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J Chin Med Assoc • March 2010 • Vol 73 • No 3 113
Introduction
Thyroid cancer is the most common endocrine ma-
lignancy and accounts for ∼1% of all cancers. In the
United States, it was estimated that 37,200 men and
women (10,000 men, 27,200 women) would be diag-
nosed with thyroid cancer in 2009.1The incidence of
thyroid cancer has increased ∼50% since 1973, and it
is the most rapidly increasing cancer among women
and the 2nd most among men. Follicular cell-derived
thyroid cancers are classified into papillary thyroid
cancer (PTC), follicular thyroid cancer (FTC), and
anaplastic thyroid cancer (ATC). PTC is the most
common type and accounts for 85–90% of all thyroid
malignancies.2,3 Differentiated thyroid cancers (DTC)
including PTC and FTC exhibit evidence of follicular
epithelial cell differentiation such as iodine uptake and
organification, and are usually treated successfully by
primary surgical excision, radioiodine therapy, and levo-
thyroxine suppression. The overall 10-year relative
survival rate of DTC is over 90%.1,4 However, up to
35% of patients suffered from disease recurrence dur-
ing a 40-year follow-up, and over 1,600 people in the
United States and 35,000 worldwide die of thyroid
cancer each year. The death rate for thyroid cancer in
the United States is ∼0.5 per 100,000 each year.1,5,6
Thyroid cancer with undifferentiated/aggressive his-
tologic variants, or loss of iodine uptake due to sub-
sequent dedifferentiation, are often inoperable and
exhibit poor response to radioiodine therapy, leading
REVIEW ARTICLE
BRAF Mutation in Papillary Thyroid Carcinoma:
Pathogenic Role and Clinical Implications
Kam-Tsun Tang1,3, Chen-Hsen Lee2,3*
Departments of 1Medical Education and Research, and 2Surgery, Taipei Veterans General Hospital, and
3National Yang-Ming University School of Medicine, Taipei, Taiwan, R.O.C.
Papillary thyroid cancer (PTC) is the most common endocrine malignancy, accounting for 85–90% of all thyroid cancers.
Genetic alternations involving the mitogen-activated protein kinase (MAPK) pathway are frequently demonstrated in PTC,
such as RET/PTC, RAS, and B-type Raf kinase (BRAF) mutations. Over 90% of BRAF mutations are T1799A, resulting in a
BRAFV600E mutation. BRAFV600E is present in ∼50% of PTC and also found in aggressive histologic variants and PTC-
derived anaplastic thyroid cancer, but is rare in follicular variants, and not found in follicular thyroid cancer. The tumori-
genic role of BRAFV600E in the development of PTC was documented in thyroid-targeted BRAFV600E transgenic mice, and
rat thyroid cells overexpressed with BRAFV600E suggested that BRAFV600E is an initiator of tumorigenesis and is required
for tumor progression in PTC. Most clinical studies have demonstrated an association of BRAFV600E mutation with aggres-
sive clinicopathologic characteristics and high tumor recurrence, although the results are controversial. The association
is also observed in patients with papillary thyroid microcarcinomas and low-risk PTC. As a highly specific and unique
mutation in PTC, testing for BRAFV600E in fine-needle aspiration specimens has been shown to refine the diagnostic accu-
racy of PTC in indeterminate cytology. Preoperative BRAFV600E analysis in low-risk patients may provide important value
for prognostication, and these patients might benefit from receiving more intensive management and frequent follow-up.
BRAF-targeted therapies have been developed to treat various human cancers including advanced thyroid cancers.
Preclinical results are encouraging, but the anticancer effects of clinical trials are disappointing. Studies of multi-kinase
inhibitors and/or combination with other regimens are underway in the treatment of advanced thyroid cancers. In this
article, we review the pathogenesis of PTC, and the clinical implications of BRAFV600E mutation in the diagnosis, prognosis
and potential targeted therapeutic strategies for thyroid cancers. [J Chin Med Assoc 2010;73(3):113–128]
Key Words: BRAF mutation, fine-needle aspiration cytology, papillary thyroid cancer
© 2010 Elsevier Taiwan LLC and the Chinese Medical Association. All rights reserved.
*Correspondence to: Dr Chen-Hsen Lee, Department of Surgery, Taipei Veterans General Hospital,
201, Section 2, Shih-Pai Road, Taipei 112, Taiwan, R.O.C.
E-mail: chlee@vghtpe.gov.tw ●Received: September 1, 2009 ●Accepted: December 11, 2009
to a high recurrence rate and unfavorable prognosis.
Traditional chemotherapy has low response rates, and
long-term efficacy is unsatisfactory.7,8 There is currently
no effective treatment for these patients.
Conventional treatment strategies for DTC are
based on various staging systems (such as the TNM
system), which are designed according to a patient’s
clinicopathological characteristics, allowing patients at
low risk to undergo less intensive therapy and less fre-
quent follow-up than patients at high risk.9,10 Unfor-
tunately, 15% of tumor recurrence after a median of
11 years’ follow-up, and over 10% of cancer deaths were
initially classified as low risk.11,12 Therefore, more
accurate risk stratification and effective treatment for
advanced thyroid cancer are important to reduce tumor
recurrence and mortality.
Recently, significant progress has been made in
the understanding of the B-type Raf kinase (BRAF)
mutation and the mitogen-activated protein kinase
(MAPK) pathway in the tumorigenesis of human
cancers.13,14 Thyroid cancers, particularly PTCs, are
frequently found to have genetic alterations. A thymi-
dine-to-adenosine transversion at exon 15 nucleotide
1799 (T1799A) of the BRAF gene, resulting in the
replacement of valine with glutamic acid at position
600 (BRAFV600E), is the most prevalent mutation in
PTC.14 As a result, BRAF mutation has recently been
the subject of intensive study to investigate its tumori-
genic role and its clinical implications.15–17 In this arti-
cle, we review the mechanism of BRAFV600E mutation
and MAPK signal transduction pathway in the patho-
genesis of PTC, the clinical implications of BRAFV600E
mutation in preoperative diagnosis and prognostic
stratification, and recent advances in the use of the
BRAFV600E mutation as a potential target of thera-
peutic strategies for thyroid cancers.
