T1796A transversion. Figure 1 shows a representative example after PCR amplification of the 124-bp sequence that spans the BRAF T1796A transversion site. Figure 2 shows the sequencing results from the positive control (NPA cell extract known to carry the BRAF mutation), the negative control (WRO cell culture extract known to carry the wild-type BRAF sequence), and a representative patient sample. For PTC, the incidence of BRAF T1796A transversion (0/14) is significantly less than that calculated from published series of older adults with PTC (270/699, p 0.0015, Fisher’s exact test) (14–22). Several, but not all, of the PTC in this study had been previously examined for the presence of ras and ret/PTC mutations (4, 7) (see Tables 1 and 2). All of the PTC that had been examined for ras mutations (n 10) were negative for mutations in H-ras and N-ras. Among the 12 PTC that had been examined for ret/PTC mutations, 5 (42%) were negative, 3 contained the ret/PTC1 rearrangement (25%), 1 (8%)
BRAF IN PTC OF YOUNG PATIENTS
323
FIG. 1. Amplified BRAF gene from papillary thyroid carcinoma (PTC) of young patients. Following 35 cycles of nested polymerase chain reaction (PTC), 2% agarose gel electrophoresis revealed the anticipated 124-bp PCR amplification product for the coding region of exon 15 of the BRAF gene that spans the T1796A transversion site. BRAF was amplified in sufficient quantity to allow sequence analysis in samples 2, 4, 5, 6, 8, and 9 (solid arrow).
contained the ret/PTC2 rearrangement, 1 (8%) contained the ret/PTC3 rearrangement, 1 (8%) contained both ret/PTC1 and ret/PTC2 rearrangements, and 1 (8%) contained both ret/PTC1 and ret/PTC3 rearrangements (25–32). Of note, the patient with both ret/PTC1 and ret/PTC3 rearrangements was the one patient known to have a history of radiation exposure. Overall, 7 of 12 (58%) contained at least one of the more common ret/PTC rearrangements. The incidence of ret/PTC rearrangements was significantly greater than that of BRAF T1796A (p 0.0012, Fisher’s exact test). Discussion
FIG. 2. BRAF gene sequence analysis. The amplified BRAF products were subjected to sequencing gel analyses using the Applied Biosystems ABI 1377 instruments (Applied Biosystems, Foster City, CA). The resultant sequences are aligned and show the positive control (NPA cell extract), the negative control (WRO cell extract), and one experimental sample (5).
This study reports an analysis of the BRAF gene sequence in thyroid cancers specifically obtained from patients younger than 21 years of age. Analysis of thyroid cancers in this age group is important because the prognosis is more favorable than in adults even when matched for similar stage of disease (1–3). An explanation for this difference could be the presence of a different repertoire of somatic mutations in thyroid cancers from young versus older patients. In support of this hypothesis, we, and others, previously found that ret/PTC rearrangements were significantly more common in young patients and that ras mutations were less common than in older patients (4,7,10,11). In our current study, we found the wild-type BRAF gene sequence and none of the most common BRAF mutation (T1796A transversion) in 20 tumors from young patients, 14 of which were PTC. For PTC, this is significantly less common (0/14, p 0.0015) than for PTC from older adults reported in multiple series in the literature (14–22). When the total number of PTC from their studies is analyzed, 270/699 PTC (38.6%) contained BRAF T1796A.
