Clinical Endocrinology (2005) 63, 461466

doi: 10.1111/j.1365-2265.2005.02367.x

ORIGINAL ARTICLE

No correlation between BRAFV600E mutation and

clinicopathological features of papillary thyroid carcinomas
in Taiwan
Blackwell Publishing, Ltd.

Rue-Tsuan Liu*, Yi-Ju Chen, Fong-Fu Chou, Chun-Liang Li, Wei-Li Wu*, Po-Chin Tsai,
Chao-Cheng Huang and Jiin-Tsuey Cheng
*Division of Metabolism, Department of Pathology and Department of Surgery, Chang Gung Memorial Hospital, Kaohsiung
and Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan, R.O.C.

Summary
Objective Genetic alterations in four oncogenes, namely RAS point
mutations, RET rearrangements (RET/PTC), NTRK1 rearrangements (TRK) and BRAF point mutations have been identified in
human papillary thyroid carcinomas (PTCs). These oncogenes
act along the RET/PTC(TRK)RASBRAFMEKMAPK kinase
pathway, mediating a number of cellular fates including growth,
proliferation and survival in thyroid cells. In this study, we analysed
mutations of BRAF in a cohort of PTCs.
Methods To screen for BRAF mutations, the genomic DNA of
105 PTCs were amplified by polymerase chain reaction (PCR) with
primers flanking exon 15 and PCR products were directly sequenced
with an automatic sequencer. These results, together with data from
our previous studies on RAS, RET rearrangements and NTRK1 rearrangements in the same tumours, were compared to determine their
individual significance in the pathogenesis of PTCs in Taiwan.
Results BRAF mutations were detected in 49 of 105 (47%) tumour
samples. All mutations involved a thymine-to-adenine transversion
at nucleotide 1799 and were heterozygous. There was no overlap
between papillary carcinomas harbouring RET rearrangements,
NTRK1 rearrangements and BRAF mutations. In this cohort,
correlation between BRAF mutations and various clinicopathological
parameters in 101 papillary carcinomas did not reveal any association
with age at diagnosis, sex, tumour size, histological variants of PTC,
multicentricity, cervical lymph node metastases, extrathyroidal
invasion, distant metastases and clinical stage.
Conclusions BRAFV600E mutation is the most prevalent oncogene in
PTCs in Taiwan. Our data did not suggest that BRAFV600E mutation
could be a potentially useful marker of prognosis in patients with
papillary carcinomas in the population studied.

Correspondence: Jiin-Tsuey Cheng, Department of Biological Sciences,

National Sun Yat-Sen University, 70 Lian Hai Road, Kaohsiung, Taiwan
80424, Republic of China. Tel.: 886 7 5252000 ext 3624; Fax: 886 7 5253624;
E-mail: tusya@mail.nsysu.edu.tw
2005 Blackwell Publishing Ltd

(Received 25 April 2005; returned for revision 20 May 2005; finally

revised 10 July 2005; accepted 11 July 2005)

Introduction
Papillary thyroid carcinoma (PTC) is a common endocrine
malignancy. The growth pattern and biological behaviour of papillary
carcinoma are variable. Certain clinical and pathological features
have been identified to predict a worse prognosis, including older
age at diagnosis, larger primary tumours, extrathyroidal invasion,
distant metastases and aggressive histological variants such as the
tall-cell variant of papillary cancer.1,2 Molecular characterization of
PTC may provide an explanation of the diverse clinical characteristics of these tumours and may be useful in identifying additional
prognostic factors at the molecular level, allowing early, aggressive
and specific therapy to improve the outcome.
Considerable progress has been made in the information on
expression of oncogenes in PTCs in the past two decades. Somatic
rearrangements of two different transmembrane receptor tyrosine
kinase genes (RET and NTRK1), fusing their carboxyl terminuscontaining tyrosine kinase domain to the amino terminus of different
activating genes, are specifically expressed in PTCs.35 The chimeric
genes resulting from RET gene rearrangements are referred to as
RET/PTC and those resulting from NTRK1 gene rearrangements as
TRK. Differences in the frequencies of RET and NTRK1 rearrangements
have been reported in PTCs collected from various geographical
areas. NTRK1 rearrangements are less frequently found in PTCs than
are RET rearrangements. Correlation of RET/PTC expression with
biological and clinical outcomes has been controversial. Some have
suggested that RET/PTC expression could serve as an indicator
of aggressive behaviour in PTC, specifically for more advanced
disease.6 8 In a multivariate analysis, it was confirmed that rearrangements of RET or NTRK1 parallel an unfavourable disease presentation,
which may correlate with a less favourable disease outcome.9
However, recent studies did not find any association between RET
10 14
activation and adverse clinical outcome.
The RAS proto-oncogenes (H-RAS, K-RAS and N-RAS) encode
membrane-associated guanosine triphosphate (GTP)-binding
461

