ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS

Significance of Allelic Percentage of BRAF c.1799T [ A (V600E)Mutation in Papillary Thyroid Carcinoma

Shih-Ping Cheng, MD, PhD1,2,6, Yi-Chiung Hsu, PhD5, Chien-Liang Liu, MD1,2, Tsang-Pai Liu, MD1,2,

Ming-Nan Chien, MD1,3, Tao-Yeuan Wang, MD1,4, and Jie-Jen Lee, MD, PhD1,2,6

1Mackay Junior College of Medicine, Nursing, and Management, Taipei, Taiwan; 2Department of Surgery,

Mackay Medical College and Mackay Memorial Hospital, Taipei, Taiwan; 3Division of Endocrinology and Metabolism,

Department of Medicine, Mackay Medical College and Mackay Memorial Hospital, Taipei, Taiwan; 4Department of

Pathology, Mackay Medical College and Mackay Memorial Hospital, Taipei, Taiwan; 5Institute of Statistical Science,

Academia Sinica, Taipei, Taiwan; 6Graduate Institute of Medical Sciences and Department of Pharmacology, Taipei

Medical University, Taipei, Taiwan

ABSTRACT

Background. Somatic BRAF mutation is frequently

observed in papillary thyroid carcinoma (PTC). Recent

evidence suggests that PTCs are heterogeneous tumors

containing a subclonal or oligoclonal occurrence of BRAF

mutation. Conflicting results have been reported concern-

ing the prognostic significance of the mutant allele

frequency. Our present aim was to investigate the associ-

ation between the percentage of BRAF c.1799T [ A

(p.Val600Glu) alleles and clinicopathological parameters

in PTC.

Methods. Genomic DNA was extracted from fresh-frozen

specimens obtained from 50 PTC patients undergoing total

thyroidectomy. The BRAF mutation status was determined

by Sanger sequencing. The percentage of mutant BRAF

alleles was quantified by mass spectrometric genotyping,

pyrosequencing, and competitive allele-specific TaqMan

PCR (castPCR).

Results. Positive rate of BRAF mutation was 72 % by Sanger

sequencing, 82 % by mass spectrometric genotying, and 84 %

by pyrosequencing or castPCR. The average percentage of

mutant BRAF alleles was 22.5, 31, and 30.7 %, respectively.

There was a good correlation among three quantification

methods (Spearman’s rho = 0.87–0.97; p \ 0.0001). The

mutant allele frequency was significantly correlated with

tumor size (rho = 0.47–0.52; p \ 0.01) and extrathyroidal

invasion. The frequency showed no difference in pathological

lymph node metastasis.

Conclusions. The percentage of mutant BRAF alleles is

positively associated with tumor burden and extrathyroidal

invasion in PTC. Relatively good correlations exist among

mass spectrometric genotyping, pyrosequencing, and cast-

PCR in quantification of mutant BRAF allele frequency.

Papillary thyroid carcinoma (PTC) is the most common

malignant tumor of the thyroid gland. Thyroid cancer

incidence has increased substantially over the last decade,

and this change is primarily attributed to an increase in

PTC.1,2 Although most PTCs are curable or controllable by

the combination of surgery, radioactive iodine ablation,

and thyrotropin suppression, a considerable number of

patients die of persistent or recurrent disease that is

refractory to conventional therapy.3 With a growing

understanding of molecular oncology, therapeutic agents

that target activating genetic alterations in thyroid cancer

have been rapidly developed.4

The v-raf murine sarcoma viral oncogene homolog B

(BRAF) mutation occurs frequently in PTC.5 The most

common BRAF mutation comprises a single base trans-

version at codon 600 in exon 15, identified as c.1799T [ A

(p.Val600Glu) and generally referred to as V600E. The

prevalence of BRAF mutation was found to be increased

significantly over a 15-year period, and it was proposed

Electronic supplementary material The online version of thisarticle (doi:10.1245/s10434-014-3723-5) contains supplementarymaterial, which is available to authorized users.

