Summary of Medical Doctoral thesis: Histopatholoy, immunohistochemistry, BRAF V600E mutation in recurrent, metastatic differentiated thyroid carcinoma

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEATH
HANOI MEDICAL UNIVERSITY

NGO THI MINH HANH

HISTOPATHOLOY, IMMUNOHISTOCHEMISTRY,
BRAF V600E MUTATION IN RECURRENT,
METASTATIC DIFFERENTIATED THYROID
CARCINOMA

Specialty: Pathology
Code: 62720105
SUMMARY OF MEDICAL DOCTORAL THESIS

HA NOI - 2020


The thesis was completed at:
HANOI MEDICAL UNIVERSITY

Scientific Supervisor: 1. Assoc. Prof. Trinh Tuan Dung, MD, PhD
2. PhD. Hoang Quoc Truong

Reviewer 1: Assoc. Prof. Nguy n

ong, MD, PhD

Reviewer 2: Assoc. Prof. Trần Vân Khánh, MD, PhD

Reviewer 3: Assoc. Prof. Tạ Văn Tờ, MD, PhD

The thesis was defended at Council of Scientists in Ha Noi Medical
University.
At

on

date

month

References the thesis in:
1. Vietnam National Library
2. Library of Ha Noi Medical University

2020


THE LIST OF ANNOUNCED RESEACHES
RELATED TO THE THESIS TOPIC
1. Ngo Thi Minh Hanh, Trinh Tuan Dung, Nguyen Duc Vinh,
Le Thi Thanh Xuan, Dao Anh Tuan, Nguyen Van Phu Thang
(2017), Histopathological characteristics of recurrent,
metastatic differentiated thyroid carcinoma. Journal of 108 Clinical Medicine and Pharmacy, Special issue 11, 423-427.
2. Ngo Thi Minh Hanh, Trinh Tuan Dung, Hoang Quoc Truong
(2018), Study on expression of some immunohistochemical
makers in recurrent, metastatic differentiated thyroid
carcinoma. Journal of 108 - Clinical Medicine and
Pharmacy, Special issue 7, 103-108.
3. Ngo Thi Minh Hanh, Trinh Tuan Dung, Hoang Quoc
Truong, Nguyen Thi Ngoc Anh, Le Minh Son (2018), The

value of BRAF V600E immunohistochemiscal expression in
recurrent, metastatic differentiated thyroid carcinoma,
Vietnam Medical Journal, 471(10), 166-172.
4. Ngo Thi Minh Hanh, Trinh Tuan Dung, Hoang Quoc Truong
(2019), BRAF V600E mutation and relationship with
histopathological characteristics of metastasic, recurrent
differentiated thyroid carcinoma, Journal of 108 - Clinical
Medicine and Pharmacy, 14, 127-133.
5. Ngo Thi Minh Hanh, Trinh Tuan Dung, Hoang Quoc Truong
(2019), BRAF V600E mutation in variants of metastatic,
recurrent and differentiated thyroid carcinoma, Ho Chi Minh
City Jounal of Medicine, 23(5), 94-98.


1
INTRODUCTION

Differentiated thyroid carcinoma (DTC) derived from follicular
cells, which are follicular thyroid carcinoma (FTC) and papillary
thyroid carcinoma (PTC). The disease tends to increase globally and
ranks number 1st in total cancers in Korea, 9th in United States and
Vietnam. The disease has good prognosis but 4 - 30% of cases recur,
metastasize, invasively progress, especially in radioactive iodine
(RAI)-refractory DTC.
Histopathology is considered as a hallmark for cancer diagnosis.
The histopathological types and variants of thyroid cancer could be
easily identified by pathologists, using routine hematoxylin - eosin
staining. However, in some difficult cases, pathologists must base on
immunohistochemistry (IHC), which is based on the principle
antibody - antigen reaction, to confirm diagnosis.

A number of studies have shown a link between BRAF V600E
mutation and pathogenesis, the risk of progresive disease and RAIrefactory mechanism. The understanding of BRAF V600E mutation
is opening the trend of a target therapy for patients with RAIrefractory DTC. With the aim to provide clinicians more
comprehensive information on the histological characteristics and
prevalence of BRAF V600E mutations in patients with recurrent,
metastatic DTC to help them choose an optimal treatment and give
better prognosis, we conducted the present study with two objectives:


2
1.