462 R.-T. Liu et al.

proteins. The most common mutational sites alter either the GTPbinding domain (codons 12 and 13 in exon 1) or the GTPase domain
(codon 61 in exon 2) of the RAS protein. RAS point mutations have
been reported in both benign and malignant histological types of
thyroid neoplasia. However, they appeared to occur selectively in follicular adenomas, follicular carcinomas, poorly differentiated and
anaplastic carcinomas, and in some papillary carcinomas.15
Activating point mutations of the BRAF gene have been recently
reported to be restricted to PTCs and poorly differentiated and
anaplastic carcinomas arising from papillary carcinomas among
various benign and malignant thyroid tumours.16,17 A high mutation
rate of this new oncogene was reported in PTCs, with a frequency
ranging from 29% to 69% in several thyroid tumour cohorts.16 24
A thymine-to-adenine transversion at nucleotide 1799 (T1799A),
formerly designated as T1796A, in exon 15 resulting in a valine-toglutamate substitution at residue 600 (V600E), formerly designated
as V599E, was the hot-spot mutational site reported in thyroid cancer.
Different mutations have recently been described in a follicular
variant of PTC25,26 and lymph node metastases from PTC.27 It was
also the most common genetic event in PTCs in studies of other
oncogenes.16,18,20,22,24
The BRAF gene encodes a cytoplasmic serine/threonine kinase
that is regulated by binding RAS. Activated RAS phosphorylates RAF,
which in turn activates a series of kinases, leading ultimately to the
activation of MAPK. The RASBRAFMEKMAPK signal transduction cascade mediating the cellular response to tyrosine kinase
receptors regulates a number of cellular fates including growth, cell
proliferation, differentiation and survival in cells.28 Thus, these four
known oncogenes (RET/PTC, TRK, RAS and BRAFV600E) act along
the RET/PTC(TRK)RASBRAFMEKMAPK signalling pathway
and contribute to the pathogenesis of PTCs.
To elucidate the molecular basis for the tumorigenesis of papillary
carcinomas in Taiwan, we systematically evaluated the known
oncogenes in a thyroid tumour cohort to determine their individual
significance in the pathogenesis of papillary carcinomas in this area.
Our previous studies on the prevalence of RAS mutations, RET/PTC
and TRK in a series of PTC samples demonstrated low occurrence
of these oncogenes, suggesting that other oncogene(s) might be
responsible for the tumorigenesis. In this study, we analysed exon
15 of the BRAF gene in this PTC cohort to determine the frequency
of the BRAFV600E mutation and correlate it with various clinicopathological parameters. The results were also combined with our
previous studies on the prevalence of RAS, RET/PTC and TRK to
investigate genetic aberrations in the RET/PTC(TRK)RASBRAF
MEKMAPK pathway in PTCs in Taiwan.

Materials and methods

Tumour samples and patient information
A total of 105 consecutive adult patients with PTC were studied. All
tumour samples were prospectively collected by one of the authors
(F.-F.C.) between 1997 and 2002 at the Department of Surgery,
Chang Gung Memorial Hospital, Kaohsiung, Taiwan. Portions of
tumour tissues were frozen in liquid nitrogen immediately after
surgical removal and subsequently stored at 70 C. At the time of

surgery, four patients were operated on for local tumour recurrence.