� Society of Surgical Oncology 2014

First Received: 31 October 2013

J.-J. Lee, MD, PhD

e-mail: surg.mmh@gmail.com

Ann Surg Oncol

DOI 10.1245/s10434-014-3723-5

that this change may contribute to the increasing incidence

of thyroid cancer.6 BRAF c.1799T [ A mutation greatly

increases the kinase activity and is associated with elevated

transcriptional output of the mitogen-activated protein

kinase (MAPK) pathway.7,8

Data from meta-analyses consistently indicate that the

presence of BRAF mutation in thyroid cancer is associated

with extrathyroidal invasion, lymph node metastasis,

advanced TNM stage, and disease recurrence.5,9–11 If thy-

roid cancer cells harboring BRAF mutation are biologically

more aggressive, tumors containing more BRAF mutation-

positive cells will theoretically have a worse outcome. In

this regard, Guerra and colleagues used pyrosequencing to

quantify the percentage of mutant BRAF alleles, demon-

strating that the mutant allele frequency correlated with age

and tumor volume, and the odds ratio of recurrent disease

was 5.31-fold higher in PTCs harboring a high ([30 %)

percentage of mutant BRAF alleles.12 On the contrary,

Gandolfi et al. 13 found no association between the per-

centage of mutant alleles and metastatic behavior. The

reason for this discrepancy remains to be elucidated.

Methods to detect BRAF mutation have not been stan-

dardized. Conventional DNA sequencing is binary (present

or absent) and takes no account of the ratio of mutated

DNA to wild type. Contemporary technological develop-

ments in genotyping are advantageous in terms of cost,

throughput, scalability, and sensitivity.14 The primary aim

of this study was to perform a multiplatform analysis to

characterize the biological significance of the percentage of

BRAF c.1799T [ A alleles in PTC. The secondary aim was

to compare different methods for quantification of mutant

BRAF allele frequency.

PATIENTS AND METHODS

Patients and Tissue Samples

This study was approved by the Institutional Review

Board of Mackay Memorial Hospital (12MMHIS175). All

patients provided written informed consent before the

procurement of tissue specimens.

A total of 50 patients who underwent total thyroidec-

tomy for PTC were consecutively selected. Five patients

who underwent lobectomy for follicular adenoma were

also included as negative controls. All patients were

euthyroid at the time of surgery and were not taking any

medications that could affect serum thyrotropin levels.

PTC tissues from the center of the lesions and corre-

sponding normal thyroid tissues from the contralateral

lobes of the same patient were obtained. All PTC tissue

samples were carefully dissected to exclude surrounding

normal tissue. Tissues were immediately snap-frozen in

liquid nitrogen and stored at -80 �C. The tissue diagnosis

was confirmed by frozen section.

Genomic DNA Extraction

Genomic DNA was extracted using the QIAamp DNA

mini kit (Qiagen, Valencia, CA, USA) according to the

manufacturer’s instructions. Serial dilutions of DNA from

B-CPAP thyroid cancer cells (homozygous BRAF mutation

?/?) were used as positive controls.15

Sanger Sequencing

Exon 15 of the BRAF gene was amplified using a spe-

cific custom-made oligonucleotide primer pair (electronic

supplementary Table 1). The PCR products were subjected

to sequencing reaction using the forward primer and Big-

Dye terminator V3.1 cycle sequencing reagents (Applied

Biosystems, Life Technologies, Carlsbad, CA, USA). DNA

sequence was read on an ABI PRISM 3730xL DNA

analyzer.

Mass Spectrometric Genotyping

The iPLEX assay is a single-base primer extension

assay.16 Locus-specific PCR primers and allele-specific

detection primers (electronic supplementary Table 1)

were designed using MassARRAY Assay Design software

(Sequenom Inc., San Diego, CA, USA). Allele detection

was performed using matrix-assisted laser desorption/

ionization time-of-flight mass spectrometer (MALDI-TOF

MS). The mass spectrograms were analyzed by the

MassARRAY TYPER software 4.0. All analyses showed a

very high call rate ([95 %, the median of call

rate [97 %), and a very high consistency rate between

blind duplicates ([97 %).

Pyrosequencing Analysis

Primer design (electronic supplementary Table 1) was

carried out using the PyroMark Assay Design software 2.0

(Qiagen). The biotinylated PCR products were attached to

Streptavidin-Sepharose beads (Amersham, GE Healthcare

Bio-Sciences, Piscataway, NJ, USA) and processed to

obtain single-stranded DNA using the PSQ 96 Sample

Preparation Kit. The sequencing-by-synthesis reaction of

the complementary strand was automatically performed

using the PyroMark Gold Q24 reagents (Qiagen) according

to kit specifications. The percentage of mutant alleles was

then calculated by PyroMark Q24 version 2.0.6 software

using the allele quantification mode.

