#: the association of the braf v600e mutation wi … [email protected] 5. forough foroughi:...
TRANSCRIPT
Journal: International Journal of Endocrinology and Metabolism
Section: General
Manuscript Type: Article Commentary
Manuscript Full Title: The association of the BRAF V600E mutation with tissue inhibitor ofmetalloproteinase-3 expression and clinicopathological features in papillary thyroid cancer;Revision: 0
Abstract:
Keywords:
Submitted by
Zarkesh Maryam [email protected],Zadeh-Vakili [email protected],Azizi Fereidoun [email protected],fanaie seyed [email protected],Foroughi Forough [email protected],Hedayati Dr. Mehdi*[email protected]
List of Uploaded Files:
cover letter docx.docx,Title page-4.4.96 dox.docx,Manuscript-4.4.96 without title pagedocX.docx
Classifications:
Suggested Reviewers:
Opposed Reviewers:
Author Final Comments:
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 1 of 25 25 June 2017 10:32:14
The Editors,� International Journal of Endocrinology and Metabolism�
Dear Modem/Sir,
We intend to publish an article entitled �The association of the BRAF V600E mutation with tissue inhibitor of metalloproteinase-3 expression and clinicopathological features in papillary thyroid cancer� in your esteemed journal as an original article. On behalf of all the contributors I will act and guarantor and will correspond with the journal from this point onward.
The authors hereby affirm that the manuscript is original, that all statements asserted as factsare based on author�s careful investigation and accuracy, that the manuscript has not beenpublished in total or in part elsewhere. The authors have full power authority to enter into thiscopyright assignment and to make the grants herein content. Each author acknowledges he/she has participated in the work in a substantive way and isprepared to take public responsibility for the work.
Yours� sincerely,
Corresponding contributor:Mehdi Hedayati (PhD.)Associate Professor,Cellular and Molecular Endocrine Research Center (CMERC), Research Institute for Endocrine Sciences of Shahid Beheshti University of Medical Sciences,Tehran, IranPo Box: 19395-4763Tel: +98(21)22432500Fax: +98(21)22416264Email: [email protected]
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 2 of 25 25 June 2017 10:32:14
Title: The association of the BRAF V600E mutation with tissue inhibitor of metalloproteinase-3 expression and clinicopathological features in papillary thyroid cancer
Authors
1. Maryam Zarkesh: Cellular and Molecular Endocrine Research Center, Research Institute forEndocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Email: [email protected]. Azita Zadeh-Vakili: Cellular and Molecular Endocrine Research Center, Research Institute for
Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, IranEmail: [email protected]
3. Fereidoun Azizi: Endocrine Research Center, Research Institute for Endocrine Sciences, ShahidBeheshti University of Medical Science, Tehran, Iran
Email: [email protected]. Ahmad S. Fanaei: Association Professor of General Surgery, Erfan Hospital, Tehran, Iran
Email: [email protected]. Forough Foroughi: Department of Pathology, Shahid Beheshti University of Medical Sciences,
Tehran, IranEmail: [email protected]
6. Mehdi Hedayati: Cellular and Molecular Endocrine Research Center, Research Institute forEndocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Email: [email protected]
Running title: BRAF V600E mutation & TIMP3 expression in PTC
Corresponding author
Mehdi Hedayati (Ph.D.)Associate Professor,Cellular and Molecular Endocrine Research Center (CMERC),Research Institute for Endocrine Sciences of Shahid Beheshti University of Medical Sciences, Tehran, IranPo Box: 19395-4763
1
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 3 of 25 25 June 2017 10:32:14
Tel: +98(21)22432500Fax: +98(21)22416264Email: [email protected]
2
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 4 of 25 25 June 2017 10:32:14
Title: The association of the BRAF V600E mutation with tissue inhibitor of metalloproteinase-3 expression and clinicopathological features in papillary thyroid cancer
Running title: BRAF V600E mutation & TIMP3 expression in PTC
1
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 5 of 25 25 June 2017 10:32:14
Abstract
Background: Papillary thyroid cancer (PTC) is the most common endocrine malignancy. The
aim of this study was to investigate the association of tissue inhibitor of metalloproteinase-3
(TIMP3) expression in thyroid tissue, based on BRAF V600E status with the clinicopathologic
characteristics of PTC.
