2019, Volume 35, Number 2, Page(s) 083-091
BRAFV600E Immunohistochemistry in Papillary Thyroid Carcinomas: Relationship Between Clinical and Morphological Parameters
Faruk Erdem KOMBAK1, Naziye ÖZKAN2, Mustafa Ümit UÐURLU3, Handan KAYA1
1Department of Pathology, Marmara University, School of Medicine, ÝSTANBUL, TURKEY
2Pathology Laboratory Techniques, Marmara University, Vocational School of Health, ÝSTANBUL, TURKEY
3Department of General Surgery, Marmara University, School of Medicine, ÝSTANBUL, TURKEY
Keywords: Thyroid, Papillary carcinoma, BRAFV600E, Immunohistochemistry, Reverse Transcriptase PCR, Prognosis
To investigate the association of the BRAFV600E mutation with papillary thyroid carcinoma using clinical, morphological and
prognostic parameters. We also intend to assess the utility of the BRAFV600E immunohistochemistry and compare it with BRAF polymerase
chain reaction (RT-PCR).
Material and Method: We applied BRAFV600E immunohistochemistry in a cohort of 107 papillary carcinomas, 19 adenomas and 13 normal
thyroid tissues that was chosen retrospectively between 2011 and 2015. Statistical analysis was based on semiquantitative immunohistochemistry
findings. We also applied BRAF RT-PCR in a subgroup of 14 papillary carcinomas, 13 metastatic lymph nodes and 4 adenomas that was chosen
Results: In regard to the comparison of BRAFV600E immunohistochemistry and BRAF RT-PCR, a 3+ nuclear and cytoplasmic immunoexpression
was considered ‘positive’. The BRAFV600E mutation was most frequently observed in classic variant cases. No mutation was detected in follicular
variant cases. The mutational status of the primary tumour and the lymph node metastasis was consistent. A significant relationship of the
BRAFV600E mutation was found with prognostic factors such as higher pT stage, classic variant, lymphatic invasion, perineural invasion, lower
mitotic index, lack of tumour capsule, intrathyroidal spread and extrathyroidal extension.
Conclusion: Immunohistochemistry, using the VE1 clone, is a reliable technique for detection of the BRAFV600E mutation. Our results with
immunohistochemistry are consistent with a previous effort. In our study, despite the correlation between some pathological prognostic
parameters and the BRAFV600E mutation; poor prognosis was found to be irrelevant overall. Morphological parameters seem to be keener than
the BRAFV600E mutation. Nevertheless, different series display different results, possibly due to environmental factors. Considering this and the
proven success of targeted therapies against the BRAFV600E mutation a thorough assessment would be important.
Papillary carcinoma of the thyroid (PTC) is one of the
most common cancers with an incidence rate of 14.42 per
100 000 person-years in 2010–2013 1
. PTCs have been
known to have a better prognosis than other malignant
tumours of the human body, although around 10% exhibit
a worse clinical course than expected in PTCs. Several
immunohistochemical stains have been used, such as
cytokeratin 19 (CK19), Hector Battifora mesothelial cell-1
(HBME-1) and Galectin-3, to diagnose and detect this small
group. The rapid development in molecular pathology has
led to deeper efforts in coming up with the privileges of
targeted therapy options.
The most relevant genetic alterations in PTC are generally
mutually exclusive and in the vast majority of cases cause
activation of the MAPK pathway; such as BRAF, RET
and RAS mutations with BRAF mutations in the centre
of attention 2. Mutations affecting the BRAF protooncogene
are point mutations, small in-frame deletions,
insertions and chromosomal rearrangements; the most
frequent of which is the BRAFV600E point mutation. As
a group, BRAF mutations activate BRAF kinase and lead to
chronic stimulation of the mitogen-activated protein kinase
pathway. In PTCs, BRAF mutations have been postulated
as a cause of tumour recurrence 3 and worse prognosis
4,5, along with initial tumour pathogenesis. Polymerase chain reaction (RT-PCR) and Sanger sequencing are
the gold standard techniques to detect BRAFV600E
mutation, whereas immunohistochemistry (IHC) needs
more scientific evidence of high specificity and sensitivity.
Some morphological findings like multicentricity, lymph
node metastasis, tumour extension beyond the thyroid
parenchyma and Psammoma bodies 6 may also predict
the BRAFV600E mutation.