S.-P. Cheng et al.

Competitive Allele-Specific TaqMan PCR

The BRAF mutation status was also determined with a

commercially available TaqMan Mutation Detection Assay

(Applied Biosystems). Competitive allele-specific TaqMan

PCR (castPCR) using the BRAF_476_mu probe for the

detection of BRAF c.1799T [ A mutation were run in

triplicates on an Applied Biosystems 7900HT Fast Real-

Time PCR System. The mutant allele frequency was

determined by comparing the cycle threshold (Ct) values of

the wild-type and mutant allele assays (DCt) in reference to

the control samples using Mutation Detector software 2.0.

The percentage of mutant BRAF alleles was calculated as

% = 1/(2DCt) 9 100.17

Statistical Analysis

Statistical analyses were performed using STATA 11.0

(Stata Corp., College Station, TX, USA) and GraphPad

Prism 6.02 (GraphPad Software, La Jolla, CA, USA).

Continuous variables were compared using the Mann–

Whitney test for two groups or Kruskal–Wallis test and

Dunn’s procedure for more than two groups. Chi square,

Fisher’s exact test, or Cochran-Armitage trend test were

used to compare categorical variables. For correlation

studies, Spearman’s rank-correlation test was used.

Throughout the analysis, p-values \ 0.05 (two-sided

hypotheses) were considered to be statistically significant.

RESULTS

Patients Characteristics

The study cohort consisted of 40 (80 %) female and 10

(20 %) male patients. Mean age at diagnosis was 47 years.

A total of 16 (32 %) patients with clinically apparent nodal

metastasis (cN1) had therapeutic central compartment neck

dissection with or without lateral neck dissection. The

remaining 34 (68 %) patients without preoperative or

intraoperative evidence of central neck metastases under-

went prophylactic central compartment dissection. Forty-

seven tumors were classical PTC (including five non-

incidental microcarcinomas), two were follicular variant

PTC, and one was solid variant PTC. Mean tumor size was

2.3 cm. Eight patients showed histological evidence of

chronic lymphocytic thyroiditis in the corresponding nor-

mal thyroid tissue. The numbers of tumor-infiltrating

lymphocytes were not specifically determined in the study.

Of 34 patients undergoing prophylactic central com-

partment dissection, 13 (38 %) showed microscopic nodal

metastasis. Therefore, a total of 29 (58 %) had pathological

nodal metastasis (pN1). The final TNM stage was deter-

mined on the basis of pathological data. One patient had

evidence of lung metastasis at the time of diagnosis, and

she was classified as TNM stage 2 because of her young

age. Tumor size was associated with clinical and patho-

logical lymph node metastasis (p = 0.016 and 0.025,

respectively).

Mutational Analysis by Sanger Sequencing

Tumor BRAF mutation was found in 36 (72 %) of 50

PTCs. No mutation was found in the contralateral normal

tissues or five follicular adenomas, indicating that this

mutation was somatically acquired and tumor-specific. As

shown in Table 1, the tumors with BRAF mutation were

associated with larger tumor size (p = 0.015), higher

probabilities of extrathyroidal invasion (p = 0.002), and

more advanced TNM stage (p = 0.003). Two (40 %) of

five microcarcinomas were positive for BRAF mutation.

Tumor BRAF mutation status was not associated with

TABLE 1 Clinicopathological parameters and BRAF c.1799T [ A

mutation status determined by Sanger sequencing

BRAF

mutation

? (n = 36)

BRAF

mutation

- (n = 14)

p Value

Female sex 29 (81) 11 (79) 1.000

Age at diagnosis

(years)a52 (37–58) 37 (32–51) 0.125

Body mass index

(kg/m2)a24.5 (21.6–28.9) 23.4 (19.4–25.8) 0.266

Body weight (kg)a 63 (52–76) 58 (56–65) 0.496

Tumor size (cm)a 2.2 (1.5–3.3) 1.4 (1.0–2.2) 0.015

Thyroiditis 4 (11) 4 (29) 0.197

Extrathyroidal

invasionb0.002

None 9 (25) 10 (71)