To

describe

characteristics

of

histopathological
recurrent,

and

metastatic

immunoshistochemical
differentiated

thyroid

carcinoma.
2. To determine the prevalence of BRAF V600E mutation, and
investigate potential association between BRAF V600E mutation and
some histopathological and clinical characteristics.

The essential of the thesis
DTC is the most common of the endocrine system and tends to
increase worldwide. The disease has a good prognosis due to
advantages of thyroid and lymph node surgery as well as RAI
therapy. However, recurrence and metastasis occur in 4 - 30% of
DTC cases, which results in poor prognosis, especially, in RAIrefractory DTC. Histopathology is gold standard for the diagnosis,
and IHC helps confirm the metastasis, progression of the disease.
BRAF V600E mutation is associated with pathogenesis, its
progression and resistance to RAI therapy, and opens a targeted
approach of cancer treatment, especially the group that no longer
respond to RAI therapy. Understanding pathogenesis, the status of
BRAF V600E mutation is essential for clinicians in selection of
appropriate therapy, management and better prognosis of DTC
patients.


3
New contribution of the thesis
- The thesis provides additional information about histopathology,
the most common variants in recurrent, metastatic DTC. These types
are classical and tall cell variant of PTC. The expression of Ki67 and
Thyroglobulin was valuable to distinguish histological variants with
intermediate malignancy with aggressive variants. The prevalence of

BRAF V600E was high (70.2%), and was more frequent in a group
of RAI-refractory DTC, extra-lymph node extension and tall cell
variants.
- The present study has demonstrated that BRAF V600E mutation
could be identified by IHC, which has high sensitivity (95.9%),
specificity (90.3%), accuracy (94.2%) compared to Realtime PCR.

The layout of the thesis
- The thesis consists of 134 pages, including 2 pages of
Introduction, a 34-page Overview, 23-page Objectives and Study
Method,

35-page

Result,

39-page

Discussion, 2

pages

Conclusions; 41 tables, 2 graphs, 13 figures and 146 references.

of


4
HƯƠNG 1: OVERVIEW
1.1. Recurrent, metastatic differentiated thyroid carcinoma

1.1.1. Mechanisms of recurrent, metastatic differentiated thyroid
carcinoma
Metastasis is comprised of a series of complex steps that
ultimately result in the presence of growing tumor implants in nonnative locations, including the stages: tumor cells detach from the
primary tumor, intravasate into vascular system, arrive within the
vasculature of an organ, extravasate from the vascular system and
proliferate in the target tissue. Many studies have explained more
about the phenomenon of metastases, including metastatic dormancy
and cancer stem cell.
Metastatic dormancy is defined as the ability of individual or
small clusters of cancer cells to migrate to a distant site and then
survive in a quiescent state for an extended period without growth.
DTC and medullary carcinoma have the most common metastatic
dormancy phenomenon in thyroid cancer. Another important concept
related to metastatic dormancy is pluripotent stem cells. Cancer can
derive from pluripotent stem cells. Stem cells are undifferentiated
cells possessing the ability for indefinite self-renewal and
pluripotency. Cancer stem cells are a group of cancer cells that have
the ability for self-renewal and differentiate into muti-lineages and
matastasis. Cells that can develop thyroid cancer in fetus from
progenitor lineages of the thyroid stem cells, thyroblasts and
prothyrocytes.


5
1.1.2. Methods for diagnosis of recurrent, metastatic differentiated
thyroid carcinoma
Patients who have undergone thyroidectomy, lymphadenopathy
and I131 treatment should be monitored periodically to detect
recurrence and metastasis. Basic test methods are quantification of

Tg and anti-serum Tg, neck ultrasound and immaging test. In case of
high Tg but negative whole body scintigraphy, CT, PET/CT or MRI
is required depending on each case, according to the guideline of
American Thyroid Association 2015.
1.2. Histopathology and immunohistochemistry in recurrent,
metastatic differentiated thyroid carcinoma
1.2.1. Histopathology
The World Health Organization (WHO) classification of thyroid
carcinoma is the most widely applied. According to the first version
of the guideline, published in 1974, thyroid carcinoma was classified
into five (5) types of histopathology. After several modifications to
accommodate the progression and prognosis, the latest 2017
classification includes several new variants and highlights the
association between genotype and histological phenotype. Some
novel types are Hobnail cell variant, Cribiform-morular variant, and
Warthin-like cell variant of PTC. The oncocytic cell variant of FTC
in previous WHO classification was isolated into a separate group
because of its association with genetic mutation.
1.2.2. Immunohistochemistry
IHC is a special staining technique that uses specific antibodies to
determine the presence of antigens on a tissue slide. This is an
effective tool to help pathologists to identify thyroid follicular cells,