CK19 is a low molecular weight cytokeratin found in simple
and complex epithelia, as well as in some carcinomas. An
increased intensity of CK19 immunostaining is used for
the diagnosis of PTC. HBME-1 is a marker of the apical
surface of the mesothelium. An apical membranous
staining of HBME-1 is also seen in PTCs. Galectin-3 is a
β-galactoside binding lectin in charge of cell adhesion.
Nuclear and cytoplasmic immunostaining is seen in PTCs.
Ki-67 detects the nuclei of cells in late G1, S, G2 and M
phases. Proliferation index in PTCs is no more than 5% in
The aim of this study is to evaluate the immunoexpression
of BRAFV600E, and its correlation with clinicopathologic
The study involved the use of formalin-fixed paraffinembedded
tissue sections of histopathologically diagnosed
cases of PTC (n = 107) and adenoma (n = 19) from the
archives of the Department of Pathology. Of the PTC
cases, 23 were microcarcinomas: 2 were follicular variants,
1 was oncocytic and 20 were classic. Twenty-three of the
PTC group had metastatic lymph nodes available. These
lymph nodes were assessed similarly. The slides that had
been routinely stained with hematoxylin and eosin, CK19,
HBME-1, Galectin-3 and Ki-67 were re-evaluated. The pT
stage, necrosis, calcification, lymphatic invasion, vascular
invasion, perineural invasion, tumour capsulation and
capsule invasion, extrathyroidal extension, multicentricity,
intrathyroidal spread and surgical margin status were
assessed as prognostic parameters. To avoid controversy,
certain criteria were used 8
as elaborated below.
Vascular invasion was defined as a direct tumour extension
into the blood vessel lumen or a tumour aggregate within
the vessel lumen. The criteria for vascular invasion are as
• The affected vessel must be located within the capsule
or immediately beyond the capsule but not within the
tumour nodule itself.
• The vessel should have a clearly identifiable wall with
• If a tumour extends directly into the vessel lumen, it
should form a polypoid mass protruding into the lumen
or exhibit thrombus formation in association with the
tumour and not just bulging into the lumen.
• The cell aggregates within the lumen should be
histologically identical to the tumour cells and be
composed of epithelial cells and not of reactive
• The intravascular tumour aggregate should be attached
to the wall of the blood vessel and covered by a layer of
Extrathyroidal extension was defined as tumour
penetration through the thyroid pseudocapsule into the
adjacent skeletal muscles or other organs.
Intrathyroidal spread was defined as an intraglandular
dissemination of a tumour via lymphatic channels, and
multiple small or larger satellite foci in the vicinity or
remotely from the main tumour mass.
Information regarding the gender and age of the patients
was obtained from the automation system of our hospital.
Clinical follow-up was provided by the general surgery
department. PTC cases were classified as either ‘good
prognosis (GP)’ or ‘poor prognosis (PP)’ upon the clinical
occurrence of lymph node metastasis, local recurrence and/
or distant metastasis. Clinicopathologic features are shown
in Table I. Serial sections (4 μm thick) were obtained from
the paraffin-embedded blocks of the selected preparations
and fixed on positively charged slides to perform IHC.
BRAFV600E IHC was performed manually using the
Novolink® Polymer Detection System (Leica, Australia).
Additional information on IHC is summarised in Table II.
Positive and negative control slides were also stained. All
31 cases (14 primary tumours (PT), 13 metastatic lymph
nodes and 4 adenomas) were selected randomly, and BRAF
mutation analysis was performed on these cases using the
Cobas® 4800 RT-PCR System (Roche Diagnostics, USA).
Assessment of Immunostaining
The assessment of BRAFV600E IHC in PTCs, melanomas
and colonic adenocarcinomas is still under debate
9. However, in our study, nuclear with or without
cytoplasmic staining was considered positive as in most
of the literature. A semiquantitative approach was used
based on the staining intensity of positively stained cells:
negative, 1+ (weak staining), 2+ (moderate staining)
and 3+ (strong staining) of any proportion of tumour
cells (Figures 1-4). Any proportion of tumour cells with
membranous and cytoplasmic staining with CK19, apical membranous staining with HBME-1 and nuclear and/
or cytoplasmic staining with Galectin-3 was considered
positive. The eyeballing technique was used for the Ki-67
labelling index, and 100 tumour cells were counted in hot
spot areas. A proportion of nuclear-stained cells with Ki-67
was recorded and divided into groups with a threshold of
Statistical assessments were performed using the SPSS
software (SPSS version 15, SPSS Inc., Chicago, IL, USA).