MAPK Signal Transduction Pathway
The MAPK pathway is an intracellular signal transduc-
tion pathway that is required for maintaining cellular
activities such as cell growth, proliferation, differenti-
ation, and apoptosis responsive to cell surface receptor
tyrosine kinase (RTK) stimulation.18,19 This pathway
relays the extracellular signals from various growth fac-
tors, hormones and cytokines to the nucleus through
the activation of signal cascades. As shown in Figure 1,
the binding of the ligands to their surface RTKs lead
to the dimerization of receptors and tyrosine residue
autophosphorylation. The activated receptors, through
adaptor proteins, activate RAS kinase. Then, RAS ki-
nase activates the phosphorylation of Raf kinases, which
in turn activate the dual-specificity protein kinases:
MAP kinase kinases (MAPKK; also known as MAP/
extracellular signal-regulated kinase, MEK) 1 and 2.
MEK1/2 phosphorylate and activate extracellular
signal-regulated kinases (ERK) 1 and 2. ERK1/2
regulate various transcription factors leading to gene
expression.
RAS kinase belongs to a family of small G-proteins
(KRAS, HRAS, NRAS) located on the inner surface
of cell membranes and function as a GTPase, switching
between active GTP-bound form and inactive GDP-
bound form. Cycling between GDP/GTP is regulated
by adaptors (e.g. growth-factor-receptor bound-2;
GRB2) and guanine nucleotide exchange factors (e.g.
son of sevenless; SOS). These proteins facilitate the RAS
active GTP-bound form formation, and RAS GTPase
catalyzes GTP hydrolysis, resulting in return to its
inactive GDP-bound form.20–22 Raf kinase was the first
identified and most characterized downstream cyto-
solic effector of RAS.23,24 It belongs to a family of
serine/threonine kinases (A-Raf, B-Raf, and C-Raf or
Raf-1), and all isoforms share 3 common conserved
regions—CR1 (RAS-binding domain and cysteine-rich
domain), CR2 (N-terminal regulatory domain), and
CR3 (C-terminal kinase catalytic domain)—as well as
several regulatory phosphorylation sites (Figure 2).25,26
The activation of Raf is a complex process taking
place at the membrane, where Raf undergoes multi-
site phosphorylation and protein interactions before
being rendered active.17,27–29 The binding of RAS to
RAS-binding domain (RBD) of Raf is regulated by di-
meric adaptors such as 14-3-3 proteins that are bound
to the phosphorylated proteins. BRAF contains con-
served phosphorylation sites at S365 and S729 that
are phosphorylated in the inactive state. Dimeric pro-
teins 14-3-3 bind to these phosphorylated sites, creat-
ing a conformation that interferes with the binding of
RAS to RBD.29 The activation of BRAF is initiated with
the recruitment of the inactive BRAF to the inner mem-
brane, where the N-terminal 14-3-3 binding site is
dephosphorylated to dissociate 14-3-3 proteins, and
followed by the phosphorylation of T599 and S602
at the activation segment (Figures 1 and 2).26 Inactive
BRAF exhibits a characteristic bilobar structure by
forming a hydrophobic interaction between residues
G596-V600 of the activation loop and residues G464-
V471 of the P loop (ATP binding sites), resulting in a
conformation that the catalytic residues cannot bind
to ATP. It is postulated that phosphorylation of T599
disrupts the hydrophobic interaction between these 2
loops, resulting in the binding of ATP and the activation
of BRAF.17 The basic mechanisms in the activation of
the 3 Raf isoforms are similar, except that A-Raf and
J Chin Med Assoc • March 2010 • Vol 73 • No 3
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K.T. Tang, C.H. Lee
J Chin Med Assoc • March 2010 • Vol 73 • No 3 115
BRAF mutation in papillary thyroid cancer
Figure 1. MAPK signal transduction pathway. In normal cells, ligands’ (L) binding of the extracellular domain of their membrane tyrosine
kinase receptors (RTK) triggers dimerization of the receptor autophosphorylation of tyrosine residues in the intracellular TK domain
activation of adaptor proteins/guanine nucleotide exchange factors (e.g. Grb2 and SOS) inactive GDP-bound form of RAS active
GTP-bound form BRAF recruitment to the membrane BRAF phosphorylation MEK phosphorylation ERK phosphorylation
nuclear translocation ERK-induced phosphorylation of nuclear transcription factors gene expression proliferation, survival,
senescence and differentiation. Raf–MEK–ERK kinase cascade is scaffolded by kinase suppressor of Ras (KSR). Protein phosphatase
2A (PP2A) is involved in the dephosphorylation of inhibitory sites of Raf kinases during their activation process.
C-Raf require additional kinases (e.g. SRC) and more
phosphorylation steps at the N-terminal side of CR3
(N-region). Negative charge within the N-region is
essential for Raf kinase activation. BRAF is constitu-
tively phosphorylated at S446, and a regulatory tyro-
sine residue is occupied by an aspartic acid at D449,
such that its constant negative charge will act like the
phosphorylation at this site.30 As a result, BRAF is
activation-ready and only requires the RAS-mediated
membrane recruitment of BRAF.
MEK1/2 are the physiological downstream effec-
tors of BRAF. BRAF has the highest basal kinase activ-
ity and is the strongest Raf activator of downstream
MEK.31,32 Activated BRAF induces phosphorylation at
2 serine residues, S217 and S221, within the activation
segment of MEK. Downstream of MEK are ERK1/2,
which are activated by phosphorylation at the T202
and Y204 residues of ERK.32 Phosphorylation of ERK
activates substrates located in the nucleus and cyto-
plasm. The majority of ERK substrates are nuclear
proteins, and the nuclear translocation of ERK phos-
phorylates various transcription factors, which in turn
regulate gene expression.33–35 Meticulous regulation
of ERK is crucial to maintain biological homeostasis
responsive to various extracellular signals. For example,
hyperactivation of the ERK pathway can induce cell
cycle arrest and senecense.36,37 In contrast, aberrant
activation of the pathway may induce tumor transfor-
mation (Figure 3).13,17 The kinetics and amplitude of
ERK signaling induced by different ligands can regulate
biological programs differentially, such as proliferation,
differentiation or apoptosis. The regulation of cellular
responses is a complicated mechanism that may in-
volve various substrates at different levels of the cascade,
such as scaffold proteins and feedback inhibitors (Figure
4).38 Apparently, tumors prefer ERK activities’ program-
ming for proliferation and survival.