Methods: Sixty fresh frozen tissues of PTC patients (15 male and 45 female) were collected
during thyroidectomy surgery. All clinicopathological information was obtained, and samples
were reviewed by a pathologist and confirmed. Exon 15 of the BRAF gene was genotyped by
sequencing; TIMP3 gene expression was assessed using SYBR-Green Real-Time PCR, and
TIMP3 protein expression was measured using ELISA.
Results: The BRAF mutation was found in 24 (40%) of the 60 PTC cases. BRAF (+) PTC had
significantly larger tumor size than BRAF (-) PTC (P= 0.039). Lymph node metastasis frequency
was significantly higher in BRAF (+) compared to the BRAF (-) group (P=0.030). No significant
reduction was seen in the tumoral tissues of the TIMP3 gene expression in BRAF (+) compared
to BRAF (-) PTC samples. However, mean TIMP3 protein level was significantly lower in
tumoral tissues compared to matched non-tumoral tissues in BRAF (+) PTC (P=0.003); TIMP3
protein level significantly was lower in tumoral tissues compare to matched non-tumoral in
BRAF (+) in subjects who had not lymph node metastasis and also subjects with lymph node
metastasis in both BRAF positive and negative.
Conclusion: The lower TIMP3 expression associated with the lymph node metastasis in PTC
patients. Our results showed that BRAF mutation was associated with larger tumor size, higher
frequency of lymph node metastasis, and lower TIMP3 protein expression.
Keywords: BRAF V600E, TIMP3 expression, papillary thyroid cancer
2
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 6 of 25 25 June 2017 10:32:14
Introduction
Papillary thyroid cancer (PTC), a follicular epithelial cell-derived thyroid cancer, is the most
common endocrine system malignancy, which accounts for approximately 85% of thyroid
cancers (1).
According to data from the Surveillance, Epidemiology, and End Results (SEER) the incidence
of PTC increased 2.9 fold from 1973 to 2002 among the American populations aged over 65
years; with a rapid increase in PTC incidence from 0 to 29.7 per 100,000 (2, 3). The prevalence
of PTC differs in various countries, and particularly among Asian populations, in whom it is
higher than other ethnicities (4). Although most of the patients with PTC have a good survival
rate, they may be prone to recurrence with increased morbidity and mortality (5, 6).
Existing evidence shows new aspect of molecular mechanisms such as genetic alterations opens
up a number of research avenues to determine pathways of progression and aggressiveness of
PTC. One of the most well-known oncogenic genetic alterations occurring in over 45% of cases
of PTC is the B-type raf proto-oncogene (BRAF) point mutation (7-9). Mutation in BRAF
(V600E) is associated with an invasive clinical period and poor clinicopathologic properties,
such as extrathyroidal invasion, lymph node metastasis, and advanced TNM stage (10), the TNM
system considers tumor size and local extent (T), lymph node metastases (N), and distant
metastases (M). However, the molecular events whereby mutation in the BRAF gene contribute
to development and progression of PTC remains unknown.
3
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 7 of 25 25 June 2017 10:32:14
Recently, regulations in the expression of various tumor suppressor genes that might be affected
by mutations in the specific gene have been investigated (11, 12). Hu et al, (12) showed that
methylation of various tumor suppressor genes such as tissue inhibitor of metalloproteinase-3
(TIMP3) may mediate progression and aggressiveness of PTC in response to a mutation in the
BRAF gene. TIMP3 is a member of tissue inhibitors of metalloproteinases that play an important
role in inhibiting growth, angiogenesis, invasion, and metastasis of various human cancers,
including thyroid cancer. It is of particular interest for therapeutic purposes due to its
characteristic of inhibiting different aspects of tumor development, mainly because it is a potent
angiogenesis inhibitor. Hu et al, (12) reported a significant association between BRAF mutation
and aberrant methylation of TIMP3. In addition hypermethylation of the TIMP3 gene is related
to thyroid cancer (13) and correlated with high-risk clinicopathological characteristics of PTC,
including extrathyroid invasion, lymph node metastasis, and advanced disease stages (III and IV)
(12). However, based on our knowledge, no study has apparently investigated the changes in
TIMP3 gene expression and protein levels in papillary thyroid tissues in relation to the BRAF
mutation and clinicopathologic features among PTC patients.
The purpose of this study was primarily to compare the association of TIMP3 gene expression
and protein levels with BRAF mutation in PTC patients. Secondly, we evaluated the association
of TIMP3 gene expression and protein levels with the clinicopathologic characteristics in PTC.