Microscopic 12 (33) 3 (21)

Macroscopic 15 (42) 1 (7)

Multifocality 13 (36) 4 (29) 0.746

Lymphovascular

invasion

12 (33) 2 (14) 0.295

Clinical lymph node

metastasis

13 (36) 3 (21) 0.501

Pathological lymph

node metastasis

21 (58) 8 (57) 1.000

TNM stageb 0.003

I 15 (42) 11 (79)

II 0 (0) 2 (14)

III 9 (25) 1 (7)

IV 12 (33) 0 (0)

Data are expressed as number (%) or median (interquartile range)a Mann–Whitney testb Cochran-Armitage trend test

Allelic Percentage of BRAF Mutation

clinical or pathological lymph node metastasis (p = 0.501

and 1.000, respectively).

Mutational Analysis by Mass Spectrometry

The percentage of mutant BRAF alleles in genomic

DNA from 50 PTCs was determined by mass spectrometric

genotyping (Fig. 1a). There was no mutant allele in nine

PTCs and five follicular adenomas. For 41 (82 %) PTCs

carrying BRAF mutation, the range of mutant allele fre-

quency was 3.2–35.2 %, with a mean and median of 22.5

and 25.2 %, respectively. Overall, five samples failed to be

assigned to positive BRAF mutation by Sanger sequencing

(90 % concordance). These discordant samples showed a

mutant allele frequency from 3.2 to 13.5 % (mean 6.6 %).

Tumor size varied from 1.3 to 4.0 cm (mean 2.4 cm).

There was no significant difference in characteristics

between the cases with concordant results and those with

discordant results, except for a lower frequency of mutant

allele in the discordant samples.

Patients were categorized on the basis of mutant allele

frequency into tertiles (Table 2). Tumor BRAF mutation

was associated with body weight (p = 0.023), tumor size

(p = 0.004), extrathyroidal invasion (p = 0.024), and

TNM stage (p = 0.029). There was no difference in

extrathyroidal invasion and TNM stage between the second

and third tertile (p = 0.347 and 0.113, respectively). A

seemingly higher disease stage in the second tertile may

reflect the relatively older age in this subgroup. As shown

in Fig. 1b, there was a strong positive correlation between

tumor size and mutant allele frequency (Spearman’s

rho = 0.47; p = 0.0005). Analyzed from another per-

spective, PTC in association with clinical nodal metastasis

showed a mutant allele percentage significantly higher than

cN0 PTC (p = 0.037; Fig. 2a). There was no difference in

the mutant allele frequency between PTC with or without

pathological nodal metastasis (p = 0.984; Fig. 2b).

Mutational Analysis by Pyrosequencing

The percentage of mutant BRAF alleles from five fol-

licular adenomas varied from 3 to 5 %. Therefore, the

cutoff was set at 5 %, consistent with previous reports.12,13

For 42 PTCs positive for BRAF mutation, the range of

mutant allele frequency was 6–46 %, with a mean and

median of 31 and 36 %, respectively. The mutant allele

frequency was highly consistent between mass spectro-

metric genotying and pyrosequencing (Fig. 3a). One

patient with a 7 % BRAF mutation allele based on py-

rosequencing analysis had no mutant BRAF allele

detectable by mass spectrometry (98 % concordance).

The percentage of mutant BRAF alleles was strongly

correlated with tumor size (Spearman’s rho = 0.52;

p = 0.0001), but not age (p = 0.782) or body weight

(p = 0.100). Comparing medians of the mutant allele fre-

quency, significant differences were observed in

extrathyroidal invasion (p = 0.034) and clinical nodal

metastasis (p = 0.041), but not pathological nodal metas-

tasis (p = 0.937). Tumor BRAF mutation determined by

pyrosequencing was associated with higher probabilities of

stage III/IV disease (p = 0.018).