Genetic Alterations in PTC
Aberrant activation of the MAPK pathway is frequently
found in human cancers (Figure 3). The consistent
finding of RAS and BRAF mutations in similar cancer
J Chin Med Assoc • March 2010 • Vol 73 • No 3
116
K.T. Tang, C.H. Lee
Figure 2. Structure of the Raf proteins. The Raf isoforms, A-Raf, B-Raf and C-Raf, share 3 conserved regions: CR1, CR2 and CR3. The
amino acids shown refer to known phosphorylation sites. CR1 contains the RAS-binding domain (RBD) and the cysteine-rich domain
(CRD), which are both required for membrane recruitment. CR2 and C-terminal contain the 14-3-3 binding sites. CR3 contains the cat-
alytic domain (including the activation segment). The negative-charge regulatory region (N-region) contains residue C-Raf (Y341), which
is conserved in A-Raf (Y302) but is replaced by aspartic acid at D449 in BRAF. S338 of C-Raf is conserved in all RAF proteins (S299 in
A-Raf and S446 in BRAF), but it is constitutively phosphorylated in BRAF (star shape). The catalytic domain contains the 2 activation-
segment phosphorylation sites C-Raf (T491 and S494), which are conserved in A-Raf (T452 and T455) and BRAF (T599 and S602).
Figure 3. Activation of MAPK signaling pathway by RAS, RET/PTC and BRAFV600E mutations. The mechanism is similar to the physiolog-
ical condition described in Figure 1, except that the signal is generated through RAS, RET/PTC and BRAFV600E mutations. The activation
of the MAPK pathway becomes constitutive to induce cell transformation.
types, and the mutation rarely involving more than 1
component of the pathway (mutually exclusive), sug-
gest that the constitutive activation of these mutants
in the pathway might be the pathogenesis of tumor
formation. They also imply that a single gene muta-
tion in the pathway is sufficient to induce cell trans-
formation.13,38–40 About 70% of patients with PTC are
found to have genetic alterations related to the MAPK
pathway, such as RET/PTC rearrangement, and RAS
and BRAF mutations, indicating that the MAPK sig-
naling pathway plays an important role in the patho-
genesis of PTC.40,41
RET protooncogene is a tyrosine kinase receptor
that is highly expressed in parafollicular C cells, but its
expression is low in thyroid follicular cells. The RET
gene can be activated in follicular cells by chromosomal
rearrangements, linking the promoter and N-terminal
domains of unrelated genes to the tyrosine kinase do-
main of the RET gene. The aberrant production of
different chimeric forms of the receptor is known as
RET/PTC. More than 11 types of RET/PTCs have
been reported, and RET/PTC1 and RET/PTC3 are
the most common rearrangements seen in PTCs, prob-
ably because RET/PTC1 and RET/PTC3 are intra-
chromosomal (chromosome 10q) paracentric inversions
by fusion of the 3’ portion of RET to the 5’ portion of
the H4 (D10S170) and NCOA4 (ELE1) genes, res-
pectively.42,43 The other RET/PTCs are rearranged by
interchromosomal translocations. As shown in Figure 3,
RET/PTCs constitutively activate the RAS/BRAF/
MAPK pathway,44–46 and the transformation of thyroid
cells could be induced by overexpression of either
RET/PTC1 or RET/PTC3 in cultured thyroid fol-
licular cells and transgenic mice. Silencing the BRAF in
RET/PTC-transformed follicular cells reversed the
tumorigenic effect of RET/PTC, confirming that sig-
naling along the BRAF-MAPK pathway is required for
its tumorigenesis.43,44,47–49 The prevalence of RET/
PTC rearrangements in PTC is varied in different geo-
graphic regions and account for ∼20% of PTC, which
is particularly common in young patients and individ-
uals with history of previous radiation exposure.50
RAS proteins are plasma membrane GTPases
switching between active GTP-bound form and inac-
tive GDP-bound form to activate downstream effector
pathways.20–22 Point mutations of RAS, either with
increased affinity for GTP (at codons 12 and 13) or
with decreased autocatalytic GTPase function (at codon
61), will withhold RAS at the active GTP-bound con-
dition leading to the constitutive activation of the
J Chin Med Assoc • March 2010 • Vol 73 • No 3 117
BRAF mutation in papillary thyroid cancer
Figure 4. Proposed model of feedback inhibition in tumor cells with RET/PTC or RTKs and with BRAFV600E. RET/PTC or RTKs activate
the tumor cell feedback mechanism and inhibit the MAPK pathway at multiple levels. Feedback mechanism or mediators such as dual-
specificity phosphatases (DUSPs) downregulate both RAF/MEK activation and ERK phosphorylation in RTK cells. BRAFV600E is constitu-
tively active and insusceptible to negative feedback.
MAPK pathway (Figure 3). Mutations of all family
members of RAS genes have been reported in differ-
ent types of thyroid follicular cell-derived tumors,
particularly in follicular adenomas and carcinomas.
However, it rarely occurs in PTCs (∼10%) and is almost
solely found in follicular variants.51–53
BRAF is highly expressed in hematopoietic cells,
neurons, testis and thyroid follicular cells.54,55 Contrary
to the extremely rare mutation found in A-Raf and C-
Raf, BRAF is the most common Raf mutation and is the
second most common somatic mutation in all human
cancers. The BRAF mutation is frequently detected in
malignant melanomas, and in colon, ovarian and thy-
roid carcinomas.13,14,17 Although >45 BRAF mutations
have been identified in human cancers, about 90% of
BRAF mutations are T A transversion in exon 15 at
nucleotide 1799 (T1799A) leading to a valine glu-
tamic acid replacement at position 600 (BRAFV600E).