Materials and methods
4
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 8 of 25 25 June 2017 10:32:14
Patients and clinicopathological data collection: In the current study, we collected primary PTC
tumor tissues, matched non-tumoral tissues, and clinicopathological data of sixty patients with
PTC who had undergone total thyroidectomy over a one-year period (2015-2016) from the Erfan
and Atiyeh hospitals in Tehran city. Clinicopathological data collected including information, i.e.
age, gender, the status of extrathyroidal invasion, lymph node metastasis, multifocality, and stage
of the tumor. A pathologist reviewed all samples, confirmed the diagnosis of PTC. To determine
the stage of tumors stage we used American Cancer Society (ACS), American Joint Committee
on Cancer (AJCC) handbook (14). All confirmed thyroid samples were snap-frozen in liquid
nitrogen and stored at -80˚C.
Ethics approval for the study was obtained from the ethical committee of the Research Institute
for Endocrine Sciences of the Shahid Beheshti University of Medical Sciences
(25ECRIES93/10/23). Written informed consent was obtained from all of the patients.
DNA extraction and BRAF mutation detection: Genomic DNA was extracted from collected
fresh frozen thyroid tissue specimens by the TRIzol reagent (Invitrogen U.S. Cat. No. 15596-
026) according to the manufacturer�s instructions after histological control. Isolated DNA was
amplified by the polymerase chain reaction (PCR) that was carried out using following specific
primers for exon 15 of the BRAF gene containing the site for the T1799A (V600E) mutation: 5�-
CTTCATAATGCTTGCTCTGATAGG-3� (Forward) and 5�-
TTCTAGTAACTCAGCAGCATCTCA-3� (Reverse); The PCR reaction was performed in
volume 20µ L with PCR master mix (Kowsar Biotech Co. Cat. No. K1140), forward and reverse
5
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 9 of 25 25 June 2017 10:32:14
primers (10pmol), genomic DNA (100ng/µ L) with annealing 62̊ C. After quality confirmation by
agarose gel electrophoresis, direct DNA sequencing of the 251bp PCR-amplified products was
carried out using the same primers by Applied Biosystems 3130/3130xl Genetic Analyzers. The
results were analyzed using Chromas software version 2.6.
RNA samples and quantitative real-time PCR: Total RNA was extracted from fresh snap-frozen
PTC tissue samples after histological control, using TRIzol reagent (Invitrogen U.S. Cat. No.
15596-026) according to the manufacturer�s instructions. RNA quantity and purity were
measured using the NanoDrop 1000 (Thermo Scientific, Waltham, and Mass). Total RNA (1µ g)
was reverse transcribed with cDNA synthesis kit (Thermo Fisher Scientific, USA) according to
the manufacturer�s protocol and stored at -20°C for future use.
The amplification condition of TIMP3 templates was optimized by quantitative reverse
transcriptase real-time PCR (qRT-PCR) using the Rotor-Gene 6000 (Corbett Research, Sydney,
Australia) with annealing 60°C. Real-time quantification was monitored by measuring the
fluorescence activity. β-actin gene was used as internal control to normalize mRNA levels. All
experiments were repeated twice. Primers were designed from the sequence of the human
cDNAs. The sequences of these primers were as follows: 5�-CTGACAGGTCGCGTCTATGA-
3� (Forward) and 5�- CCGATAGTTCAGCCCCTTGC-3� (Reverse) for TIMP3, 5�-
GATCAAGATCATTGCTCCTCCT-3� (Forward) and 5�- TACTCCTGCTTGCTGATCCA-3�
6
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 10 of 25 25 June 2017 10:32:14
(Reverse) for β-actin. PCR amplification was performed in 25 µL volumes, using the SYBR
Green master mix (Thermo Fisher Scientific, USA).
Enzyme-linked immune sorbent assay (ELISA) analysis of TIMP3 protein expression: Total
protein was extracted from snap-frozen thyroid tissues. The samples were incised and weighted
(totally 100mg tissue/ 1mL buffer). A certain amount of PBS (pH 7.4, 100mM) was added and
homogenized thoroughly using a homogenizer (QIAGEN, Germany), and then, centrifuged (at
4000-6000 RPM) for approximately 10 minutes; supernatants were collected and frozen at -20°C
for later use. ZellBio GmbH assay kit (Cat. No: ZB-11267-H9648, Germany) was used to
quantitatively assay human TIMP3 based on the Biotin double antibody sandwich technology.
Statistical analysis: Normal distribution of quantitative data was determined by Kolmogorov-
Smirnov test. Group comparisons of categorical variables were performed using the x2 test or
linear-by-linear association. Categorical variables were expressed as frequency and percentage.