Mutational Analysis by castPCR

Five follicular adenomas were in the range of 0.03–

1.7 % mutant allele. A conservative 2.5 % cutoff was

chosen to avoid unreliable estimates due to stochastic

fluctuations in low copy number situations.18 In concor-

dance with the results obtained by pyrosequencing, 42

PTCs were positive for BRAF mutation based on castPCR

assay. The range of mutant allele frequency was 3.0–

61.6 %, with a mean and median of 30.7 and 29.1 %,

respectively. The mutant allele frequency was moderately

correlated between mass spectrometric genotying and

castPCR assay (Fig. 3b). A similar correlation coefficient

was observed between pyrosequencing and castPCR

(Spearman’s rho = 0.87; p \ 0.0001).

0

10

20

BR

AF

c.1

799T

>A (

% a

llele

s)

30

40

00

10

20

30

40

1 2 3

Tumor size (cm)

Mut

ant

alle

le f

requ

ency

(%

)

4 5 6

(a)

(b)

FIG. 1 a The percentage of mutant BRAF c.1799T [ A (p.Val600-

Glu) alleles detected by mass spectrometric genotyping in 50

papillary thyroid carcinomas. b Scatter plot showing the correlation

between tumor size and the percentage of mutant BRAF alleles. Lines

represent simple linear regression ±95 % confidence interval. Spear-

man’s rho = 0.47, p = 0.0005; simple regression adjusted r2 = 0.21,

p = 0.001

S.-P. Cheng et al.

The percentage of mutant BRAF alleles was positively

correlated with tumor size (Spearman’s rho = 0.47;

p = 0.006). In addition, the mutant allele frequency was

associated with extrathyroidal invasion (p = 0.010) and

stage III/IV disease (p = 0.009), but not clinical or patho-

logical nodal metastasis (p = 0.075 and 0.602, respectively).

DISCUSSION

The occurrence of BRAF mutation in PTC has been

extensively investigated. It appears that the development of

BRAF mutation in the thyroid gland is associated with

environmental factors and iodine intake.19,20 The preva-

lence of BRAF mutation in PTC varies according to the

detection methods, ranging from 27 to 90 % in the litera-

ture.9,10,21 Among the mutation detection methods, Sanger

sequencing is least sensitive and capable of detecting

samples having 20–30 % mutant alleles.22 Additionally,

the mutation detection may be influenced by DNA

extraction methods. Kim et al. 23 showed that direct DNA

sequencing and pyrosequencing to detect BRAF mutation

were more accurate in fresh-frozen than formalin-fixed and

paraffin-embedded tissue sections. In this study, we

confirmed that Sanger sequencing may fail to detect BRAF

mutation in a small portion of frozen PTC specimens which

contain a low allelic fraction.

BRAF mutation can be found in a substantial number of

microcarcinomas.23,24 In transgenic mouse models, mice

with targeted expression of BRAFV600E in thyroid cells

developed PTC characteristics of the human disease.25,26

These findings indicate that BRAF mutation represents an

early, initiating event in tumor development. However,

discordant patterns of BRAF mutation were found in about

40 % of the multifocal PTCs.27,28 By analyzing the rela-

tionship between genome-wide allelic imbalances and

BRAF mutation, Jovanovic and collaborators reported that

BRAF mutation did not represent the earliest transforming

event.29 Furthermore, de novo BRAF mutation was

observed in metastatic lymph nodes from mutation-nega-

tive primary tumors.30 These results imply that BRAF

mutation may be a late genetic change heterogeneously

occurring in the tumor bulk. As such, the role of BRAF

mutation in pathogenesis of thyroid cancer appears com-

plicated and conflicting, and is being actively debated.31

Resolving these contradicting views is important to

understanding the oncologic and therapeutic significance of

TABLE 2 Clinicopathological parameters and BRAF c.1799T [ A mutation status determined by mass spectrometric genotyping

Mutant allele 0–10 %

(n = 16)

Mutant allele 10–27.5 %

(n = 16)

Mutant allele [ 27.5 %

(n = 18)

p-Value

Female sex 13 (81) 14 (88) 13 (72) 0.615

Age at diagnosis (years)a 37 (32–58) 56 (52–59) 41 (33–50) 0.053

Body mass index (kg/m2)a 23.4 (20.1–26.8) 24.0 (20.5–26.0) 26.6 (23.0–29.4) 0.191