6
distinguish benign from malignant tumors, determine malignant
degree, the metabolic function of folicular cells, identify the invasion
of tumor and detect BRAF V600E mutation using mutation-specific
antibody (VE1).
1.3. BRAF V600E mutation in recurrent, metastatic

differentiated thyroid carcinoma
The BRAF gene (B-type Raf kinase) is located on the
chromosome 7 with a coding region containing 18 exons, size of
2478 bp which codes an 84,436 dalton protein consisting of 766
amino acids. A mutation of the T1799A position of BRAF gene
increases enzyme kinase activity and activates the MAPK signaling
pathway, plays a role in cell proliferation, differentiation and death.
Activation of the BRAF V600E gene in thyroid carcinoma was first
reported in 2003. The BRAF V600E mutation plays an important role
not only in causing thyroid cancer, but also in promoting invasion
and progression of the tumour. Studies have also linked the role of
BRAF V600E mutation is associated with the loss of NIS, and
susbequent radioiodine-refractory.


7
CHAPTER 2. STUDY SUBJECTS AND METHOD
2.1. Study subjects
104 patients with recurrent, metastatic DTC who were operated
recurrent, metastatic lesions at Military Central Hospital 108 between
March 2012 and July 2018.
* Inclusion criteria
- DTC patients who had total thyroidectomy, I131 treatment, and
were periodically monitored.
- Presence of recurrent, metastatic lesions based on immaging
diagnosis (ultrasound, CT, PET/CT) 6 months after the first I131
treatment.
- Histopathological confirmation of recurrence and metastasis.
- Availability of clinical data and good quality formalin-fixed
paraffin-embbed tissues.

* Exclusion criteria
- Patients with distant metastasis at the diagnosis time of primary
DTC.
- Patients with recurrent, metastatic DTC, but not suitable for
surgical treament.
- Patients who withdrew from treatment and follow up.
- Patients had an other cancer apart from DTC.
- Patients who refused to participate in the study.
2.2. Method
2.2.1. Study design: cross-sectional study


8
- Sample size: calculated using the formula for estimating a
sample size for prevalence.

N= Z21-α/2 p(1 2- p)

(1)

d

n: study sample size,

α (level of significance) : 0.05

p: estimated prevalence. According to Barollo (2010), the prevalence
of BRAF V600E mutation among PTC recurrence was 73%. In the
present study, p = 0.73.

Z (21 / 2)

: alpha level’s z-score, with  = 0.05,

Z (21 / 2)

=

1.962 for the confident interval of 95%
d: margin of error, in the present study, d = 0.1
Using the formula (1), the minimum number of patients needed to
be recruited for the study was: n = 76 patients.
2.2.2. Data collection tools and method
* Extract clinical data from medical records
- Classificaiton of TNM was based on AJCC 2010.
- Recurrent risk was assessed using ATA 2009.
- Classification of RIA-refractory DTC was based on ATA 2015.
* Histological studies
- Histopathological types were determined according to WHO
2017.
- Evaluation of intravascular tumor cells should be adherent to
the vessel walls, either covered by endothelium or in a context of
thrombus or fibrin.


9
- Evaluation of extranodal extension when tumor cells extending
beyond the lymph-node capsule into the perinodal fibroadipose
tissue.