Comparisons of means were evaluated by the independent samples t-test. The intergroup
differences were assessed with the one-sample paired t-test (tumoral versus matched non-tumoral
tissues). All normal distributed continuous data were expressed as mean ± standard deviation
(SD). All analyses were performed using SPSS software (version 20.0 for Windows; SPSS Inc.,
Chicago, IL). Probability values <0.05 were used as the cutoff for statistical signi� cance. The
relative gene expression was assessed using the relative expression software tool (REST)
software (version 2009).
7
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 11 of 25 25 June 2017 10:32:14
Results
BRAF mutation status in relation to clinicopathological characteristic: Mean age of males and
females was 39.6±15.6 and 36.9±11.7 years, respectively; the BRAF V600E mutation was found
in 24 of 60 (40%) of the PTC cases. Figure 1 shows a sample of the sequencing results for a
BRAF (+) and (-). The frequencies of classic, follicular, oncocytic, and sclerosing variants of
PTC were 83.3, 11.7, 1.7, and 1.7%, respectively. There was no significant difference in
frequency of variants in BRAF mutation status (P=0.322); prevalence of unifocal, multifocal,
and no multicentricity tumors was 27.5, 47.5, and 25%, respectively, which did not differ
significantly in BRAF mutation status (P=0.953).
We analyzed the clinicopathological characteristics of the PTC cases with BRAF mutation
(Table 1), and found no extrathyroidal invasion in cases. BRAF (+) PTC had significantly larger
tumor size than BRAF (-) PTC (mean size 1.98±0.82 vs. 1.31±1.35 cm, respectively; P= 0.039).
Lymph node metastasis frequency was significantly higher in the BRAF (+) compared to the
BRAF (-) group (62.5 vs. 34.3%, respectively, P= 0.030).
Expression of TIMP3 in PTC: The expression of TIMP3 gene was lower in tumoral tissues than
those in matched non-tumoral ones (1.79±1.76 vs. 2.42±2.83; P=0.152) (Figure 2a), mean
TIMP3 protein level was significantly lower in tumoral tissues compared to the matched non-
tumoral tissues (7.36±2.79 vs. 10.77±5.57 ng/mL, respectively; P=0.001) (Figure 2b).
Differential expression of TIMP3 in BRAF V600E PTC: Moreover, the expression of TIMP3
gene was lower in BRAF (+) PTC compared to BRAF (-) PTC patients (1.21±1.09 vs.8
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 12 of 25 25 June 2017 10:32:14
1.76±2.13; P=0.274) (Figure 3a). Mean TIMP3 protein level was significantly lower in tumoral
tissues compared to the matched non-tumoral tissues (6.97±2.37 vs. 11.4±5.59 ng/mL,
respectively; P= 0.003) in BRAF (+) PTC; protein level was 7.77±3.19 and 10.0±5.61 ng/mL in
tumoral and matched non-tumoral tissues, respectively in BRAF (-) PTC, which was marginally
significant (P= 0.084) (Figures 3b and 3c).
TIMP3 protein level and lymph node metastasis in BRAF V600E (+) PTC: No association was
found between TIMP3 protein level and clinicopathological features, except with lymph node
metastasis. In all PTC cases the protein level of TIMP3 was lower in tumoral tissues compared to
the matched non-tumoral tissues of subjects with positive lymph node metastasis (7.55±2.63 vs.
12.4±6.78; P= 0.004). After splitting the file into BRAF positive and negative, results showed
that protein level of TIMP3 remained significantly lower in tumoral tissues in BRAF (+) in
subjects without lymph node metastasis and those with lymph node metastasis (Table 2).
Discussion
Data shows that the most frequent genetic variation occurring in PTC is a mutation in the
nucleotide 1796 of the BRAF gene, giving rise to an activated form, BRAF V600E (15).
Overactivation of the RAF/MEK/MAPK signaling pathway meddles with proliferation,
differentiation, and programmed cell death (apoptosis) (16). Furthermore, it has been suggested
that the BRAF mutation is associated with a weak prognosis in PTC patients (17). On the other
hand, there is increasing evidence suggesting that TIMP3 protects against tumor development by
suppressing tumor growth, metastasis, angiogenesis, and apoptosis (18). All genetic alternations
9
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 13 of 25 25 June 2017 10:32:14
found in thyroid cancer need to be evaluated for the pathogenesis of this disease, its accurate
diagnosis, precise prognosis and the appropriate management of these patients.