324 Several clinical features of the 14 PTC in our study are worth noting. First, the mean age of the patients (15.6 0.65 years) is typical for young patients with thyroid cancer (3). The average tumor size (1.4 0.21 cm) is relatively small, suggesting a favorable prognosis (3). Tumors greater than 2 cm in diameter have been shown to have a significantly higher risk of recurrence in this age group (3). The majority of these PTC are DeGroot class 1 or 2, which are also associated with a favorable prognosis (1). Consistent with these lower DeGroot classes and MACIS scores less than 4, none of the PTC recurred with a mean follow-up of 67 11 months (33). If the BRAF T1796A mutation were associated with more aggressive clinical features, its paucity in young patients might help explain the more favorable prognosis for PTC in this age group (1–3). Among the 14 PTC in our study, ret/PTC rearrangements remained the most common (7/12, 58%) compared to mutations in BRAF (0/14) and ras (0/10). This difference was significant when comparing the incidence of BRAF and ret/PTC rearrangements (p 0.0012), but none of the PTC contained either a BRAF T1796A or ras mutation. The one patient with a known history of radiation exposure had both ret/PTC1 and ret/PTC3 rearrangements. The presence of ret/PTC3 is consistent with the finding by Nikiforov et al. (11) that ret/PTC3 is the most frequent type of rearrangement in radiation-induced PTC in children. In previous studies of BRAF mutations in older patients, the BRAF T1796A transversion has only been reported in PTC, and not in other histologic variants of thyroid cancer (14–20). None of the other tumors in our study (4 FTC, 1 MTC, and 1 benign adenoma) were found to harbor the BRAF T1796A mutation. Although our findings are in agreement with these previous studies, the number of FTC, MTC, and benign tumors was small. Also in agreement with several previous studies, we failed to find a BRAF T1796A mutation in any of the PTC that contained a ret/PTC rearrangement (15,17,22). Nor did we find an overlap with ras mutations. In addition, previous studies have reported an association of this common BRAF mutation in adults with classic (15,16) and tall cell histotypes (22) of PTC. Of our 14 PTC, only 5 (36%) were of the classic (typical) type, and 1 (7%) was of the tall cell variant. The low numbers of these associated histotypes may also help explain the paucity of BRAF T1796A mutations detected, if these variants are associated with the mutation in children as well as older adults. Our data are limited by several factors in addition to the small sample size. First, the group did not contain any PTC with distant metastases or any patients under 10 years of age. Studies in adult PTC have shown an association of the BRAF T1796A mutation with more advanced stage of disease (19,20). Second, although we had previously analyzed many of these PTC for ret/PTC rearrangements and ras mutations, we only amplified the more common ras and ret/PTC mutations (ret/PTC1, ret/PTC2, and ret/PTC3) (25–32). It is possible that some of these PTC might contain additional ret/PTC rearrangements, but we lack sufficient material to examine this possibility. Similarly, we only examined these PTC for the most common BRAF mutation (T1796A). Although this particular mutation accounts for the vast majority of BRAF mutations in PTC from older adults, it is possible that PTC from young
PENKO ET AL. patients might harbor a different mutation in BRAF. A final limitation is that the recurrence data used in this study are retrospective and did not include serum thyroglobulin measurements for detection of recurrent disease. For these reasons, the clinical implications of our data must be validated in larger series of more diverse tumors from young patients. In summary, we report a BRAF T1796A mutational analysis in PTC from young patients. Our data show that the BRAF T1796A mutation is uncommon in this age group, while ret/PTC rearrangements occur in a slight majority of PTC. References 1. DeGroot LJ, Kaplan EL, McCormick M, Straus FH 1990 Natural history, treatment, and course of papillary thyroid carcinoma. J Clin Endocrinol Metab 71:414–424. 2. McClellan DR, Francis GL 1996 Thyroid cancer in children, pregnant women, and patients with Graves’ disease. Endocrinol Metab Clin North Am 25:27–48. 3. Welch Dinauer CA, Tuttle RM, Robie DK, McClellan DR, Svec RL, Adair C, Francis GL 1998 Clinical features associated with metastasis and recurrence of differentiated thyroid cancer in children, adolescents and young adults. Clin Endocrinol (Oxf) 49:619–628. 