* Immunohistochemistry studies
- IHC staining was conducted using Ventana's automated
BenchMark Ultra machine, with Thyroglobulin, TTF1, CK19,
HBME1, P53, Ki67, CD56, Galectin-3, BRAF V600E.
- Result evaluation: negative when the nucleus presented only
blue color, positive when the nucleus presented yellow brown color.
+ No staining of tumor cells is considered negative
+ Less than 25% of tumor cells stainted was considered positive
(+)
+ Between 25 - 50% of tumor cells stainted was considered
positive (++)
+ Equal or over 50% of tumor cells stainted was considered
positive (+++)
- Ki67 was identified in the largest proliferating region, showing
the percentage of positive tumor cells after counting at least 1,000
cells in each region.
- P53 was considered positive when ≥ 5% of nucleus presented
yellow brown colour.
* Detection of BRAF V600E mutation


10
- DNA extraction was conducted using the CE-IVD biological kit,
Invisorb® Spin Tissue Mini Kit (Invisorb) manufactured by Stratec,
Germany.
- Detection of BRAF V600E mutation: by PCR technique using
CE-IVD biological kit, Thyroid Cancer Mutation Analysis Kit
manufactured by EntroGen, USA.
2.3. Statistical analysis: using statistical software SPSS, version
20.0. Evaluation: p < 0.05 difference was statistically significant, p >

0.05: difference was not statistically significant
2.4. Ethical consideration: The present study was approved by the
Ethics Committee of Hanoi Medical University.
Study design


11
104 study subjects

Clinical characteristics

Objective
2

Histopathological studies

Detect BRAF

Immunohistochem

Objective

V600E mutation

ical study

1

Expression of
BRAF V600E

Expression of
Thyrolobulin, TTF1,
Galectin-3, HBME1,
Ki67, P53, CD56,
CK19


12
CHAPTER 3: RESULTS

3.1. Characteristics of histopathology and immunohistochemistry
of recurrent, metastatic DTC
3.1.1. Histopathological characteristics
- The most frequent type was PTC (96.1%), meanwhile the FTC
accounted for only 3.9%. In particular, classical PTC accounted for
53.8%, and tall cell variant accounted for 20.2%.
- The proportion of vascular invasion, necrosis, extranodal
extension was respectively 19.2%, 14.4%, 55.3%.
- Aggressive variants of PTC was more prevalent in age group ≥
45 years old, compared to the group < 45 years old (p = 0.022), and
more prevalent in males than females (p < 0.001). This variant was
also more prevalent at late stage (p = 0.013), and I131 resistant cases
(p = 0.018). The risk of recurrence and metastasis was higher in the
aggressive variants than in intermediate variants of PTC (p = 0.045).
- Aggressive variants were associated with higher risk of necrosis
(OR = 6.33, 95% CI: 1.62 - 24.8), and extranodal extension (OR =
3.42, 95% CI: 1.36 - 8.59), compared to intermediate variants of
PTC.
- No association was found between variants of recurrent,

metastatic PTC and tumor size, node metastases at primary diagnosis,
vascular invasion and calcification.


13
3.1.2. Immunohistochemistry characteristics
Table 3.9. Expression of immunohistochemistry in recurrent,
metastatic differentiated thyroid carcinoma
Histological

PTC

FTC

(N = 104)

(N = 100)

(N = 4)

(n, %)

(n, %)

(n, %)

CK19

93 (89.4)

93 (93.0)

0

Thyroglobulin

93 (89.4)

90 (90.0)

3 (75.0)

TTF1

90 (86.5)

87 (87.0)

3 (75.0)

Ki67

36 (34.6)

34 (34.0)

2 (50.0)

HBME1

102 (98.1)

99 (99.0)

3 (75.0)

5 (4.8)

5 (5.0)

0

Galactin-3

34 (32.7)

34 (34.0)

0

P53

15 (14.4)

14 (14.0)

1 (25.0)

types
Markers

CD56

DTC

- Recurrent, metastatic DTC has positive expression of CK19
(89.4%), Thyroglobulin (89.4%), TTF1 (86.5%), HBME1 (98.1%),
but lower expression of Ki67 (34.6%), Galectin-3 (32.7%) and P53
(14.4%).
- CK19, Thyroglobin, TTF1, HBME1 show diffuse destiny were
80.6%; 93.5%; 96.7%, 94%, respectively. The intensity expression of
CK19, Thyroglobin, TTF1, HBME1 was mainly at moderate and
strong level, with respective percentage of 94.6%, 100%, 94.4%,
98.0%.
- Galectin-3 was found positive mostly in the foci (47.1%), with a
weak to moderate intensity (44.1% to 47.1%).