In this study, we evaluated the most common mutation found in PTC as well as a tumor
suppressor gene in tumoral and matched non-tumoral tissues of PTC patients that had not yet
been tested. Results showed that the BRAF mutation was related to tumor size and lymph node
metastasis. Moreover, the gene expression of TIMP3 was downregulated in tumoral tissues
compared to the matched non-tumoral one. Moreover, protein level was downregulated in
tumoral tissues in BRAF (+) PTCs; in addition, results indicated that TIMP3 protein level was
associated with lymph node metastasis in subjects who had the BRAF V600E (+) mutation.
In agreement with our findings, a review by Leonardi GC et al (19) from 19 studies reported a
positive correlation between the BRAF V600E mutation with the clinicopathological features,
including tumor size, extrathyroidal invasion, lymph node metastasis, and advanced stages in a
number of reports of PTC. However, some studies found no such association (20, 21), an
inconsistency which may be due to various factors, such as different diagnostic criteria and the
small number of cases analyzed in different studies.
Like our results, Anania MC et al (18), presented downregulation of TIMP3 in PTC with respect
to normal thyroid tissues using analysis of gene expression data and in silico analysis; they
demonstrated that TIMP3 exerts a negative regulatory role in thyroid carcinogenesis.
Downregulation of TIMP3 gene has been demonstrated in melanoma, kidney, brain,
choriocarcinoma, renal cell carcinoma, esophageal adenocarcinoma, and other human cancers
10
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 14 of 25 25 June 2017 10:32:14
(22-25). Moreover, TIMP3 promoter hypermethylation is observed in many tumors such as
gastric, esophageal, and thyroid carcinoma, and causes loss of TIMP3 protein expression (26,
27). However, downregulation of TIMP3 protein not resulting from TIMP3 promoter
hypermethylation has also been reported in pancreatic adenocarcinoma, an occurrence also
known to be associated with microRNA and the lack of heterozygosity (28, 29).
In this study, we found no significant reduction of TIMP3 gene expressed in tumoral tissues and
matched non-tumoral tissues but it associated significantly with diminished TIMP3 protein
expression. Consistent with previous studies (30), our results indicate that the expression of
TIMP3 in protein levels was reduced in tumoral tissues compared to matched non-tumoral one.
In this study, the observed decline in TIMP3 gene expression level in tumoral tissues, however
not significant, but was in concordance with reduction in protein expression. May be reduced
protein levels depends on other regulatory mechanisms which are acting during neoplastic
transformations.
The positive association of TIMP3 protein expression with BRAF mutation and lymph node
metastasis confirm that expression of this metastasis suppressor gene may play a role in the
aggressiveness of PTC, conferred by the BRAF mutation. This result reveals the biological
relevance of the positive correlation between BRAF mutation and expression of TIMP3 which is
accompanied by MAPK/ERK signaling pathway (31).
To our knowledge, this is the first report indicating a relationship between TIMP3 expression
and BRAF mutation status in tumoral and matched non-tumoral tissues of PTC patients.
11
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 15 of 25 25 June 2017 10:32:14
However, as a limitation for this cross-sectional study we had not access to thyroid samples from
healthy people (without thyroid carcinoma) to evaluate the probable constitutional changes in
TIMP3 expression.
Even though we found an association between BRAF mutation and TIMP3 expression with PTC
invasiveness, we did not have a follow-up study to investigate their prognostic role and/or
recurrence to give more credibility to the results. BRAF mutation, as the most frequent genetic
irregularity in PTC, is not completely understood into long-term prognosis due to the
contradictory findings documented. In the future, the prognostic importance of BRAF V600E
and low expression of TIMP3 should be evaluated based on long-term outcomes.
In conclusion, our results showed that BRAF mutation was associated with larger tumor size,
higher lymph node metastasis, and lower TIMP3 protein expression. Moreover, lymph node
metastasis was seen in the lower TIMP3 protein expression.
Sources of funding: This research did not receive any specific grant from funding agencies in
the public, commercial, or not-for-profit sectors.
Disclosure: The authors do not report any relevant conflicts of interest.
Acknowledgements: This work is related to Maryam Zarkesh Ph.D. by research thesis and
supported by Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical
Sciences, Tehran, Iran. The authors wish to express their gratitude Ms. Afsoon Danesh-afrooz,
Ms. Laleh Hoghoughi Rad, Ms. Hoda Ghadaksaz, Dr. Adeleh.S Razavi, Mr. Emad Yuzbashian,
12
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 16 of 25 25 June 2017 10:32:14
and Dr. Zahra Nozhat for data collecting and preparing. The authors wish to acknowledge Ms.