Body weight (kg)a,b 58 (54–69) 55 (47–65) 74 (58–80) 0.023

Tumor size (cm)a,c 1.3 (1.1–2.0) 2.3 (1.5–3.2) 2.5 (2.0–3.5) 0.004

Thyroiditis 5 (31) 2 (13) 1 (6) 0.140

Extrathyroidal invasion 0.024

None 10 (63) 4 (25) 5 (28)

Microscopic 5 (31) 3 (19) 7 (39)

Macroscopic 1 (6) 9 (56) 6 (33)

Multifocality 4 (25) 9 (56) 4 (22) 0.111

Lymphovascular invasion 1 (6) 6 (38) 7 (39) 0.062

Clinical lymph node metastasis 2 (13) 5 (31) 9 (50) 0.070

Pathological lymph node

metastasis

9 (56) 9 (56) 11 (61) 1.000

TNM stage 0.029

I 11 (69) 4 (25) 11 (61)

II 2 (13) 0 (0) 0 (0)

III 2 (13) 5 (31) 3 (17)

IV 1 (6) 7 (44) 4 (22)

Data are expressed as number (%) or median (interquartile range)a Kruskal–Wallis testb Dunn’s post hoc tests: tertile 1 vs. tertile 2, p = 1.000; tertile 2 vs. tertile 3, p = 0.021; tertile 1 vs. tertile 3, p = 0.256c Dunn’s post hoc tests: tertile 1 vs. tertile 2, p = 0.098; tertile 2 vs. tertile 3, p = 0.855; tertile 1 vs tertile 3, p = 0.003

Allelic Percentage of BRAF Mutation

BRAF mutation. In the hierarchical model of clonal evo-

lution, early somatic mutations tend to be propagated in

many or all clones, whereas later events occur only in some

clones.32 A 50 % percentage of mutant BRAF alleles would

be the expected finding, if all PTC cells carry one mutant

and one wild-type BRAF allele. To test this concept, Guerra

and colleagues first demonstrated that most PTCs consist of

a mixture of tumor cells carrying wild-type and mutant

BRAF.33 Gandolfi et al.13 also found an average mutated

allele percentage of 27.4 % with a range between 7.5 and

49.8 %. In line with their observations, our data showed

that the mutant allele frequency was less than 25 % in 18

(44 %) of 41 PTCs positive for mutation. To further

examine the biological relevance of clonality of BRAF

mutation, we studied the correlation between the mutant

allele frequency and clinicopathological parameters. Our

results indicate that larger tumors and tumors with extra-

thyroidal invasion had a significantly higher mutant allele

frequency. These findings are consistent with recent meta-

analyses.5,9–11 Of note is the fact that a strong correlation

existed between tumor size and mutant allele frequency. In

agreement with the present observations, Guerra and

colleagues demonstrated that tumor volume directly cor-

related with the mutant allele frequency.12 A reasonable

interpretation of these results is that BRAF mutation pro-

vides cancer cells a survival and growth advantage,

resulting in selection of BRAF mutant alleles during tumor

progression. These data also refute an alternative hypoth-

esis which proposed that BRAF mutation occurs as a

primary genetic event but is later removed by the DNA

repair machinery.31

In contrast to the results of previous meta-analy-

ses,5,9–11 our data indicate that BRAF mutation was not

associated with lymph node metastasis. It is worth noting

that in most studies, neck dissections were performed for

suspicious lymphadenopathy.9 We observed a small and

inconsistent difference in the mutant allele frequency

between clinically node-positive and node-negative

tumors. When prophylactic central compartment dissection

was performed, the mutant allele frequency was not

associated with pathological nodal status. In accordance,

Gandolfi et al.13 found that the average mutated allele

0

cN0

cN1

10

20

Mut

ant

BR

AF

(%

alle

les)

30

40

P = 0.037

0

pN0

pN1

10

P = 0.984

Mut

ant

BR

AF

(%

alle

les)

20

30

40

(a)

(b)

FIG. 2 Vertical scatter plots of the percentage of mutant BRAF

alleles grouped by a clinical and b pathological lymph node

metastasis. Lines represent median values. Mann–Whitney tests were

performed to evaluate the difference between groups

00

10

20

30

40

50

10 20

Mass genotyping (%)

Pyr

oseq

uenc

ing

(%)