4. Fenton C, Anderson J, Lukes Y, Dinauer CA, Tuttle RM, Francis GL 1999 Ras mutations are uncommon in sporadic thyroid cancer in children and young adults. J Endocrinol Invest 22:781–789. 5. Fenton CL, Patel A, Tuttle RM, Francis GL 2000 Autoantibodies to p53 in sera of patients with autoimmune thyroid disease. Ann Clin Lab Sci 30:179–183. 6. Fenton C, Patel A, Dinauer C, Robie DK, Tuttle RM, Francis GL 2000 The expression of vascular endothelial growth factor and the type 1 vascular endothelial growth factor receptor correlate with the size of papillary thyroid carcinoma in children and young adults. Thyroid 10:349–357. 7. Fenton CL, Lukes Y, Nicholson D, Dinauer CA, Francis GL, Tuttle RM 2000 The ret/PTC mutations are common in sporadic papillary thyroid carcinoma of children and young adults. J Clin Endocrinol Metab 85:1170–1175. 8. Gydee H, O’Neill JT, Patel A, Bauer AJ, Tuttle RM, Francis G 2004 Differentiated thyroid carcinomas from children and adolescents express insulin-like growth factor-1 (IGF-1) and the IGF-1 receptor (IGF-1-R). Cancers with the most intense IGF-1-R expression may be more aggressive. Pediatr Res 55:1–7. 9. Straight A, Patel A, Fenton C, Dinauer C, Tuttle RM, Francis G 2002 Thyroid carcinomas that express telomerase follow a more aggressive clinical course for children and adolescents. J Endocrinol Invest 25:302–308. 10. Bongarzone I, Fugazzola L, Vingeri P, Mariani L, Mondellini P, Pacini F, Basolo F, Pinchera A, Pilotti S, Pierotti MA 1996 Age-related activation of the tyrosine kinase receptor protooncogenes RET and NTRK1 in papillary thyroid carcinoma. J Clin Endocrinol Metab 81:2006–2009. 11. Nikiforov YE, Rowland JM, Bove KE, Monforte-Munoz H, Fagin JA 1997 Distinct pattern of ret oncogene rearrangements in morphological variants of radiation-induced and sporadic thyroid papillary carcinomas in children. Cancer Research 57:1690–1694. 12. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A,
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Address reprint requests to: Gary Francis, M.D., Ph.D. Department of Pediatrics Uniformed Services University of the Health Sciences 4301 Jones Bridge Road Bethesda, MD 20814 E-mail:
[email protected]
This article has been cited by: 1. Gary Francis, Steven G Waguespack. 2011. An individualized approach to the child with thyroid cancer. Expert Review of Endocrinology & Metabolism 6:1, 85-92. [CrossRef] 2. Steven G. Waguespack , Steven I. Sherman , Michelle D. Williams , Gary L. Clayman , Cynthia E. Herzog . 2009. The Successful Use of Sorafenib to Treat Pediatric Papillary Thyroid CarcinomaThe Successful Use of Sorafenib to Treat Pediatric Papillary Thyroid Carcinoma. Thyroid 19:4, 407-412. [Abstract] [PDF] [PDF Plus] 3. M SOBRINHOSIMOES, V MAXIMO, A ROCHA, V TROVISCO, P CASTRO, A PRETO, J LIMA, P SOARES. 2008. Intragenic Mutations in Thyroid Cancer. Endocrinology & Metabolism Clinics of North America 37:2, 333-362. [CrossRef] 4. Catherine A Dinauer, Christopher Breuer, Scott A Rivkees. 2008. Differentiated thyroid cancer in children: diagnosis and management. Current Opinion in Oncology 20:1, 59-65. [CrossRef] 5. C DINAUER, G FRANCIS. 2007. Thyroid Cancer in Children. Endocrinology & Metabolism Clinics of North America 36:3, 779-806. [CrossRef] 6. Heejei Yoon, Huiling He, Rebecca Nagy, Ramana Davuluri, Saul Suster, Daniel Schoenberg, Natalia Pellegata, Albert de la Chapelle. 2007. Identification of a novel noncoding RNA gene, NAMA , that is downregulated in papillary thyroid carcinoma with BRAF mutation and associated with growth arrest. International Journal of Cancer 121:4, 767-775. [CrossRef] 7. Shunichi Yamashita, Vladimir Saenko. 2007. Mechanisms of Disease: molecular genetics of childhood thyroid cancers. Nature Clinical Practice Endocrinology & Metabolism 3:5, 422-429. [CrossRef] 8. Doris Mayr, Astrid Hirschmann, Udo Löhrs, Joachim Diebold. 2006. KRAS and BRAF mutations in ovarian tumors: A comprehensive study of invasive carcinomas, borderline tumors and extraovarian implants. Gynecologic Oncology 103:3, 883-887. [CrossRef] 9. Paola Collini, Franco Mattavelli, Alessandro Pellegrinelli, Marta Barisella, Andrea Ferrari, Maura Massimino. 2006. Papillary Carcinoma of the Thyroid Gland of Childhood and Adolescence: Morphologic Subtypes, Biologic Behavior and Prognosis. The American Journal of Surgical Pathology 30:11, 1420-1426. [CrossRef] 10. Barbara J. Collins , Arthur B. Schneider , Richard A. Prinz , Xiulong Xu . 2006. Low Frequency of BRAF Mutations in Adult Patients with Papillary Thyroid Cancers Following Childhood Radiation ExposureLow Frequency of BRAF Mutations in Adult Patients with Papillary Thyroid Cancers Following Childhood Radiation Exposure. Thyroid 16:1, 61-66. [Abstract] [PDF] [PDF Plus]
