Overexpression of epidermal growth factor receptor and its downstream effector, focal adhesion kinase, correlates with papillary thyroid carcinoma progression

Summary

Epidermal growth factor receptor (EGFR) and its downstream effector, focal adhesion kinase (FAK), have been shown to be overexpressed frequently in human malignancies and implicated in tumour aggressiveness. We aimed to investigate the relationship between EGFR and FAK expression and their possible correlation with the clinical phenotype of patients with papillary thyroid carcinoma (PTC). Expression profiles of EGFR and FAK were analysed in PTC tissue samples (n = 104) by immunohistochemistry and Western blotting. Additionally, EGFR and FAK were immunohistochemically analysed in 20 primary tumours paired with their metastatic tissue in lymph nodes. High expression of EGFR and FAK was found in 55.77% and 57.69% cases, respectively, with a strong positive association between them (P < 0.0001, Spearman's correlation coefficient = 0.844). Expression of each molecule and their coexpression correlated significantly with the presence of lymph node metastasis (LNM), degree of tumour infiltration, extrathyroid invasion and pT status of the patients. Western blot analysis confirmed that coexpression of high levels of EGFR and FAK correlated with adverse clinicopathological features. When compared to the corresponding primary tumour, increased or maintained high levels of EGFR and FAK were found in LNM, indicating their concordant expression during lymphatic spread. In conclusion, high levels of EGFR and its downstream effector, FAK, in association with lymphatic spread and tumour infiltration indicate their involvement in PTC progression and suggest that both molecules may predict its aggressive behaviour. Furthermore, FAK could be a potential target for anticancer therapy in patients with advanced thyroid cancer.

Introduction

Thyroid cancer is the most frequent endocrine malignancy. Papillary thyroid carcinoma (PTC) – the most common histological type of thyroid cancer, accounting for more than 80% of all thyroid malignancies – has shown a fast‐rising incidence during the last few decades (Ito et al. 2013). In general, PTC can be effectively treated by surgical resection, can often be combined with radio‐iodine therapy and has a favourable prognosis with an overall 5‐year survival rate of about 95%. However, survival drops precipitously in the setting of locally advanced, metastatic and/or radio‐iodine‐refractory disease. Advanced PTC is generally resistant to systemic chemotherapy and external beam radiation. Once distant metastases occur, the 10‐year survival rate is <20% (Siironen et al. 2005; Ito & Miyauchi 2009; Zhao et al. 2012). Thus, a subset of patients thought to be disease‐free after initial treatment will develop recurrence and/or metastases and die as a direct consequence of this carcinoma. It is therefore of great importance to identify patients with PTC at high risk for a poor outcome. Some patient‐related and clinical factors, such as old age, male sex, lymphovascular invasion and extrathyroid extension, have been associated with an increased risk of disease progression, but they lack the precision to tailor surveillance and anticipate prognosis accurately (Sipos & Mazzaferri 2010; Choi et al. 2015). In addition, there is a clear need for novel treatments for patients with advanced thyroid cancer. Consequently, analysing the molecular characteristics of PTC and exploring new targets for therapy are major scientific and clinical concerns in the field of thyroid oncology.

Epidermal growth factor (EGF) and its receptor (EGFR) have been implicated in the pathogenesis of many different types of cancer and thus provide attractive targets for molecular therapy. Epidermal growth factor receptor (EGFR) is a 170‐KDa cell‐surface glycoprotein consisting of an extracellular ligand binding domain, a transmembrane domain and an intracellular domain with intrinsic tyrosine kinase activity (Mendelsohn & Baselga 2000; Yarden 2001). Upon ligand binding, EGFR activates intracellular signal transduction pathways including Ras/Raf/mitogen‐activated protein kinase (MAPK) pathway and the phosphoinositol 3‐kinase (PI3K)/Akt pathway, which are involved in promoting proliferation, survival, angiogenesis and migration (Ullrich & Schlessinger 1990; Katz et al. 2007). Deregulation of EGFR signalling, caused by receptor overexpression, autocrine ligand stimulation or activating mutations, has been frequently implicated in several types of human cancers and associated with an advanced stage of malignancy characterized with metastatic competence and poor prognosis (Nedergaard et al. 2012).

Focal adhesion kinase (FAK) is a 125‐KDa cytoplasmic tyrosine kinase that plays important roles in integrin‐mediated signal transduction and also participates in signalling by other cell‐surface receptors, such as EGFR (Parsons et al. 1994; Schlaepfer et al. 1999). Upon integrin binding to extracellular matrix proteins or activation of EGFR, FAK is autophosphorylated and activated by forming a complex with Src kinase. This initiates multiple downstream signalling pathways to regulate different cellular functions. In cancer cells, FAK enhances the effects of both MAPK and PI3K signalling pathways activated by EGFR, promoting in that way uninhibited proliferation and survival under anchorage‐independent conditions and increasing their ability to migrate and metastasize (Hauck et al. 2002; Zhao & Guan 2011; Sulzmaier et al. 2014). Elevated levels of FAK have been correlated with invasive properties in a variety of human tumours (Chatzizacharias et al. 2008; Golubovskaya et al. 2009; Tai et al. 2015; Yoon et al. 2015), including thyroid cancer (Owens et al. 1996).

To gain better insight into the clinical significance of deregulated expression of EGFR and its downstream effector, FAK, in thyroid carcinoma progression, we have analysed the expression profiles of both proteins in a series of clinical PTC samples in relation to clinicopathological parameters as well as their changes during tumour progression via metastatic spreading.