Niloofar Shiva for critical editing of English grammar and syntax of the manuscript.
Authors� contribution
Study concept and design: Hedayati & Zarkesh; acquisition of data: Fanaei & Foroughi; analysis
and interpretation of data: Zarkesh; drafting of the manuscript: Zadeh-Vakili, & Zarkesh; critical
revision of the manuscript for important intellectual content: Azizi & Hedayati; statistical
analysis: Zarkesh; study supervision: Hedayati
13
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 17 of 25 25 June 2017 10:32:14
References
1. Sipos JA, Mazzaferri EL. Thyroid cancer epidemiology and prognostic variables. Clinical oncology
(Royal College of Radiologists (Great Britain)). 2010;22(6):395-404. doi: 10.1016/j.clon.2010.05.004.
2. Hundahl SA, Fleming ID, Fremgen AM, Menck HR. A National Cancer Data Base report on 53,856
cases of thyroid carcinoma treated in the U.S., 1985-1995 [see commetns]. Cancer. 1998;83(12):2638-48.
DOI: 10.1002/(SICI)1097-0142(19981215)83:12<2638::AID-CNCR31>3.0.CO;2-1
3. Morris LG, Sikora AG, Tosteson TD, Davies L. The increasing incidence of thyroid cancer: the
influence of access to care. Thyroid. 2013;23(7):885-91. doi: 10.1089/thy.2013.0045.
4. Kilfoy BA, Zheng T, Holford TR, Han X, Ward MH, Sjodin A, et al. International patterns and trends
in thyroid cancer incidence, 1973-2002. Cancer causes & control : CCC. 2009;20(5):525-31. doi:
10.1007/s10552-008-9260-4.
5. Hay ID, Bergstralh EJ, Goellner JR, Ebersold JR, Grant CS. Predicting outcome in papillary thyroid
carcinoma: development of a reliable prognostic scoring system in a cohort of 1779 patients surgically
treated at one institution during 1940 through 1989. Surgery. 1993;114(6):1050-7; discussion 7-8. PMID:
8256208.
6. Sherman SI, Brierley JD, Sperling M, Ain KB, Bigos ST, Cooper DS, et al. Prospective multicenter
study of thyroiscarcinoma treatment: initial analysis of staging and outcome. National Thyroid Cancer
Treatment Cooperative Study Registry Group. Cancer. 1998;83(5):1012-21. PMID: 9731906.
7. Xing M. BRAF mutation in thyroid cancer. Endocrine-related cancer. 2005;12(2):245-62. doi:
10.1677/erc.1.0978.
8. Nikiforova MN, Kimura ET, Gandhi M, Biddinger PW, Knauf JA, Basolo F, et al. BRAF mutations in
thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas
arising from papillary carcinomas. The Journal of clinical endocrinology and metabolism.
2003;88(11):5399-404. doi: 10.1210/jc.2003-030838.
9. Xing M, Westra WH, Tufano RP, Cohen Y, Rosenbaum E, Rhoden KJ, et al. BRAF mutation predicts
a poorer clinical prognosis for papillary thyroid cancer. The Journal of clinical endocrinology and
metabolism. 2005;90(12):6373-9. doi: 10.1210/jc.2005-0987.
10. Cohen Y, Xing M, Mambo E, Guo Z, Wu G, Trink B, et al. BRAF mutation in papillary thyroid
carcinoma. Journal of the National Cancer Institute. 2003;95(8):625-7. doi:
https://doi.org/10.1093/jnci/95.8.625.
11. Vasko V, Espinosa AV, Scouten W, He H, Auer H, Liyanarachchi S, et al. Gene expression and
functional evidence of epithelial-to-mesenchymal transition in papillary thyroid carcinoma invasion.
Proceedings of the National Academy of Sciences. 2007;104(8):2803-8. doi: 10.1073/pnas.0610733104.
14
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 18 of 25 25 June 2017 10:32:14
12. Hu S, Liu D, Tufano RP, Carson KA, Rosenbaum E, Cohen Y, et al. Association of aberrant
methylation of tumor suppressor genes with tumor aggressiveness and BRAF mutation in papillary
thyroid cancer. International journal of cancer. 2006;119(10):2322-9. doi: 10.1002/ijc.22110.
