no correlation with clinicopathological taiwan

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Clinical Endocrinology (2005) 63, 461466 doi: 10.1111/j.1365-2265.2005.02367.x

2005 Blackwell Publishing Ltd461

O R I G I N A L A R T I C L E

BlackwellPublishing,Ltd.

No correlation between BRAFV600E

mutation and

clinicopathological features of papillary thyroid carcinomas

in Taiwan

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

rearrange-ments (

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 andNTRK1

re-

arrangements 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 BRAF

V600E

mutation is the most prevalent oncogene in

PTCs in Taiwan. Our data did not suggest that BRAF

V600E

mutationcould be a potentially useful marker of prognosis in patients with

papillary carcinomas in the population studied.

(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 characteris-

tics 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

andNTRK1

), fusing their carboxyl terminus-

containing 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 fromNTRK1

gene rearrangements as

TRK

. Differences in the frequencies of RET

andNTRK1

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 withbiological 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 rearrange-

ments of RET

orNTRK1

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

activation and adverse clinical outcome.

1014

The RAS

proto-oncogenes (H-

RAS

, K-

RAS

and N-

RAS

) encode

membrane-associated guanosine triphosphate (GTP)-binding

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: [email protected]


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462

R.-T. Liu et al.

2005 Blackwell Publishing Ltd, Clinical Endocrinology

, 63

, 461466

proteins. The most common mutational sites alter either the GTP-

binding 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 fol-

licular 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 andanaplastic 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.

1624

A thymine-to-adenine transversion at nucleotide 1799 (T1799A),

formerly designated as T1796A, in exon 15 resulting in a valine-to-

glutamate 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 PTC

25,26

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 trans-

duction 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 BRAF

V600E

) 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 BRAF

V600E

mutation and correlate it with various clinico-

pathological 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 fash-

ion, by two pathologists (Y.-J.C. and C.-C.H.) and subtyped into spe-

cific 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 beenanalysed previously for the presence of RET

andNTRK1

rearrange-

ments 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 10-

m-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

; reverseprimer, 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 Purifica-

tion Kit (Boehringer-Mannheim, Germany) and sequenced on an


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BRAF mutation in papillary thyroid carcinomas

463

2005 Blackwell Publishing Ltd, Clinical Endocrinology

, 63

, 461466

automated sequencer (ABI PRISMTM 310, Applied Biosystems,

USA) using the Bigdye terminator Kit according to the standard pro-

tocol of the manufacturer. PCR products were sequenced unidirec-

tionally 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 andclinicopathological features

The subjects were characterized by age at diagnosis, sex and the follow-

ing 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

Cancer

30

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

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

andNTRK1

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 RETorNTRK1

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 clinico-

pathological 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

RET/PTC TRK RAS BRAF

Kimura et al.18

16% (11/67) ND 16% (11/67) 33% (22/67)

Soares et al.20

18% (7/39) ND 7% (2/27) 46% (23/50)

Fukushima et al.22

ND ND 0% (0/76) 53% (40/76)

This study 8% (8/105) 1% (1/105) 0% (0/20) 47% (49/105)

ND, not determined.

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

and RET/PTC mutations in PTCs

Table 2. Correlation of BRAF mutation with clinicopathological

parameters of PTC

BRAF mutation

(+) n(%)

BRAF mutation

() n(%) P

Age 0422

< 45 years 25 (5319) 33 (6111)

45 years 22 (4681) 21 (3889)

Gender 09862

Female 33 (7021) 38 (7037)

Male 14 (2979) 16 (2963)

Tumour size* 0883

< 10 mm 1 (217) 1 (185)

1040 mm 41 (8913) 44 (8148)

> 40 mm 4 (870) 6 (1111)

Regional nodal metastases 03983

No 30 (6383) 30 (5556)

Yes 17 (3617) 24 (4444)

Extrathyroidal invasion 0472

No 21 (4468) 28 (5185)

Yes 26 (5532) 26 (4815)

Stage (AJCC) 06312I 25 (5319) 32 (5926)

II 3 (638) 5 (926)

III 19 (4043) 14 (2593)

IV 0 (000) 3 (556)

Distant metastases 0058

No 47 (10000) 50 (9259)

Yes 0 (000) 4 (741)

Histological variants of PTC* 47 53

Classic 33 (7021) 30 (5660) 01627

Tall-cell 9 (1915) 5 (943) 01656

Follicular 2 (426) 6 (1132) 01974

Microcarcinoma 1 (213) 2 (377) 06342

Solid/trabecular 0 (000) 2 (377) 01821

Diffuse sclerosing 0 (000) 1 (189) 03489

Encapsulated 1 (213) 6 (1132) 00734

Oncocytic 1 (213) 1 (189) 09324

Multicentricity* 09124

No 19 (4043) 22 (4151)

Yes 28 (5957) 31 (5849)

*Less than 101 cases were available for statistical analysis.


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464 R.-T. Liu et al.

2005 Blackwell Publishing Ltd, Clinical Endocrinology, 63, 461466

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. RASpoint 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 RASmutations were studied in only 20 out of 105

PTCs in this series, no RASgene 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 RASmutations 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 tech-

nique, and demonstrated that nine of 60 PTCs (15%) presentedRET/

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 RASmutation. Using immuno-

histochemical staining to detect expression of the RET tyrosine

kinase (TK) domain, it was found that a large number of BRAF-

mutated PTCs (8/21) also expressed RET, suggesting the possibility

of an overlap between BRAFmutations and RET/PTC rearrange-

ments.21

However, multiple lines of evidence have demonstrated that

both expressions of RET-TK mRNA and c-RET mRNA are com-

monly detected in PTCs without RET/PTC rearrangements.12,3638

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 carcinomasharbouring 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, RASor BRAF mutations in PTCs in this study con-

firmed 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 BRAFmutations 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 BRAFmutations with a higherfrequency of RASmutations.

18In 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. BRAFmutations

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 stageand were not associated with older age and extrathyroidal extension

in another study.17

Both studies did not demonstrate significant

association between BRAFmutations and sex, tumour size and cervical

lymph node metastases. BRAFmutations 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 BRAFmutations 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 BRAFmutation 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 BRAFmutations 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 BRAFmutations

in both classic and tall-cell variants,16

two clinically and biologically

distinct variants of papillary carcinoma.2A statistically significant

correlation between BRAFmutations and development of PTCs ofthe classic papillary histotype has also been reported.

24In our study,

the mutation of BRAFwas not predominant in any histological

variant of papillary carcinomas. Although the association between

mutated BRAFand tall-cell morphology provides evidence for the

association between this genetic event and more aggressive tumour

behaviour, the finding of an association between BRAFmutation and

classic PTCs suggests the possibility that BRAFmutations reduce the

risk of a histological progression. The fact that BRAFmutations were

frequently detected in PTCs and were found in different stages and

different variants of PTC suggests that BRAFmutations alone are not


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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 BRAFmutation 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 BRAFmutations. In our study 59% of patients presented with

multicentricity. Therefore, defining the BRAFstatus in differentprimary 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 clinico-

pathological 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 BRAFmutations in papillary carcinomas.

In conclusion, the BRAFV600E

mutation is the most prevalent onco-

gene in PTCs in Taiwan. Our data did not suggest that the BRAFV600E

mutation could be a potentially useful marker of prognosis ofpatients 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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