Except for the rare BRAF mutations (K601E, AKAP9-
BRAF, V600E +K601del, V599ins, V600D +
FGLAT601-605ins) reported in thyroid cancer, over
90% of BRAF mutation in PTCs are BRAFV600E.13,56–60
The prevalence of BRAFV600E in PTCs varies from
29% to 83%, and is ∼60% in classic PTC, ∼77% in tall-
cell variant, and ∼25% in PTC-derived ATC, but it is
rare (0–12%) in follicular-variant PTC and is not
found in FTC.15 An in vitro study showed that the
replacement of a negative-charge residue glutamate at
V600 adjacent to T599 induces an effect similar to
phosphorylation at T599 and S602, which disrupts
the hydrophobic interaction between the P loop and
the activation loop.17 The kinase activity of BRAFV600E
is 460-fold higher than the wild-type BRAF, and this
active conformation can constitutively activate its down-
stream effectors to transform normal cells or induce
cancer proliferation without the need of RAS for acti-
vation.13,17
BRAFV600E mutation is frequently detected in pap-
illary thyroid microcarcinomas (PTMC), suggesting
that the mutation is an early event during PTC devel-
opment.61–65 The tumorigenic role of BRAFV600E in
the development of PTC was documented in thyroid-
targeted BRAFV600E transgenic mice, and tumors devel-
oped from these transgenic mice were found to
progressively transform into poorly differentiated can-
cers with aggressive characteristics.66 In vitro,
BRAFV600E-overexpressed rat thyroid cells grown on
MatrigelTM showed an increase in migration of thyroid
cells. It has also been reported to be associated with
the upregulation of metalloproteinases (MMPs), par-
ticularly matrix MMP3, MMP9 and MMP13 genes,
which are related to tumor invasion.45,67 The prolifer-
ation of BRAFV600E-harbored or transfected cell lines
could be inhibited by MAPK pathway inhibitors or
siRNA specific BRAF knockdown.68,69 The above ob-
servations suggest that BRAFV600E is an initiator of
tumorigenesis through the MAPK pathway, and is
required for the progression of PTC.70 Both RET/
PTC and BRAFV600E mutations can constitutively acti-
vate MAPK pathways, resulting in follicular cell transfor-
mation. However, several studies have shown that RET/
PTC-expressed rat thyroid cells induce a weaker tumor-
igenic effect than BRAFV600E mutation.71 Conditional
expression of BRAFV600E in thyroid cells markedly
increased the MatrigelTM invasion of the transformed
thyroid cells, which is more invasive than RET/PTC
expressed cells.67 Furthermore, microarray studies have
also shown that human PTCs harbor BRAFV600E and
RAS mutation, and RET/PTCs exhibit different gene
expression profiles, suggesting that different mutants
may affect the pathway outcomes differently. Among
them, BRAFV600E mutation is the most potent activa-
tor in the stimulation of MAPK pathway output.72
Interestingly, the high kinase activity of BRAFV600E
mutation seems to be not effectively translated to ERK
activity; it only increased ERK activity 2- to 4.6-fold,
suggesting the existence of regulatory mechanisms in
the controlling of the signal output.17,27 Several pos-
sible feedback mechanisms have been reported to
inhibit the ERK pathway output.73–76 ERK stimulates
gene expression of feedback regulators (e.g. dual-
specificity phosphatases; Sprouty) to inhibit RAS ac-
tivating proteins (e.g. SOS), and also ERK’s own
activities (Figure 4).73,74 Phosphorylated-ERK has been
demonstrated to phosphorylate Raf directly to induce
hyperphosphorylation of Raf, leading to conforma-
tional changes that might interfere with the binding
of Raf to RAS, MEK or scaffold proteins.77 The phys-
iological role of these feedback loops is unclear, and it
may aim to prevent cell cycle arrest and senescence
due to hyperactivation of the ERK pathway. The con-
stitutive activation of the MAPK signal pathway by
RAS and BRAFV600E mutations was also found to
induce feedback downregulation similar to the physi-
ological condition.78 Conceivably, these oncoproteins
might take steps to minimize the effects of feedback
inhibition by either insensitivity of the mutant protein
to normal negative feedback or directly affecting the
mediators of the feedback mechanism. Recent investi-
gation demonstrated that feedback inhibition of
Raf/MEK signaling was found to downregulate ERK
output in RTK cells, but not in BRAFV600E cells
(Figure 4). The evasion of the feedback mechanism in
BRAFV600E cells was evidenced by the increase in
transcriptional output and MEK/ERK-dependent
transformation. This phenomenon may partly explain
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K.T. Tang, C.H. Lee
the stronger tumorigenic effect observed in the
BRAFV600E-expressed cells than in the RET/PTC-
expressed cells.78
Tumorigenesis is a complex process involving mul-
tiple signaling networks instead of a single linear uni-
directional cascade of the MAPK pathway. The high
prevalence of BRAFV600E mutation in tall-cell variant
and PTC-derived ATC suggests that BRAFV600E muta-
tions may play a role in the progression of PTC to
more aggressive thyroid carcinomas.15 Recent studies
have shown that induction of BRAFV600E expression
in rat thyroid cells facilitated the acquisition of sec-
ondary genetic events through induction of genomic
instability, but not in RET/PTC-expressed cells.79
Genetic alternations in the PI3K/Akt pathway and
PTEN have also been found in thyroid cancers such
as ATC and metastatic tumors from radioactive iodine-
refractory (RAIR) PTC, particularly in the later stages
of cancer progression.80 Aberrant activation of the
PI3K/Akt pathway is often coexistent with BRAFV600E
in ATC and RAIR tumors, suggesting that the genetic
instability induced by the primary BRAFV600E mutation
in PTC may facilitate the secondary genetic alterna-
tions involving the PI3K/Akt pathway. The secondary
mutation might lead to the progression of DTC to the
more aggressive thyroid cancer, and dedifferentiation
of the cancer cells.81 This hypothesis is supported by
observations that tumors in thyroid-targeted BRAFV600E
transgenic mice progressed to more aggressive phe-
notype, and BRAFV600E mutation is associated with
advanced patient age and not frequently detected in
childhood PTCs.15,66,82 Further investigations of the
feedback and alternation pathways are important in
understanding the mechanisms involved in the tu-
morigenesis of PTC, which might provide valuable
information regarding clinical implications.