30 40

00

10

20

30

40

50

60

10 20

Mass genotyping (%)

cast

PC

R (

%)

30 40

(a)

(b)

FIG. 3 a Scatter plot showing the correlation of the mutant BRAF

allele frequency determined by mass spectrometric genotying and

pyrosequencing analysis. Spearman’s rho = 0.97, p \ 0.0001; simple

regression adjusted r2 = 0.98, p \ 0.001. b Scatter plot showing the

correlation of the mutant BRAF allele frequency determined by mass

spectrometric genotying and competitive allele-specific TaqMan PCR

(castPCR). Spearman’s rho = 0.87, p \ 0.0001; simple regression

adjusted r2 = 0.85, p \ 0.001

S.-P. Cheng et al.

percentage was significantly lower in corresponding met-

astatic nodes compared with the primary PTCs (18.6 vs.

28.6 %). Therefore, it is likely that cancer cells harboring

BRAF mutation do not really have enhanced metastatic

capacity. More frequent lymph node metastasis observed

in BRAF mutation-positive tumors may result from a lar-

ger tumor burden and more extensive extrathyroidal

invasion. It was recently showed that BRAF mutation was

the only independent predictor of central compartment

lymph node metastasis in PTC;34 however, the design of

the study was biased by including BRAF status as a cri-

terion for prophylactic nodal dissection.

A plethora of genetic and phenotypic heterogeneity

comes from ongoing genetic instability within a cancer.

Most of the spontaneous mutations are evolutionarily

neutral or deleterious. Rare mutations that confer compet-

itive advantage can be selected for by Darwinian forces and

will eventually become predominant. Nonetheless,

achieving clonal dominance can take a long time, partly

because of spatial constraints.35 Our findings have impor-

tant biological implications in that they point out the

possibility that activating BRAF mutation may not neces-

sarily be an initiating prerequisite in PTC tumorigenesis,

but this secondary genetic alteration provides a survival

advantage to cancer cells, and accumulates. The fact that

the mutant allele frequency was associated with extrathy-

roidal invasion also makes it unlikely that expansion

emerges from fixation of neutral mutations over time

(genetic drift). Our results seemingly suggest that quanti-

tative assessment of mutation frequency provides no

additional information on clinical significance of BRAF

mutation (Tables 1 and 2). However, even a small portion

of mutation-negative tumor cells may hamper efficacy of

BRAF-targeted therapy. In RAS/RAF wild-type tumors,

RAF kinase inhibitors may paradoxically activate the

MAPK pathway in an RAS-dependent manner, thus

enhancing tumor growth in xenograft models.36 Further

studies may be undertaken to determine the effects of

clonality measures on clinical response to BRAF-targeted

therapy.

A limitation of our study is the inability to exclude

stromal contamination. Tumor samples always have some

degree of contamination by normal stromal, endothelial,

and inflammatory cells. The mutant allele frequency in

unselected cells would be lower than that in laser-captured

PTC cells.33 Nonetheless, the average percentage of mutant

BRAF alleles analyzed by different methods in the present

study (22.5 to 31 %) was compatible with those of recent

reports (21.9 to 27.4 %).12,13 Using next-generation

sequencing, Nikiforova and colleagues also demonstrated

that the mutant allele frequency was 18–44 % (mean 34 %)

in PTCs, corresponding to 36–88 % of cells with hetero-

zygous mutation.37

CONCLUSIONS

The percentage of mutant BRAF alleles in PTC is sig-

nificantly associated with tumor burden and extrathyroidal

invasion. A positive correlation between the mutant allele

frequency and tumor size implies that BRAF mutation may

play a role in tumor progression.

ACKNOWLEDGMENT The authors would like to thank the

Translational Core Laboratories of National Translational Medicine

and Clinical Trial Resource Center for technical assistance in

MALDI-TOF MS experiments. This work was supported by the

National Science Council of Taiwan (100-2314-B-195-001-MY3) and

Mackay Memorial Hospital (MMH-10206 and MMH-E-102-10).

DISCLOSURES Shih-Ping Cheng, Yi-Chiung Hsu, Chien-Liang

Liu, Tsang-Pai Liu, Ming-Nan Chien, Tao-Yeuan Wang, and Jie-Jen

Lee have nothing to disclose.

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