13. Hoque MO, Rosenbaum E, Westra WH, Xing M, Ladenson P, Zeiger MA, et al. Quantitative
assessment of promoter methylation profiles in thyroid neoplasms. The Journal of clinical endocrinology
and metabolism. 2005;90(7):4011-8. doi: 10.1210/jc.2005-0313.
14. Edge S. American Cancer Society. AJCC cancer staging handbook: from the AJCC cancer staging
manual. New York: Springer; 2010. http://www.springer.com/kr/book/9780387884424.
15. Tang KT, Lee CH. BRAF mutation in papillary thyroid carcinoma: pathogenic role and clinical
implications. Journal of the Chinese Medical Association : JCMA. 2010;73(3):113-28. doi: 10.1016/S1726-
4901(10)70025-3.
16. Zhang W, Liu HT. MAPK signal pathways in the regulation of cell proliferation in mammalian cells.
Cell Res. 2002;12(1):9-18. doi:10.1038/sj.cr.7290105.
17. Pelizzo M, Dobrinja C, Ide EC, Zane M, Lora O, Toniato A, et al. The role of BRAF (V600E) mutation
as poor prognostic factor for the outcome of patients with intrathyroid papillary thyroid carcinoma.
Biomedicine & Pharmacotherapy. 2014;68(4):413-7. doi: 10.1016/j.biopha.2014.03.008.
18. Anania MC, Sensi M, Radaelli E, Miranda C, Vizioli MG, Pagliardini S, et al. TIMP3 regulates
migration, invasion and in vivo tumorigenicity of thyroid tumor cells. Oncogene. [Article].
2011;30(27):3011-23. doi: 10.1038/onc.2011.18.
19. Leonardi GC, Candido S, Carbone M, Raiti F, Colaianni V, Garozzo S, et al. BRAF mutations in
papillary thyroid carcinoma and emerging targeted therapies (Review). Molecular medicine reports.
2012;6(4):687-94. DOI: 10.3892/mmr.2012.1016.
20. Liu RT, Chen YJ, Chou FF, Li CL, Wu WL, Tsai PC, et al. No correlation between BRAFV600E
mutation and clinicopathological features of papillary thyroid carcinomas in Taiwan. Clinical
endocrinology. 2005;63(4):461-6. DOI: 10.1111/j.1365-2265.2005.02367.x.
21. Yasuhiro I, Yoshida H, Maruo R, Morita S, Takano T, Hirokawa M, et al. BRAF mutation in papillary
thyroid carcinoma in a Japanese population: its lack of correlation with high-risk clinicopathological
features and disease-free survival of patients. Endocrine journal. 2009;56(1):89-97. doi:
http://doi.org/10.1507/endocrj.K08E-208.
22. Feng H, Cheung AN, Xue WC, Wang Y, Wang X, Fu S, et al. Down-regulation and promoter
methylation of tissue inhibitor of metalloproteinase 3 in choriocarcinoma. Gynecol Oncol.
2004;94(2):375-82. DOI: 10.1016/j.ygyno.2004.04.019.
15
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 19 of 25 25 June 2017 10:32:14
23. Masson D, Rioux-Leclercq N, Fergelot P, Jouan F, Mottier S, Théoleyre S, et al. Loss of expression
of TIMP3 in clear cell renal cell carcinoma. European Journal of Cancer. 2010;46(8):1430-7. DOI:
10.1016/j.ejca.2010.01.009.
24. Darnton SJ, Hardie LJ, Muc RS, Wild CP, Casson AG. Tissue inhibitor of metalloproteinase 3 ‐(TIMP 3) gene is methylated in the development of esophageal adenocarcinoma: Loss of expression ‐correlates with poor prognosis. International journal of cancer. 2005;115(3):351-8. DOI:
10.1002/ijc.20830.
25. Das AM, Bolkestein M, van der Klok T, Oude Ophuis CMC, Vermeulen CE, Rens JAP, et al. Tissue
inhibitor of metalloproteinase-3 (TIMP3) expression decreases during melanoma progression and
inhibits melanoma cell migration. European Journal of Cancer. 2016;66:34-46. doi:
10.1016/j.ejca.2016.06.020.
26. Guan Z, Zhang J, Song S, Dai D. Promoter methylation and expression of TIMP3 gene in gastric
cancer. Diagnostic pathology. 2013;8(1):110. doi: 10.1186/1746-1596-8-110.
27. Brait M, Loyo M, Rosenbaum E, Ostrow KL, Markova A, Papagerakis S, et al. Correlation between
BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer. Epigenetics.