Clinical Implications Associated With
BRAFV600E Mutation in PTC
Recently, BRAFV600E has taken center stage due to the
findings that it may be associated with tumorigenesis
and aggressiveness. Many studies have been done to
investigate the clinicopathologic characteristics, and
the potential utility of BRAFV600E mutation on the
diagnostic, prognostic and therapeutic aspects of PTC.
Unlike the highly consistent results obtained from
in vitro studies, the current clinical data show con-
troversial results regarding BRAFV600E mutation as a
genetic prognostic marker of PTC.
Environmental and various predisposing factors have
been reported to increase the risk of thyroid cancer,
such as radiation exposure, dietary iodine, genetics
and life style.83 Radiation-associated PTCs are usually
associated with RET/PTC and to a lesser extent with
NTRK1, but not with BRAFV600E mutation.84–86
Ciampi et al57 reported that AKAP9-BRAF fusion
was more commonly found in radiation-induced PTC
than sporadic PTC. AKAP9-BRAF is caused by para-
centric inversion of chromosome 7q, resulting in an in-
frame fusion between exons 1–8 of the AKAP9 gene
and exons 9–10 of BRAF.87 This implies that radiation-
induced PTCs are likely caused by the chromosome-
paracentric inversion linked to constitutive activators,
while sporadic PTCs are predominantly activated by
point mutations on the effector kinases of the MAPK
pathway. In Italy, a higher incidence of thyroid cancer
and BRAFV600E-PTC were found in people residing
in Eastern Sicily, including the volcanic area of Etna
(45.9%), than people in Western Sicily (22.7%). Iodine
deficiency was not found to be the cause of the differ-
ence, and the unidentified carcinogens were suspected
to be the volcanic soil, water or atmosphere.88 A large
Chinese epidemiological study of 1,032 conventional
PTCs demonstrated that cities with high iodine con-
tent had significantly higher incidence of BRAFV600E
mutation (69%) than cities with normal iodine content
(53%). The results suggest that high iodine intake is a
risk factor for BRAFV600E mutation and may therefore
be a risk factor for PTC development.89
Over 30 studies on the relationship between
BRAFV600E and the clinicopathological characteristics
in PTC have been reported worldwide. The majority
of them suggested that BRAFV600E mutation was
associated with advanced disease stages and aggressive
phenotype, while others did not find this association;
the results remain controversial to date. Several stud-
ies reported a significant association of BRAFV600E
mutation with high-risk clinicopathological character-
istics such as older age,65,90–96 male sex,97,98 tumor
size,88,98–101 and aggressive subtype.63,65,92,94,102–104
Many studies found that extrathyroidal invasion, lymph
node metastasis, and advanced stages III/VI are the 3
most common risk factors consistently associated with
BRAFV600E mutation.63,65,87,88,90–96,98–100,102,105,106
Oler et al60 and Vasko et al107 observed that BRAFV600E
mutation in lymph node metastasis was occasionally
not found in their primary lesion, suggesting that tu-
mor cells that acquire the mutation de novo are proba-
bly prompted to metastasis. Rodolico et al95 further
demonstrated that metastatic lymph nodes harboring
the BRAFV600E mutation were larger in size and had a
higher prevalence of extracapsular invasion than those
without the mutation. However, other studies on
paired primary and lymph node metastatic lesions did
J Chin Med Assoc • March 2010 • Vol 73 • No 3 119
BRAF mutation in papillary thyroid cancer
not find discordant mutation in most of the lesion
pairs, indicating that the acquisition of BRAFV600E
mutations are not a requirement in the progression
from localized to metastatic PTC.92,105,108 In a large
Italian cohort study, Lupi et al63 found that BRAFV600E
mutation was associated with the absence of tumor
capsule, particularly in follicular- and micro-PTC vari-
ants, but not in conventional variant. Two meta-
analyses also reported an association of BRAFV600E
mutation with extrathyroidal invasion, aggressive his-
totype and advanced disease stages, but not with age,
sex, or tumor size, and the association of BRAFV600E
mutation with lymph node metastasis is not a uniform
finding.15,109 In fact, a recent large Chinese cohort
study investigating the association of iodine intake
with BRAFV600E mutation in different cities demon-
strated that overall results of extrathyroidal invasion,
lymph node metastasis and advanced disease stages were
significantly associated with BRAFV600E mutation, but
the association with extrathyroidal invasion and lymph
node metastasis were not seen in all cities when ana-
lyzed city by city.89
Clinicopathological characteristics and staging sys-
tems are designed to predict tumor recurrence and
disease prognosis.9Three studies, including a multi-
center study of 219 patients, an American study of
245 conventional PTC cases, and an Italian study of
102 patients, demonstrated that BRAFV600E mutation
was associated with aggressive clinicopathological fea-
tures, and was also an independent predictor of tumor
recurrence after a median of 15 months, 6 years, and
15 years of follow-up, respectively.92,100,106 Kim et al98
reported that BRAFV600E mutation was associated with
tumor recurrence in 203 conventional PTC follow-ups
for a median of 7.3 years, but was not an independent
predictor after adjustment for clinicopathological prog-
nostic factors. More importantly, the association was
also observed in patients with low disease stages I/II,
and conventional PTC.92,98,106 Another finding from
a study of 54 recurrent PTCs showed that 77.8% of
tumors were found to harbor BRAFV600E mutation and
9.3% had both BRAF and RET/PTC mutations.110
The results give further support to the premise that
secondary mutations may cause tumors to progress to
a more aggressive status. In contrast to the studies
above, in a recent large Japanese cohort study of 631
patients with PTC who were followed-up for a median
of 83 months, neither clinicopathological character-
istics nor tumor recurrence was associated with
BRAFV600E mutation.111 A number of studies from
various ethnic groups and geographic regions also did
not find any association of BRAFV600E mutation with
the aggressive clinicopathological features.62,64,112–119
The conflicting results of these studies might be due
to variations in the study populations in terms of size,
age distribution, histological variants, genetic factors,
environmental factors, disease stages at the time of
initial diagnosis, and the methods or criteria used.