Patient information, including demographic data, tumour size and
distant metastases, was obtained by chart review. Histological slides
from 105 papillary carcinomas were re-examined, in a blinded fashion, by two pathologists (Y.-J.C. and C.-C.H.) and subtyped into specific histological variants according to the histopathological typing
of the World Health Organization.29 The presence of cervical lymph
node metastases, multicentricity and extrathyroidal invasion was
determined histologically. The same cohort of 105 PTCs have been
analysed previously for the presence of RET and NTRK1 rearrangements by reverse transcription polymerase chain reaction (RT-PCR)
from frozen tissues. Twenty of them were also randomly selected for
the study of RAS point mutations. At the time when BRAF mutations
were investigated, snap-frozen tissue samples were available for 90
patients and paraffin-embedded tissue samples were obtained from
the pathology files of Chang Gung Memorial Hospital for the
remaining 15 patients. The study protocol was approved by the
Medical Ethics Committee of Chang Gung Memorial Hospital. All
patients gave their informed consent.
DNA extraction
For frozen tumour samples, genomic DNA was isolated from at
least 20 mg of tissue using the QIAamp DNA Mini Kit (QIAGEN
Inc., CA, USA) according to the manufacturers instructions. For
paraffin-embedded tissues, paraffin blocks were sectioned to obtain
tissue for DNA extraction. To localize the area of tumour tissue for
histological examination, 5-m-thick sections were stained with
haematoxylin and eosin. Tumour tissue was separated from the
surrounding normal tissue by microdissection. At least three 10m-thick sections of tumour tissue were used for genomic DNA
extraction. In brief, the microdissected paraffin-embedded tissues
were xylene-deparaffinized and digested in proteinase K (04 g/l
in 30 l of digestion buffer: 10 m TrisHCl, pH 80, 1 m EDTA,
and 1% Tween-20) overnight. After heat inactivation of the enzyme,
the sample was subject to phenolchloroform extraction followed by
ethanol precipitation. The ethanol-precipitated genomic DNA was
resuspended in 30 l of distilled sterile water. Of this, 5 l was used
as the DNA template for PCR amplification.
Detection of BRAF mutations
Genomic DNA, extracted from frozen tissues or paraffin-embedded
specimens, was used as the template for amplification of exon 15 of
the BRAF gene by PCR. The primer sequences were as follows:
forward primer, 5-TCATAATGCTTGCTCTGATAGGA-3; reverse
primer, 5-GGCCAAAAATTTAATCAGTGGA-3. Extracted DNA
was amplified in a Progene thermocycler (Techne Inc., NJ, USA) for
40 cycles using the cycling conditions: 96 C for 1 min, 56 C for
1 min and 72 C for 1 min. PCR products visualized in 2% agarose
gel were purified for automatic sequencing.
Sequencing
PCR products were purified with a High Pure PCR Product Purification Kit (Boehringer-Mannheim, Germany) and sequenced on an
2005 Blackwell Publishing Ltd, Clinical Endocrinology, 63, 461466

BRAF mutation in papillary thyroid carcinomas 463

automated sequencer (ABI PRISMTM 310, Applied Biosystems,
USA) using the Bigdye terminator Kit according to the standard protocol of the manufacturer. PCR products were sequenced unidirectionally or, if in doubt, in both directions. To ascertain the results,
sequencing was repeated from the same or different PCR products
in 35 tumour samples.
Correlation analysis between BRAF mutations and
clinicopathological features
The subjects were characterized by age at diagnosis, sex and the following attributes of tumour pathology: size, histological variants of
PTC, number of foci, cervical lymph node metastases, extrathyroidal
invasion, and distant metastases. A tumournodemetastasis (TNM)
classification system adopted by the American Joint Committee on
Cancer30 was used for clinical staging of thyroid cancer. This TNM
system is based primarily on pathological findings. It separates
patients into four stages, with progressively poorer survival with
increasing stage. The clinicopathological features were correlated
with the status of BRAF in the tumour.
Statistical analysis
Data were analysed using the MannWhitney U-test or 2 for
independence test. Students t-test was performed for analysis of
histological variants. A P-value < 005 denoted the presence of a
significant difference.