14
- The level of Ki67 expression is low (> 5% positive, accounted
for only 8.3%), strong intensity (72.2%).
- The level of P53 expression at the moderate level accounted for
66.7 %, weak intensity (86.7%).
- Aggressive variants of PTC had lower prevalence of negative
thyroglobulin than intermediate variants of PTC, with OR = 0.14,
95% CI: 0.03 - 0.69, higher prevalence of positive Ki67 (OR = 4.0,
95% CI: 1.67 - 9.57).
3.2.

BRAF

V600E

mutation

in

recurrent,

metastatic

differentiated thyroid carcinoma
3.3.1. Prevalence of BRAF V600E mutation in recurrent,
metastatic differentiated thyroid carcinoma
Table 3.18. Value of immunohistochemistry versus Realtime-PCR
in detection of BRAF V600E mutation
Immunohistochemistry

Realtime-PCR

Total

Positive

Negative

Positive

70

3

73

Negative

3

28

31

Total

73

31

104

- The proportion of BRAF V600E protein expression determined
by IHC was 73/104 (70.2%) and the BRAF V600E mutation
proportion was 73/104 (70.2%).
- The sensitivity of IHC: 70/73 x 100% = 95.9%
- The specificity of IHC: 28/31 x100% = 90.3%
- Positive predictive value: 70/73 x 100% = 95.9%


15
- Negative predictive value: 28/31 x 100% = 90.3%

- The accuracy of IHC: (70 + 28) / 104 x 100% = 94.2%
- The proportion of mutation of BRAF V600E in PTC was 73.0%,
there was no mutation in FTC.
- BRAF V600E mutation occurred in most PTC in which classical
PTC and tall cell variants accounted for 64.3% and 95.3%,
respectively.
3.3.2. Relation between BRAF V600E mutation and some
clinicohistological characteristics
Table 3.21, 322, 323, 324, 325. Relation between BRAF V600E
mutation and clinical characteristics
BRAF V600E mutation
Characteristics

Positive

Negative

n (%)

n (%)

OR
(95%CI)

Age

≥ 45

42 (76.4)

13 (23.6)

1.87

(N = 104)

< 45

31 (63.3)

18 (36.7)

(0.80 – 4.40)

Sex

Male

20 (80.0)

5 (20.0)

1.96

(N = 104)

Female

53 (67.1)

26 (32.9)

(0.66 – 5.82)

Tumour

T3-4

15 (55.6)

12 (44.4)

T1-2

45 (81.8)

10 (18.2)

Yes

39 (68.4)

16 (31.6)

No

34 (72.3)

12 (27.7)

(0.36 – 1.94)

III- IV

44 (68.8)

20 (31.2)

1.25

size
(N = 82)
Nodal
metastasis
(N = 104)
Stage

0.28
(0.10 – 0.77)

0.83

p

0.145

0.22

0.012

0.664

0.651


16
(N = 95)

I - II

22 (73.3)

8 (26.7)

Distant

Yes

10 (58.8)

7 (41.2)

No

63 (72.4)

24 (27.6)

Yes

62 (81.6)

14 (18.4)

No

11 (39.3)

17 (60.7)

metastasis
(N = 104)
Resistance
131

to I

(N = 104)

(0.48 – 3.29)
0.54
(0.19 – 1.60)

0.263

6.84

<

(2.63 – 17.7)

0.001

BRAF V600E mutation was more frequent in T1-2 tumours,
compared to T3-T4 ones (p = 0.012). The percentage of this mutation
was also higher in the group of RAI-refractory DTC, compared to the
group of non-RAI-refractory DTC (p < 0.001), but was not related to
age, gender, disease stage, lymph node and distant metastasis.
Table 3.26, 3.27, 3.28. Relation between BRAF V600E mutation
and some histopathological characteristics
BRAF V600E
mutation
Characteristics

> 50%
Structure
(N=104)

papillary

Positive

Negative

n (%)

n (%)

50 (76.9)

15 (23.1)

OR
(95%CI)

2.32
(0.98 – 5.48)

solid

23 (59.0)

16 (41.0)

PTC variants

Tall cell

20 (95.2)

1 (4.8)

11.11

(N=104)

Classical

36 (64.3)

20 (35.7)

(1.39 – 89.1)

p

0.053

0.008


17
Vascular

Yes

14 (70.0)

6 (30.0)

No

59 (70.2)

25 (29.8)

(0.34 – 2.87)