Some studies demonstrated that BRAFV600E-
mutated PTC is associated with high recurrence rate,
and a decrease in radioiodine uptake in the recurrent
tumor.102,106 These observations were supported by a
recent study of patients with RAIR-differentiated PTC,
in which 62% of patients were found to be BRAFV600E-
positive, and 54% were 18F-fluorodeoxyglucose pos-
itron emission tomography (18-FDG-PET)-positive.
Interestingly, all of the 18-FDG-PET-positive patients
were found to be BRAFV600E-positive.80 In the process
of thyroid hormone synthesis, inorganic iodine is
actively transported into the thyroid cells via a basal
membrane protein—sodium iodide symporter (NIS).
Iodide is, in turn, transported into the follicle via an
apical protein—pendrin, where iodide is oxidized by
thyroid peroxidase (TPO) and incorporated into thy-
roglobulin in the synthesis of thyroid hormone. This
process is regulated by thyroid-stimulating hormone
(TSH) through the binding of the membranous TSH
receptor (TSHR). Recent immunohistochemistry study
showed that tumor tissues with BRAFV600E mutation
had lower NIS expression, and failure of NIS targeted
to the membrane when compared with PTC without
the mutation.102 BRAFV600E mutation was also found
to be associated with a decrease in gene expression of
TSHR, TPO, NIS, thyroglobulin and pendrin in pri-
mary or recurrent tumors.72,99,102,113,120,121 Instead
of iodide-metabolizing gene silencing, glucose trans-
porter-1 (GLUT-1) expression was found to be in-
creased in PTC, which was significantly higher in
tumors with BRAFV600E mutation than wild-type.120
These data support the biological basis for the clinical
use of 18-FDG-PET to detect recurrent/metastatic
lesions in patients with RAIR PTC. Recently, Romei
et al119 reported that there was neither association of
BRAFV600E mutation with clinicopathological charac-
teristics nor with GLUT-1/3 expression in PTC, but
there was consistently lower expressions of NIS and
TPO in BRAFV600E-mutated PTC; the lower expres-
sions of NIS and TPO were not seen in PTC with
RET/PTC rearrangement. A recent Brazilian study
also reported that decreased NIS gene expression was
found in conventional PTC and PTMC harboring
BRAFV600E mutation.99 In vitro, conditional BRAFV600E
expression in rat thyroid cell lines suppressed iodide-
metabolizing genes. Inhibition of MAPK pathway or
silencing BRAF using inhibitors or siRNA restored the
expression of the iodide-metabolizing genes.79,102,122
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K.T. Tang, C.H. Lee
The results indicate that the MAPK pathway plays an
important role in the regulation of iodide-metabolizing
genes, and several studies further demonstrated that
the MAPK pathway promotes expression of DNA
methyltransferase, which silences these genes through
promoter methylation.122–124
The current treatment strategies for PTC allow
patients at low risk to undergo less intensive postop-
erative adjunctive therapy and less frequent follow-up
than patients at high risk, whereas patients in advanced
stages, with aggressive histotype, or at high risk are
managed by more aggressive therapy such as total/
near total thyroidectomy, lymph node dissection, adju-
vant radioiodine ablation and thyroid hormone sup-
pression therapy.10,125,126 For patients at high risk, the
preoperative diagnosis of BRAFV600E mutation seems
to have little additional value in the current treatment
and follow-up protocols. However, the majority of
patients with DTC are classified as low risk, and many
of them suffer from tumor recurrence in later years.
Testing for BRAFV600E mutation might be helpful in
tailoring the therapeutic strategies for these patients,
in case BRAFV600E mutation is proven to be a marker
of poor prognosis.
The incidence of PTMC (tumor size <1 cm in
diameter) has dramatically increased since the intro-
duction of high-resolution ultrasound-guided fine-
needle aspiration (FNA) biopsy for patients with
nodular thyroid disease. The prevalence of PTMC has
increased approximately 2- to 4-fold in various coun-
tries during the last 2 decades, and the rapid rise of
PTC in recent years is mainly due to the increased rate
of diagnosis of PTMC.127–130 PTMC accounts for ap-
proximately a quarter of thyroid cancers.131 It is gen-
erally considered to be a low-risk cancer, and most cases
are classified as stages I/II. Recommended treatment
for these small low-risk tumors, in the absence of known
risk factors or lymph node metastasis, is lobectomy
with or without isthmectomy.10,125 However, multi-
focality, extrathyroidal extension, and lymph node me-
tastasis are often reported in PTMC, with incidences
of 7.1–56.8%, 2–62.1%, and 0–64%, respectively.131
Several studies have compared the clinical and histo-
logic characteristics between PTC and incidental or
nonincidental PTMC, and found that the prevalence
of multifocality, extrathyroidal extension and lymph
node metastasis are similar in PTMC and PTC. The
aggressive phenotypes are more frequently found in
nonincidental PTMC and PTMC with size larger than
5 mm and 8 mm, respectively.132–135 BRAFV600E muta-
tion is also the most common genetic alteration in
PTMC, accounting for 17–65.6%, with incidence lower
than that of PTC in general.61–65,88,136 Recently, several
studies have investigated the relationship between clin-
icopathological characteristics and BRAFV600E-mutated
PTMC. Park et al136 reported a surprisingly high preva-
lence of extrathyroidal invasion (52.2%) and lymph
node metastasis (32.9%) in PTMC; the frequency of
BRAFV600E mutation and the recurrence rate of PTMC
were similar to those of PTC. Lupi et al63 and Frasca
et al88 observed that BRAFV600E-PTMC was associ-
ated with extrathyroidal extension and advanced dis-
ease stages. Their findings are consistent with the recent
report from Lee et al137 that more BRAFV600E mutation
was detected in PTMC with advanced disease stages,
extrathyroidal extension, and nodal metastasis than in
those without these aggressive clinicopathological char-
acteristics. Ugolini et al61 reported an association of
BRAFV600E mutation with the lack of tumor capsule in
PTMC. Rodolico et al95 further found that BRAFV600E
mutation was associated with lymph node metastases,
a wider diameter of the largest metastatic area, a higher
number of involved lymph nodes, and a higher percent-
age of metastatic lesions with extracapsular extension
in PTMC. Together, the clinicopathological charac-
teristics of BRAFV600E-PTMC seem to be no different
from those of its larger counterpart, and BRAFV600E-
PTMCs exhibit signs of greater aggressiveness and
higher recurrence rate than wild-type. Most patients
with PTMC are classified as stages I/II. According to
current treatment strategies, these patients might re-
ceive “inadequate” treatment and less frequent follow-
up. Thus, some investigators have suggested evaluating
BRAFV600E mutation in these patients preoperatively,
and treating patients with positive BRAFV600E muta-
tion more aggressively.