Results

Table 2. Correlation of BRAFV600E mutation with clinicopathological

parameters of PTC

Age
< 45 years
45 years
Gender
Female
Male
Tumour size*
< 10 mm
10 40 mm
> 40 mm
Regional nodal metastases
No
Yes
Extrathyroidal invasion
No
Yes
Stage (AJCC)
I
II
III
IV
Distant metastases
No
Yes
Histological variants of PTC*
Classic
Tall-cell
Follicular
Microcarcinoma
Solid/trabecular
Diffuse sclerosing
Encapsulated
Oncocytic
Multicentricity*
No
Yes

BRAF mutation
(+) n (%)

BRAF mutation
() n (%)

25 (5319)
22 (4681)

33 (6111)
21 (3889)

33 (7021)
14 (2979)

38 (7037)
16 (2963)

1 (217)
41 (8913)
4 (870)

1 (185)
44 (8148)
6 (1111)

30 (6383)
17 (3617)

30 (5556)
24 (4444)

21 (4468)
26 (5532)

28 (5185)
26 (4815)

25 (5319)
3 (638)
19 (4043)
0 (000)

32 (5926)
5 (926)
14 (2593)
3 (556)

47 (10000)
0 (000)
47
33 (7021)
9 (1915)
2 (426)
1 (213)
0 (000)
0 (000)
1 (213)
1 (213)

50 (9259)
4 (741)
53
30 (5660)
5 (943)
6 (1132)
2 (377)
2 (377)
1 (189)
6 (1132)
1 (189)

19 (4043)
28 (5957)

22 (4151)
31 (5849)

P
0422

09862

0883

03983

0472

06312

0058

01627
01656
01974
06342
01821
03489
00734
09324
09124

The details of clinical data and morphological features of these

tumours are described elsewhere.31 Of 105 cases of papillary carcinomas,
49 (47%) have heterozygous mutations T1799A in exon 15 of the
BRAF gene. In our previous studies the same cohort of tumours was
analysed for the presence of RET and NTRK1 rearrangements; 20 of
them were also studied for mutations in the known hot-spots in the
three RAS genes. Eight of 105 PTCs (8%) had RET rearrangements.
Of these tumours, three involved RET/PTC1, four involved RET/
PTC3 and one involved ELKS-RET rearrangement.31 One of 105
PTCs (1%) had a TRK-T2 rearranged transcript (manuscript in
preparation). None of these nine PTCs harbouring RET or NTRK1
rearrangements had the BRAFV600E mutation. We did not find RAS
mutations in the 20 PTCs studied.14 The results of mutations of these
genes in this sample cohort are shown in Table 1. We examined the
correlation between the BRAFV600E mutation and various clinicopathological parameters in 101 patients, excluding four cases operated

on for local tumour recurrence (Table 2). In these four cases, BRAF
mutations were found in two. No significant correlation was found
between the BRAFV600E mutation and sex, age at diagnosis, tumour
size, histological variants of PTC, multicentricity, cervical lymph

Table 1. Lack of overlap among BRAF, RAS, TRK

and RET/PTC mutations in PTCs

RET/PTC

TRK

RAS

BRAF

16% (11/67)
18% (7/39)
ND
8% (8/105)

ND
ND
ND
1% (1/105)

16% (11/67)
7% (2/27)
0% (0/76)
0% (0/20)

33% (22/67)
46% (23/50)
53% (40/76)
47% (49/105)

Kimura et al.18
Soares et al.20
22
Fukushima et al.
This study
ND, not determined.
2005 Blackwell Publishing Ltd, Clinical Endocrinology, 63, 461466

*Less than 101 cases were available for statistical analysis.

