Documente Academic
Documente Profesional
Documente Cultură
doi: 10.1111/j.1365-2265.2005.02367.x
ORIGINAL ARTICLE
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.
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
Results
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
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
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
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
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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