Use of BRAF Mutation for
Preoperative Diagnosis
Preoperative evaluation of nodular goiter is based on
FNA cytology to select patients for surgical treatment
or medical follow-up.138 As BRAFV600E mutation and
RET/PTC rearrangements are exclusively found in
PTC, examining these markers in DNA specimens
obtained from FNA can make a diagnosis in most
PTCs. Salvatore et al139 detected 38% and 18% of
BRAFV600E and RET/PTC in FNA samples, respec-
tively. The identification of BRAFV600E mutation and
RET/PTC refined the diagnosis of PTC in 5 of 15
samples that were considered either indeterminate or
insufficient at cytology. Using FNA BRAFV600E analy-
sis, Cohen et al140 confirmed the BRAFV600E mutation
in 72% of carcinomas within the malignant group,
and established the diagnosis of PTC in 16% of the
J Chin Med Assoc • March 2010 • Vol 73 • No 3 121
BRAF mutation in papillary thyroid cancer
indeterminate group. No BRAFV600E mutation was de-
tected in the benign group. Two recent studies from
Marchetti et al141 and Zatelli et al142 demonstrated
that combining traditional cytology and molecular
analysis of BRAFV600E mutation on FNA specimens
improved the diagnostic accuracy of PTCs from 62.3%
to 82.2% and from 77.3% to 86.7%, respectively. How-
ever, BRAFV600E mutation is only positive in ∼50% of
PTCs, and negative results cannot exclude malig-
nancy. Therefore, the sensitivity of BRAFV600E muta-
tion analysis for PTC diagnosis is limited, although the
specificity is high. Most of the indeterminate speci-
mens are follicular neoplasm and follicular variant of
PTC, which are rarely found to harbor BRAFV600E
mutation. Inadequate FNA may lead to insufficient
tumor DNA recovery from the nucleic acid prepara-
tions, which might lead to false-negative results. In
fact, traditional FNA cytology by expert pathologists
can provide reliable information on PTCs with an
overall accuracy >90%.138 Therefore, the value of rou-
tine BRAFV600E mutation analysis for PTC diagnosis in
FNA is marginal. It is more reasonable to reserve the
test for patients with indeterminate/inadequate FNA
cytology, which may improve the diagnostic yield in
these patients.
A recent study reported that RAS and PAX8/PPAR
gene analysis in addition to BRAFV600E and RET/PTC
analysis in FNA specimens enhanced the diagnostic
accuracy of FNA cytology, particularly in indetermi-
nate cytology. In the study, 97% of nodules with posi-
tive mutations were ultimately found to be malignant,
suggesting that additional RAS and PAX8/PPAR analy-
sis improved the diagnostic accuracy of indeterminate
cytology which are predominantly follicular neoplasm
and follicular variant of PTC.143 Xing et al144 investi-
gated the utility of BRAFV600E mutation analysis of
FNA specimens for preoperative risk stratification in
PTC. Their results showed a significant association of
BRAFV600E mutation with poor clinicopathological
outcomes, and BRAFV600E mutation predicted extra-
thyroidal extension, thyroid capsular invasion, and
lymph node metastasis. More importantly, 36% of
PTCs with BRAFV600E mutation were found to have
tumor persistence/recurrence, compared with 12% of
PTCs without BRAFV600E mutation, after a median of
3 years’ follow-up (odds ratio, 4.16). The positive and
negative predictive values for the test to predict tumor
persistence/recurrence were 36% and 88% for all PTCs,
and 34% and 92% for conventional PTCs, respec-
tively.144 Preoperative FNA for BRAFV600E diagnosis
seems to be helpful for tailoring the treatment strate-
gies for PTC with low grade and for PTMC. However,
further investigations in large randomized, controlled,
prospective trials are necessary to confirm the role of
BRAFV600E mutation as a clinically useful prognostic
marker.
Use of BRAF Mutation for Therapeutic
Decisions
As mentioned above, the constitutive activation of
BRAFV600E mutation in the MAPK pathway seems to
be the cause of tumorigenesis and progression in
PTC.13,14,17 Using inhibitors that target BRAF kinase
or its downstream effectors is the logical therapeutic
approach to inhibit tumor growth and progression of
PTCs. Different trials have evaluated the anticancer
effects of BRAF inhibitors, and the preclinical results
are encouraging. Nonselective BRAF inhibitors AAL-
881 and LBT-613 are isoquinolone compounds that
have been shown to inhibit cell cycle progression from
S-phase to G2-M phase and G0-G1 arrest, resulting
in growth reduction and apoptosis in several thyroid
cancer cell lines and in xenograft tumors.68 Specific
knockdown of BRAFV600E by siRNA inhibited the
growth of ATC cell lines, the growth and transfor-
mation of BRAFV600E-mutated PTC cells, and prolif-
eration and tumorigenesis in xenograft tumors.69,70
PLX4032, a small-molecule-specific BRAF inhibitor,
arrested the cell growth of ATC cells and NPA human
thyroid cancer cell lines harboring BRAFV600E mu-
tation.145 Prolonged treatment of ATC cells with
PLX4032 induced the re-expression of NIS. In thy-
roid cancer cell lines bearing the RET/PTC1 and
wild-type BRAF, PLX4032 showed an approximately
50-fold higher IC50 value than BRAFV600E cell lines,
indicating that PLX4032 has selective growth in-
hibitory effect on BRAFV600E-mutated thyroid cancer
cells.145 U0126 is a MEK inhibitor that has been
reported to inhibit the growth of the thyroid cell-
expressed BRAFV600E and restore the expression of
iodide-metabolizing genes.122 AZD6244 is a potent
MEK1/2 inhibitor that has been demonstrated to
inhibit ERK phosphorylation in thyroid cancer cell
lines regardless of the status of BRAFV600E mutation,
and the dose required to inhibit the cell growth in 4
BRAFV600E mutant cell lines is lower than that for the
2 wild-type cell lines. AZD6244 has also been shown to
inhibit the growth of xenograft tumors derived from
BRAFV600E mutant ATC cell lines.146 Another small-
molecule potent MEK1/2-selective inhibitor, CI-1040
(PD-184352), inhibited cancer cell proliferation and
tumor xenografts derived from various cancer cells
harboring BRAFV600E or RAS mutations.147 CI-1040
inhibited the growth and induced re-expression of