464 R.-T. Liu et al.

node metastases, extrathyroidal invasion, clinical stage, or distant
metastases at presentation.

Discussion
In this study we demonstrated that a heterozygous point mutation
of the BRAF gene was detected in 49 of 105 (47%) sporadic
(nonradiation-induced) adult PTCs, a frequency similar to most
reports from different geographical areas.
Table 1 summarizes those studies demonstrating the frequencies
of both the BRAFV600E mutation and any other genetic alterations
along the RET/PTC(TRK)RASBRAFMEKMAPK pathway in
the same cohort of PTCs. RAS point mutations were present in 16%
and 7% of papillary carcinomas in two studies.18,20 However, no
mutations were identified in the Japanese series22 and in this current
study. Although RAS mutations were studied in only 20 out of 105
PTCs in this series, no RAS gene mutation was found in 42 cases of
PTCs (including these 20 cases) in our previous study.15 Even though
several large studies also failed to identify any RAS mutations in
PTCs,3234 the possibility of RAS mutations in our studied cohort
cannot be excluded.
The prevalence of RET/PTC1, RET/PTC2 and RET/PTC3 has been
found to vary between 0% and 20% in most series of sporadic PTCs
analysed by type-specific RT-PCR alone or Southern blot analysis of
genomic DNA from frozen tissues.12,35,36 As shown in Table 1,
Kimura et al. detected 16% of RET/PTC by Southern blot analysis
of genomic DNA from 67 PTCs.18 Using type-specific RT-PCR to
detect RET/PTC1, RET/PTC2 and RET/PTC3, we identified 7% of
RET/PTC in PTC samples as opposed to 18% in the Portugal series.20
Puxeddu et al. screened 60 Italian PTCs for the presence of RET/
PTC1 and RET/PTC3 chimeric transcripts, using the RT-PCR technique, and demonstrated that nine of 60 PTCs (15%) presented RET/
PTC expression.24 In these four studies including our series, as shown
in Table 1, none of the tumours showed an overlap between a BRAF
mutation, RET/PTC rearrangement or RAS mutation. Using immunohistochemical staining to detect expression of the RET tyrosine
kinase (TK) domain, it was found that a large number of BRAFmutated PTCs (8/21) also expressed RET, suggesting the possibility
of an overlap between BRAF mutations and RET/PTC rearrangements.21 However, multiple lines of evidence have demonstrated that
both expressions of RET-TK mRNA and c-RET mRNA are commonly detected in PTCs without RET/PTC rearrangements.12,36 38
Furthermore, wild-type and alternatively spliced RET transcripts
might coexist with RET/PTC rearrangements.39 These observations
raised the question about the specificity of RET-TK expression in PTC
samples. At present, there was no overlap between papillary carcinomas
harbouring BRAF mutations and RET rearrangements, which
were detected by either Southern blot analysis of genomic DNA or
type-specific RT-PCR. In our series we also demonstrated that
tumours with NTRK1 rearrangements do not harbour BRAF
mutations. The failure to demonstrate an overlap between RET/
PTC, TRK, RAS or BRAF mutations in PTCs in this study confirmed and extended previous findings, in which alteration of
any component in the RET/PTC(TRK)RASBRAFMEKMAPK
signalling pathway was shown to be sufficient for the initiation of
sporadic PTCs and may provide a more reliable means for further