Continuous variables were expressed as mean ± standard
deviation together with a range (minimum–maximum).
The comparison of categorical variables was performed
using the chi-square test. Fisher’s exact test was used to
compare BRAFV600E IHC and RT-PCR. A P-value of less
than 0.05 was accepted as statistically significant.
Of the PTC cases, 43% (n = 46) was pT1, 27% (n = 29) pT2
and 30% (n = 32) pT3. The mean tumour size was 20.98 mm
(min. 3 mm and max. 75 mm) in PTCs and 25.79 mm (min.
9 mm and max. 67 mm) in adenomas. Of the PTC cases, 45%
(n = 48) was classic variant, 23% (n = 25) follicular, 10% (n
= 11) oncocytic and 22% (n = 23) microcarcinomas (Figures
5-8). Of the microcarcinomas, 87% (n = 20) was classic
variant, 9% (n = 2) follicular and 4% (n = 1) oncocytic. Of
the adenomas, 15.8% (n = 3) was oncocytic.
Twelve of the PTC cases were negative for HBME-1, one
was negative for CK19 and six were negative for Galectin-3.
In BRAFV600E IHC, 31 of the PTC cases were negative.
Twelve cases showed a Ki-67 proliferation index higher
than 5%. For BRAFV600E IHC, positive cases exhibited
varying percentages of staining as shown in Table III.
The results of BRAF RT-PCR of randomly selected cases
are shown in Table IV. For comparison of IHC and RTPCR,
the highest likelihood ratio (8.18) was obtained
with the hypothesis ‘Only 3+ IHC of BRAFV600E is truly
positive’. The sensitivity of BRAFV600E IHC was calculated at 90.9%, whereas the specificity was 88.8%. In addition,
the positive predictive value was 95.2%, and the negative
predictive value was 80%. Given the likelihood ratio at
8.18, 1 of every 10 tests was meant to be wrong. In this
respect, the BRAFV600E IHC findings are reconsidered
and summarised in Table V.
In cases where a metastatic lymph node (MLN) is present,
the BRAFV600E positivity was 11% in PT. Two of those
showed no staining in MLN, whereas PT was positive.
Another two cases showed a lower percentage of positive
tumour cells in MLN (Table VI). However, PTs and
conjugate MLNs were statistically correlated upon the
BRAFV600E mutation (Phi = 83.7%, p = 0.0001).
Click Here to Zoom
|Table VI: BRAFV600E positive cells (3+ intensity) in PTs and conjugate MLNs.
The BRAFV600E mutation was found to be statistically
correlated with a higher pT stage (p = 0.003), classic
morphology (p = 0.003), lower mitotic index (p = 0.020),
lymphatic invasion (p = 0.013), perineural invasion (p =
0.006), a lack of tumour capsule (p = 0.016), extrathyroidal
extension (p = 0.0001) and intrathyroidal spread (p =
0.0001). Noassociation was found between the BRAFV600E
mutation and the patient’s age, sex, synchronous lymph
node metastasis, necrosis, calcification, vascular invasion,
tumour capsule invasion, multicentricity, expressions of
CK19, HBME-1, and Galectin-3 and the Ki-67 proliferation index. Of the BRAFV600E-mutated cases, 52.9% exhibited
nodular hyperplasia in the non-tumoural parenchyma,
whereas 23.5% showed lymphocytic thyroiditis. Neither
had a significant association.
The clinical prognosis was assessed in two separate groups
as described earlier. The poor prognostic group was found
to be statistically correlated with a higher pT stage (p =
0.005), classic morphology (p = 0.011), calcification (p =
0.017), lymphatic invasion (p = 0.008), vascular invasion (p
= 0.0001), lack of tumour capsule (p = 0.004), extrathyroidal
extension (p = 0.0001), intrathyroidal spread (p = 0.001)
and positive surgical margin (p = 0.002). No association
was found with the patient’s age, sex, necrosis, mitotic
index, perineural invasion, tumour capsule invasion and
multicentricity. HBME-1 positive cases were found to be
correlated with PP (p = 0.049), whereas CK19 expression,
Galectin-3 expression and Ki-67 proliferation index were
In cases where the BRAFV600E mutation was present, poor
prognostic incidents such as lymph node metastasis, local
recurrence and distant metastasis were more frequent.