Yes

42 (80.8)

10 (19.2)

2.59

No

26 (61.9)

16 (38.1)

Necrosis

Yes

11 (73.3)

4 (26.7)

1.2

(N=104)

No

26 (61.9)

27 (30.3)

(0.35- 4.10)

Calcification

Yes

21 (70.0)

9 (30.0)

0.99

(N=104)

No

52 (70.3)

22 (29.7)

(0.40 – 2.50)

invasion
(N= 104)
Extranodal
extension
(N=94)

0.99

(1.02 – 6.55)

0.983

0.042

0.774

0.978

The percentage of BRAF V600E mutation was higher in tall cell
variant of PTC, compared to classical PTC (p = 0.008). This
percentage was also higher in extranodal extension compare to nonextranodal extension (p = 0.042).

CHAPTER 4. DISCUSSION
4.1. Characteristics of histopathology and immunohistochemistry
- PTC was the most frequent among recurrent, metastatic DTC,
accounted for 96.1%, while FTC accounted for only 3.9%. PTC
usually spreads via lymphatic system, whereas FTC spreads via
blood vessels. Our study focused on recurrent, metastatic cases,
which its lesions were removed. Therefore, PTC was more frequent
in the present study.


18
- We found 19.2% of recurrent, metastatic DTC having vascular
invasion. This percentage reported by other studies ranged from 7.6
to 33%. Necrosis rate was 14.4%. Necrosis is one of the criteria to
classify poorly DTC thyroid carcinoma. Therefore, when observing
signs of necrosis, pathologists should pay more attention along with

other criteria to better evaluate the histopathology variants.
- The latest WHO 2017 guideline classifies PTC into 15 variants,
which are subsequently categorized into three (3) groups of low,
intermediate and high malignancy. In a study of Rivera, the
aggressive variants were more frequent in RAI-refractory DTC,
accounted for 63%.
The aggressive variants had higher rate of extranodal extension
than the intermediate group (p = 0.008). Extranodal and peri-thyroid
invasion was associated with an increased incidence of relapse and
death. In recurrent DTC, lymphatic metastasis was more invasive of
fibroadipose tissue than those in primary DTC. This might be partly
due to the difficulty in removing cervical lymph nodes at the first
surgery, especially for those having multiple and large lymph nodes.
- IHC is a useful tool in pathology. In most cases of thyroid
tumors, pathologists can easily make a diagnose of a biopsy
specimen, by using routine HE staining. But in some cases,
especially

recurrent

and

metastatic

DTC,

determination

of

histopalogical type can be difficult.
The proportion of 86.5% positive TTF1 expression was 87.0% in
PTC, in general, and this proportion was 75% in FTC. Positive


19
expression of TTF1 was strong and diffuse in most variants. This is
an important marker in the differential diagnosis from other
carcinoma, determing the origin of cell tumor, especially from
metastatic cancer. Thyroglobulin expression was less frequent in
aggrasive variants than in intermediate variants (p = 0.006). The
degree of malignancy of tumor cells can affect the ability to
synthesize Thyroglobulin, and this abnormality can be detected by
IHC method. Furthermore, thyroglobuin expression is also influenced
by the degree of tumor differentiation. Thyroglobulin expression was
more highly positive in well differentiated tumor.
CK19, HBME1 and Gelectin 3 are effective markers for the
diagnosis of PTC, but rarely express together in FTC. The expression
intensity of CK19, HBME1 was strong and diffuse while Galectin-3
was usually at foci and moderate intensity.
P53 expression was less prevalent in DTC (14.4%) and there were
no strong positive cases. In fact, P53 reveals through IHC in poorly
thyroid carcinoma and anaplactic thyroid carcinoma . On the other
hand, overexpression of the P53 protein is not always due to the
TP53 mutation.
Ki67 is an indicator to evaluate cell proliferative activity. In our
study, Ki67 positive (> 1%) accounted for 34.6%, and this result was
similar to the study of Siironen: Ki67 positive (>1%) was observed in
30% of PTC. The expression of Ki67 was higher in agressive
variants, compared to the intermediate ones.