2012;7(7):710-9. doi: 10.4161/epi.20524.
28. Liu W, Li M, Chen X, Zhang D, Wei L, Zhang Z, et al. MicroRNA-373 promotes migration and
invasion in human esophageal squamous cell carcinoma by inhibiting TIMP3 expression. American
journal of cancer research. 2016;6(1):1. PMCID: PMC4759392.
29. Fendrich V, Slater EP, Heinmöller E, Ramaswamy A, Celik I, Nowak O, et al. Alterations of the
tissue inhibitor of metalloproteinase-3 (TIMP3) gene in pancreatic adenocarcinomas. Pancreas.
2005;30(2):e40-e5. PMID: 15714128.
30. Terayama Y, Matsuura T, Ozaki K. Lack of Correlation between Aberrant p16, RAR-β2, TIMP3,
ERCC1, and BRCA1 Protein Expression and Promoter Methylation in Squamous Cell Carcinoma
Accompanying Candida albicans-Induced Inflammation. PloS one. 2016;11(7):e0159090. doi:
https://doi.org/10.1371/journal.pone.0159090.
31. Xing M. Molecular pathogenesis and mechanisms of thyroid cancer. Nature reviews Cancer.
2013;13(3):184-99. doi: 10.1038/nrc3431.
16
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 20 of 25 25 June 2017 10:32:14
Table 1. Clinicopathological characteristics of PTC patients in BRAF mutation
Variables Total
BRAF V600E mutation
Positive NegativeP
value
Age (year) 40.9±13.7 38.8±13.1 36.7±12.4 0.527
Male, n(%)Female, n(%)
15(25.0)45(75.0)
8(33.3)16(66.7)
7(19.4)29(80.6)
0.180
Tumor size (cm) 1.58±1.20 1.98±0.82 1.31±1.35 0.039
Extracapsular invasion 14(23.7) 6(25) 8(22.9) 0.544
Lymph node metastasis 27(49.1) 15(62.5) 12(34.3) 0.030
Lymphovascular invasion 8(13.6) 4(16.7) 4(11.4) 0.418
Tumor stage (AJCC), n (%)IIIIIIIVA
46(79.3)3(5.2)7(12.1)2(3.4)
17(70.8)1(4.2)5(20.8)1(4.2)
29(85.3)2(5.9)2(5.9)1(2.9)
0.153
Data in parentheses are percentage.
17
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 21 of 25 25 June 2017 10:32:14
Table 2. Protein levels of TIMP3 in BRAF V600E and lymph node metastasis
Lymph nodemetastasis status
Samplestatus
TIMP3 protein level (ng/mL) PvalueTumoral Matched non-
tumoral
NoAll PTCs
7.17±3.02* 9.09±3.35 0.086
Yes 7.55±2.63 12.4±6.78 0.004
NoBRAF (-) 7.47±3.16 7.58±2.55 0.939
BRAF (+) 6.87±3.04 10.6±3.49 0.028
YesBRAF (-) 8.08±3.39 12.5±6.84 0.043
BRAF (+) 7.08±1.74 12.2±7.10 0.048*Mean±SD
18
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 22 of 25 25 June 2017 10:32:14
Figure 1. The sequence of BRAF V600E (+) and (-) by Chromas software
19
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 23 of 25 25 June 2017 10:32:14
Figure 2. a) Relative expression of TIMP3 gene and b) Protein level of TIMP3 in tumoral tissuescompare to matched non-tumoral tissues; *P< 0.05
20
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Relat
ive ex
press
ion of
TIM
P3 (F
old ch
ange
)
Matched Non_tumoral Tumoral
Prot
ein le
vels
of T
IMP3
(ng/
mL)
a b
*
*
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 24 of 25 25 June 2017 10:32:14
0.0
0.5
1.0
1.5
2.0
2.5
BRAF-V600E Status
Relat
ive ex
press
ion of
TIM
P3 (F
old ch
ange
)
BRAF Negative BRAF Positive
Figure 3. a) Relative expression of TIMP3 gene in BRAF mutation status, b) Protein level of
TIMP3 in BRAF (+), and c) Protein level of TIMP3 in BRAF (-); *P< 0.05
21
a*
a
b c
Prot
ein le
vels
of T
IMP3
(ng/
mL)
Prot
ein le
vels
of T
IMP3
(ng/
mL)
*
BRAF (+) BRAF (-)
#: The association of the BRAF V600E mutation wi... Revision 0
Journal: International Journal... Page 25 of 25 25 June 2017 10:32:14