However, we could not reveal any statistical significance
(p = 0.255). On the other hand, BRAFV600E positive cases
constituted a minority of 40% in the poor prognostic group.
PTCs are malignant tumours with an increasing incidence
. The widespread use of radiological techniques and
fine-needle aspiration biopsy has been partly responsible
for this increasing incidence, although the vast majority
of new cases are microcarcinomas 10
. Lobectomy or
total thyroidectomy with subsequent radioactive iodine
ablation in selected patients is usually more effective;
however, approximately 10% of PTC patients suffers from
recurrence, lymph node or distant metastasis and hence
require further intervention. BRAF mutations are the
major promising development for PTC recently, providing
a highly efficient anti-tyrosine kinase therapy.
Several studies have compared BRAFV600E IHC with
molecular techniques. For instance, Qiu et al. 11 assessed
BRAFV600E IHC by distinguishing samples as positive
and negative without considering staining intensity
and percentage of stained tumour cells. The study also
compared IHC with RT-PCR and Sanger sequencing. Jung
et al. 12 also compared IHC with RT-PCR and BRAF
RNA in situ hybridisation. Zagzag et al. 13 accepted
3+ BRAFV600E staining as positive and compared IHC
with direct sequencing. Ilie et al. 14 accepted the results
as positive if 100% of the tumour cells stained 3+ and
compared IHC with direct sequencing. To sum up, the
sensitivity and specificity of BRAFV600E IHC ranges from
89% to 100% and from 61% to 100%, respectively. In our
study, we accepted 3+ BRAFV600E staining as positive,
disregarding the percentage of tumour cells, and compared
IHC with RT-PCR. The sensitivity of BRAFV600E IHC was
90.9%, whereas the specificity was 88.8%. It is important to
note that the RT-PCR system we used detects V600D and
V600K mutations along with V600E, thus giving a nondiscriminatory
Using the criteria 3+ nuclear and cytoplasmic staining in
the PTC group, the BRAFV600E mutation rate was 31.8%.
This rate increased up to 59.5% in classic variant cases, but
it decreased to 26% in papillary microcarcinomas (PMCs)
and 9% in oncocytic variant cases. In follicular variant and
adenoma cases, no mutation was detected. In the recent
literature, the BRAFV600E mutation rate has been reported
between 35% and 70%, and the mutations were more often associated with a classic variant, tall cell variant and poorly
differentiated/anaplastic carcinomas that arise from welldifferentiated
PTCs 15. The mutation rate is much lower
in follicular carcinomas 16, which is similar to our results.
Intratumoural heterogeneity is a substantial phenomenon
for understanding pathogenesis and its clinicopathologic
role. As in other BRAF-harbouring tumours such as
malignant melanomas and colorectal and pulmonary
adenocarcinomas, PTCs have been shown to exhibit
heterogeneously mutated tumour cells. Guerra et al. 17
showed BRAF-mutated tumour cells in MLNs of cases with
BRAF negative primary, prompting that BRAF mutations
constitute a subclonal alteration and may arise de novo
in BRAF negative tumours later on. On the other hand,
de Biase et al. 16 revealed a direct proportion between
tumour size and percentage of BRAF-mutated tumour cells,
suggesting that BRAF mutation is an early period alteration.
Walts et al. 18 stated 100% concordance of BRAF
mutation between PT and MLN and 92.3% concordance
between different areas of PTs. In their experience, two
BRAF-mutated PT cases exhibited BRAF-negative MLNs
and recurrent tumours afterward. We observed a range
of 80–90% BRAF-mutated tumour cells in PTs, two of
which exhibited BRAF-negative MLNs and the other two
showed a lower percentage of BRAF-mutated tumour cells
in conjugated MLNs. The existence of such subclones
disturbs the efficacy of targeted therapies. In this regard,
quantitative BRAF mutation analysis may be suggested in
PT, MLN, distant metastasis or recurrent tumour samples.