20
In general, in the diagnosis of recurrent, metastatic DTC, there is
no specific markers for a particular cancer type. The diagnosis of
histological type of thyroid carcinoma has to rely on a combination
of multiple markers, depending on their sensitivity, specificity, and
molecular manifestation of recurrent, metastatic DTC.
4.2. The prevalence of BRAF V600E mutation and its relation to
some clinical and histopathological characteristics
- The IHC method for BRAF V600E mutation has a sensitivity of
95.9%, a specificity of 90.3%, a positive predictive value of 95.9%, a
negative predictive value of 90.3%, and an accuracy of 94.2%. In
Ventana's study, the sensitivity and specificity of IHC method to
detect genetic mutation in PTC were 100% and 75%, respectively.
87.7% BRAF V600E protein expressed at moderate and strong
indensity. Weak, foci or unclear positive may be uncertain for
diagnosis, and an another molecular test will be required to determine
the status of BRAF V600E.
- The proportion of BRAF V600E mutation in recurrent,
metastatic DTC was 70.2%. This proportion in recurrent, metastatic
PTC was 73.0%, and no BRAF V600E mutation was found in FTC.
According to previous published studies, BRAF V600E mutation
accounted for 29-83%.
The highest proportion of BRAF V600E mutation was observed
in tall cell variants (95.2%), followed by classical PTC (64.3%). In a
number of other studies, authors also reported that the BRAF V600E
mutation was the most frequent in aggressive variants as tall cell

21
variants, classical PTC, but less frequent in FTC, with the respective
proportion of 77%, 60% and 12%.
The proportion of BRAF V600E mutation was higher in T1 - T2
stage patients than in T3-T4 stage ones (Table 3.21). In Xing's study,
the average tumour size of patients with BRAF V600E mutation was
2 cm (1.4 to 3.0 cm), while this size in those without the mutation
was 2.4 cm (1.5 - 3.5 cm), p = 0.009. It can be explained that the
occurrence of BRAF V600E mutation activates the MAPK pathway,
which initiates the process of thyroid cell growth and proliferation. In
fact, the progression of disease is related to aggressive variants (e.g.
hobnail cell, solid and diffuse sclerosing variant). In these variants,
the size of the tumor is larger but the BRAF mutation rate is lower.
BRAF V600E mutation of RAI- refractory DTC in our study was
81.6%. In other studies, this proportion ranged from 43 % to 66 % in
recurrent, metastatic DTC, meanwhile, it is higher, up to 94% of RAIrefractory DTC.
Tall cell variant of PTC had a higher rate of BRAF V600E
mutation than classical PTC (p = 0.008). In other studies, the rate of
BRAF V600E mutation observed in tall cell variant (even PTC which
consist of 10% tall cells) was higher than classical PTC.
- BRAF V600E mutation was higher in patients with extranodal
extension (p = 0.042). In the study of Ricate-Fihlo, this mutation was
not related to age, gender, tumor size, extra-thyroid and vascular
invasion, as well as extranodal extention, but the prognostic value of
survival time significantly increased when combining BRAF V600E


22
mutation with the presence of extranodal extention. The survival
predictive value of this combination (p = 0.004) was higher than that of

BRAF V600E mutation (p = 0.02) or extranodal extention (p = 0.12)
alone.
- There was no association between BRAF V600E mutation and
age, tumour stage, node and distant metastasis, vascular invasion,
presence of necrosis and calcification.

CONCLUSION
1. Characteristics of histopathology and immunohistochemistry
in recurrent, metastatic differentiate thyroid carcinoma
1.1. Histopathological characteristics
- Recurrent, metastatic location: the highest proportion is cervical
node metastasis (90.4%), followed by local recurrence (13.5%).
- PTC accounted for the highest proportion (96.1 %), followed by
classical PTC (53.8 %), and tall cell variant (20.2 %).
- The proportion of vascular invasion, necrosis, extranodal extension
and calcification was 19.2%, 14.4%, 55.3% and 28.8%, respectively.
- The aggressive variants were associated with necrosis,
extranodal extension, sex, RAI-refractory DTC, more frequent in
patients ≥ 45 years old (compared to < 45 years old).
1.2. Immunohistochemical characteristics
- The proportion of recurrent, metastatic DTC which were positive
with HBME1 was 98.1%, followed by CK19 (89.4%), Thyroglobulin
(89.4%), TTF1 (86.5%), Ki67 (34.6%), Galectin-3 (32.4%) and P53
(14.4%).