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122
K.T. Tang, C.H. Lee
some thyroid genes in thyroid cancer cell lines with
BRAFV600E mutation, but not in cells with RET/PTC
or wild-type alleles.148 The results suggest that the
inhibition effects of CI-1040 on tumor cell prolifera-
tion are BRAF or RAS mutation-selective. CI-1040 is
the first MEK inhibitor to enter clinical trials to evalu-
ate its performance in the treatment of lung, colon,
breast and pancreatic cancers. However, no significant
clinical anticancer effect was observed.149,150
BAY43-9006 (sorafenib), is the most studied
multi-kinase inhibitor for targeting BRAF and angio-
genesis-related RTK. It has been shown to inhibit the
proliferation of ATC lines and tumor xenografts. How-
ever, the effect seems to be caused by blocking angio-
genesis via VEGFR signaling rather than by inhibiting
BRAF selectively.151 Several clinical trials that studied
sorafenib monotherapy for the treatment of various
malignancies including iodine-resistant thyroid can-
cer have been completed recently. Although phase I
trials showed encouraging results that sorafenib was a
well tolerated agent, phase II trials showed little or no
antitumor effects in advanced melanoma patients when
sorafenib was used as a single-agent therapy.152 Re-
cently, a longer than 16-week phase II trial of sora-
fenib in 30 patients with metastatic iodine-refractory
thyroid carcinoma showed an overall clinical benefit
of 77%, 70% with thyroglobulin reduction, and a me-
dian 79-week progression-free survival.153 Another
phase II trial of sorafenib in patients with metastatic
thyroid cancer also showed a similar antitumor activity,
with a median progression-free survival of ∼64 weeks,
and a reduction in the levels of VEGFR phosphoryla-
tion, ERK phosphorylation, and VEGF expression in
tumor biopsies.154 To date, there is no evidence to show
that the antitumor effects of sorafenib are through
the inhibition of BRAF. Sorafenib is a multi-kinase
inhibitor that may also target other kinase pathways
such as VEGFR to inhibit tumorigenesis. Indeed, anti-
tumor effects were also observed with other kinase
inhibitors such as axitinib and motesanib that inhibit
VEGFRs and PDGFRs.155,156 Factors other than BRAF
mutation may affect tumor response to sorafenib, and
the combination of different kinase inhibitors and/or
chemotherapy may be a potential therapeutic strategy
in the future.
Conclusions and Perspectives
The discovery that BRAFV600E is the most common
mutation in PTC, and molecular studies demonstrating
its tumorigenic role in PTC, suggest that BRAF muta-
tion is the initiator of PTC. The notion of the inherited
strong kinase potency of BRAFV600E with the ability to
induce genetic instability, silence iodide-metabolizing
genes, and evade the feedback mechanisms which
may promote the progression and aggressiveness of
PTCs is comprehensible. Indeed, the majority of clin-
ical studies that support the association of BRAFV600E
with aggressive clinicopathological characteristics and
higher tumor recurrence make BRAFV600E mutation a
potential diagnostic and prognostic marker, although
quite a number of studies did not find any association.
Using BRAFV600E mutation in FNA specimens as a
diagnostic marker to improve the diagnostic accuracy
of PTC in indeterminate and inadequate cytology is
feasible. However, the high frequency of follicular neo-
plasm and follicular-variant PTCs in indeterminate
samples rarely being BRAFV600E-positive, and the low
tumor DNA yield in inadequate specimens, limit the
clinical use of the test. Multiple-genotype analysis, such
as for RAS and PAX8-PPAR, improves the diagnostic
accuracy in indeterminate cytology but may cause false-
positive results because RAS mutation is also positive
in follicular adenomas. It seems that future discovery
of more specific markers is the ultimate solution for
this issue.
PTC is a relatively benign cancer when compared
with other malignancies, and the majority of patients
can be cured after the initial treatment with an opti-
mistic outcome. Preoperative BRAFV600E analysis might
have value for predicting which patients with low-grade
disease classified by the current staging systems will
eventually have an aggressive clinical course. It would
be beneficial for these patients (including BRAFV600E-
mutated PTMC) to receive more intensive primary
treatment, higher dose of radio-iodide ablation, and
frequent follow-up to reduce the risk of tumor metas-
tasis and recurrence later in the course of tumor pro-
gression. It might also be clinically indicated to use
18-FDG-PET to detect early recurrence of RAIR PTC.
It is noteworthy that there is currently no effective
therapy for tumors that are inoperable or lose iodine
avidity. However, current available data regarding the
negative clinical implications of BRAFV600E mutation in
low-risk PTC are inconclusive. Further investigations in
large randomized, controlled, prospective trials are nec-
essary to confirm the prognostic role of BRAFV600E
mutation before it is applied in routine clinical practice.
To date, the understanding of in vitro molecular
pathways involved in thyroid carcinogenesis supports
the rationale to develop BRAF-targeted therapies for
PTC. Although the preclinical results are encouraging,
the anticancer effects of clinical trials for specific BRAF-
targeted therapies are unsatisfactory. Tumorigenesis is
a complex process that involves other signal pathways
J Chin Med Assoc • March 2010 • Vol 73 • No 3 123
BRAF mutation in papillary thyroid cancer
to effect tumor aggressiveness and progression. As new
targets are disclosed, future research might use selective
target inhibitors or multi-kinase inhibitors alone or in
combination with other regimens (e.g. cytotoxic drugs,
radioiodine therapy after re-expression of thyroid-
specific genes) for the treatment of advanced thyroid
cancers, with the hope that these approaches can pro-
vide satisfactory results.
Acknowledgments
This work was supported by a grant (V99C1-171) from
Taipei Veterans General Hospital.
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