To start with associations between the BRAFV600E
mutation and clinicopathologic parameters, we found
noassociation with the patient’s age and sex, as in the largescale
meta-analysis of Wang et al. 19 and series of Shin
et al. 20. In our experience, BRAFV600 mutation was
found to be correlated with a higher pT stage, lymphatic
invasion, perineural invasion, lack of tumour capsule,
extrathyroidal extension and intrathyroidal spread. Several
studies have stated various morphological findings, and
their combinations are correlated with the BRAFV600E
mutation, interestingly having extrathyroidal extension in
Surprisingly, the BRAFV600E mutation rate was higher in
tumours with a lower mitotic index, as in tumours with a
lower Ki-67 proliferation index, despite its incoherency. No
effort has been found in the English literature that addresses
theassociation between BRAF mutations and mitotic
index or the Ki-67 proliferation index. Nevertheless, welldifferentiated
PTCs are known to have a lower proliferation
index than other malignancies. We observed that the Ki-67 proliferation index is higher than 5% in 19.6% of the PTC
cases, reaching up to 15%. In addition, we did not find any
significant association between mitotic/Ki-67 index and
worse clinical and/or pathologic prognostic parameters.
Guerra et al. 23 showed a higher rate of CK19 expression
in BRAF-mutated tumours, whereas Galectin-3 was not
associated with BRAF. In terms of HBME-1 expression and
BRAF, our effort needs to be published first. However, in
our series, no significantassociation was found between the
BRAFV600E mutation and expression of CK19, Galectin-3
In cases where follow-up data are available, a survival
analysis could not be made because there was no death by
disease. The cases were assessed in two separate groups:
GP and PP, as described earlier. The patient’s age and sex
were not found to be correlated with the prognosis. This
is despite the fact that Howell et al. 24 stated that the
BRAFV600E mutation and older age (≥ 65 years) predict
recurrence and Suman et al. 25 associated younger age (≤
45 years) with central lymph node metastasis.
In our series, PP was found to be associated with a higher pT
stage, classic morphology, calcification, lymphatic invasion,
vascular invasion, lack of tumour capsule, intrathyroidal
spread, extrathyroidal extension, positive surgical margin
and loss of HMBE-1 expression. Likewise, Rossi et al. 26
have associated PP in poorly differentiated and anaplastic
thyroid carcinomas with loss of HBME-1 expression.
The association between the BRAFV600E mutation and PP
can be properly summarised by the meta-analysis of Wang
et al. 19. In contrast to what has been reported recently.
Pelttari et al. 27, with their lengthy follow-up duration,
have shown that the BRAFV600E mutation is not correlated
with lymph node metastasis and/or recurrence. Zheng et
al. 28 have revealed that the BRAFV600E mutation and
the recurrence within PMCs are not related. Nam et al.
21 have also shown that the BRAFV600E mutation is not
significantly associated with lymph node metastasis. In these
series, despite the overall concern, some morphological
findings such as extrathyroidal extension are interestingly
correlated with the BRAFV600E mutation. For instance,
Shin et al. 20 revealed that the BRAFV600E mutation
does not seem to be associated with the overall prognosis
but morphological parameters are associated solely and
together with aggressive behaviour. We also did not find
any association between the BRAFV600E mutation and the
overall prognosis but with such morphologic parameters.
In conclusion, BRAFV600E IHC with VE1 clone can
be accepted as a reliable technique for detecting the BRAFV600E mutation. Our series of well-differentiated
PTCs has exhibited a rate of BRAFV600E mutation similar
to recent literature. With our effort, morphological findings
may be considered keener than the BRAFV600E mutation
in predicting aggressive behaviour. However, demographic,
clinical and morphological findings and genetic alterations
should be assessed together to estimate a more precise
prognosis. Although further therapeutic interventions are
needed, it is better to look for the BRAFV600E mutation in
PT, lymph node metastasis, recurrent tumour and distant
metastasis, if available.
We would like to express our sincere gratitude to Prof.
Nural Bekiroðlu (Marmara University, School of Medicine,
Department of Biostatistics) for her supervision in our
CONFLICT of INTEREST
All authors declare that there is no conflict of interest that
could be perceived as prejudicing the impartiality of the
This work was supported by the BAP (Scientific Research
Projects Unit of Marmara University, grant number SAGC-
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