confirmation of genetic alterations along this signalling pathway in

individual PTCs.
In contrast to the wide variation of reported frequencies of RAS
mutations or RET/PTC in papillary carcinomas in the literature, the
prevalence of BRAF mutations reported from different populations
was fairly consistent, detected in 36 46% of papillary carcinomas in
most series.16,18,20,21,23 It is of interest to note that in the four series
studying both BRAF and RAS mutations (Table 1), Kimura et al.
observed a lower prevalence of BRAF mutations with a higher
frequency of RAS mutations.18 In this regard, studies from different
populations examining the relative distribution of genetic alteration
of any component in the RET/PTC(TRK)RASBRAFMEKMAPK
pathway may provide insights into the tumorigenesis in a specific
geographical area.
Controversy exists as to the biological characteristics, pathological
features and clinical behaviour of the tumours harbouring BRAF
mutations, compared with those that are negative. BRAF mutations
were associated with older age, extrathyroidal extension, and more
frequent presentation at clinical stages III and IV but were not
associated with distant metastases in one study,16 but correlated
significantly with distant metastases and advanced clinical stage
and were not associated with older age and extrathyroidal extension
in another study.17 Both studies did not demonstrate significant
association between BRAF mutations and sex, tumour size and cervical
lymph node metastases. BRAF mutations have also been associated
with higher prevalence of extrathyroidal invasion, cervical lymph
node metastases and more advanced pathological stage, and a higher
incidence of cancer recurrence.40 The above observations suggest
that BRAF mutations could be a useful marker of poor prognosis of
patients with papillary cancer. Conversely, Xu et al. found that BRAF
mutations occurred at a significantly higher frequency in male
patients than in female patients but were not associated with patient
age or tumour stage.21 A geneticclinical association analysis failed
to find any association between BRAF mutation and age at diagnosis,
gender, dimension, and local invasiveness of the primary cancer, the
presence of lymph node metastases, tumour stage, and multifocality
of the disease.24 Our data revealed no correlation between BRAF
mutation and any clinical or pathological characteristics examined
(Table 2). Thus, our results, like these two previous studies,21,24 did not
suggest that BRAF mutations have prognostic value in patients with
papillary thyroid cancer. Of note, in our study all four tumours with
distant metastases contained wild-type BRAF (P = 0058) (Table 2).
Nikiforova et al. reported preferential occurrence of BRAF mutations
in both classic and tall-cell variants,16 two clinically and biologically
distinct variants of papillary carcinoma.2 A statistically significant
correlation between BRAF mutations and development of PTCs of
the classic papillary histotype has also been reported.24 In our study,
the mutation of BRAF was not predominant in any histological
variant of papillary carcinomas. Although the association between
mutated BRAF and tall-cell morphology provides evidence for the
association between this genetic event and more aggressive tumour
behaviour, the finding of an association between BRAF mutation and
classic PTCs suggests the possibility that BRAF mutations reduce the
risk of a histological progression. The fact that BRAF mutations were
frequently detected in PTCs and were found in different stages and
different variants of PTC suggests that BRAF mutations alone are not
2005 Blackwell Publishing Ltd, Clinical Endocrinology, 63, 461466

BRAF mutation in papillary thyroid carcinomas 465

a major determinant for the heterogeneity of clinicopathological
features observed in PTCs. Furthermore, the intriguing findings
of a BRAF mutation in the regional lymph nodes and its absence
in primary tumours, as well as that different BRAF genotypes
were recognized in distinct lymph nodes,27 raise the possibility
that each metastasis spreads from a different primary focus,
which further complicates the clinicopathological interpretation
of BRAF mutations. In our study 59% of patients presented with
multicentricity. Therefore, defining the BRAF status in different
primary focus, regional and distant metastases in patients with
multicentric PTC is an important area for future investigation.
It is unclear at this stage whether the discrepancy of clinicopathological correlation depends on the population studied. The
relatively small sample size of most series, including ours, raises
the possibility that statistical variability might, at least in part,
account for the discrepant results. Future studies on a greater number
of patients with an adequate period of follow-up will be required to
elucidate the consequence of BRAF mutations in papillary carcinomas.
In conclusion, the BRAFV600E mutation is the most prevalent oncogene in PTCs in Taiwan. Our data did not suggest that the BRAFV600E
mutation could be a potentially useful marker of prognosis of
patients with papillary carcinomas in this cohort.

Acknowledgements
This work was supported in part by the University Integration
Programme from the Ministry of Education (to J.-T.C.) and the
Chang Gung Medical Research Project CMRPG8008 (to R.-T.L.).

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