Materials and methods

Tissue specimens and clinical data

One hundred and four formalin‐fixed and paraffin‐embedded thyroid specimens were obtained from surgical resections at the Center for Endocrine Surgery, Clinical Center of Serbia, Belgrade, Serbia.

Haematoxylin–eosin‐stained histological slides were re‐evaluated by a pathologist to confirm the diagnosis according to the World Health Organization classification of thyroid tumours (DeLellis et al. 2004). Out of 104 cases, 59 were classical, 27 were follicular, and 12 were solid tumours, while six cases were classified as other PTC histotypes (tall cell, Warthin‐like, diffuse sclerotic). Personal and clinical data of the patients were retrieved by reviewing pathology reports and included age, sex, tumour size, presence of lymph node metastasis, tumour/thyroid capsule infiltration, extrathyroid extension, pT status and TNM stage. Patients were staged according to the pathologic TNM staging system in accordance with the American Joint Committee on Cancer (Edge et al. 2010). In addition, all cases were subsequently re‐evaluated for the degree of tumour infiltration, as described previously (Basolo et al. 2010): A – totally encapsulated tumours; B – non‐encapsulated tumours without thyroid capsule invasion; C – tumours with thyroid capsule invasion; and D – tumours with extrathyroid invasion.

Additionally, tissue sections from 20 primary tumours with paired metastatic tissue from lymph nodes of the same patient were also examined immunohistochemically. In parallel with archival specimens, fresh PTC tissues obtained after thyroidectomy were collected, snap‐frozen and stored at −80°C to be used later for Western blot analysis.

All procedures performed in studies were in accordance with the ethical standards of the institutional and national research committee and with the Declaration of Helsinki.

The study protocol was approved by the Ethics Committee of the Clinical Center of Serbia, Belgrade, Serbia.

Immunohistochemistry

Immunohistochemical analysis was performed using an established protocol. Formalin‐fixed, paraffin‐embedded tissue sections (4–6 μm thick) were subjected to deparaffinization with xylene and rehydration with ethanol. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 30 min following blocking of non‐specific binding. The specimens were further incubated overnight at 4°C with primary antibodies against EGFR (rabbit polyclonal sc‐03; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or FAK (rabbit polyclonal sc‐558; Santa Cruz Biotechnology, Inc.) at 1:100 dilution for both. This was followed by incubation with biotinylated anti‐rabbit IgG for 35 min at room temperature and thereafter with the avidin–biotin–peroxidase complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) for 30 min. 3,3′‐Diaminobenzidine tetrahydrochloride (DAB) solution was used as the chromogen for visualization (Peroxidase Substrate Kit; Vector Laboratories) followed by counterstaining with haematoxylin. Slides were dehydrated in ethanol, cleared in xylene and mounted with coverslips using permanent mounting medium. Staining was analysed using an Axio Imager 1.0 microscope (Carl Zeiss, Jena, Germany) supplied with a Canon A640 Digital Camera System (Canon, Tokyo, Japan). Tissue samples receiving no primary antibody (anti‐EGFR, anti‐FAK) – that is, the antibody was replaced with PBS during staining procedure – exhibited no staining and served as negative controls.

Immunohistochemical scoring

The tumour sections were examined by two individual researchers with no knowledge of the clinical findings. Both intensity and distribution were included in the semiquantitative evaluation of immunoreactivity. Staining was scored as follows: 0 – absence of staining; 1 – weak widespread or focal (up to 40%) staining of tumour cells; 2 – moderate staining in more than 40% of tumour cells; and 3 – strong diffuse staining in tumour cells. Cases scored as (0) and (1) were considered as low‐expressing, and cases scored as (2) and (3) as high‐expressing.

Statistical analysis

The software package spss 21 (SPSS, Chicago, IL, USA) was used for statistical analysis. Correlation of EGFR and FAK expression with clinicopathological parameters (age, sex, tumour size, presence of lymph node metastasis, degree of tumour infiltration, extrathyroid invasion, pT status, TNM stage and histotype) was evaluated with the two‐tailed chi‐square or Fisher exact test. Probability values <0.05 were considered as statistically significant. The Spearman correlation test was employed to determine correlation between EGFR and FAK expression.

Protein extraction

Proteins were extracted from snap‐frozen thyroid carcinoma tissue using RIPA buffer (50 mM Tris‐HCl pH 8.0, 150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS) with Protease Inhibitor Cocktail (p8340; Sigma‐Aldrich, St Louis, MO, USA). After 30 min of homogenization on ice, samples were centrifuged at 11,000 g for 20 min at 4°C. The supernatant was aliquoted and protein concentration determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA).

Western blot

Variation of EGFR and FAK expression was examined in 20 cases of PTC with different clinical parameters by Western blot analysis. Proteins (50 μg) in the PTC supernatant were separated by SDS‐PAGE on 7.5% gels and thereafter transferred to nitrocellulose membrane (Amersham™ Protran™, 0.45‐μm NC; GE Healthcare Life Science, Freiburg, Germany), blocked in 5% casein in Tris‐buffered saline–Tween (TBST) for 2 h at room temperature and incubated with primary antibodies against EGFR and FAK at 1:1000 dilution overnight at 4°C. Antibodies were the same as those used for immunohistochemistry. After washing the membrane with TBST, biotinylated anti‐rabbit IgG antibodies were added at 1:2000 dilution and then incubated with ABC reagent for 30 min at room temperature. Bands were visualized by chemiluminescence upon incubation with ECL Substrate (Pierce).

Results

Immunohistochemical expression of EGFR and FAK in PTC

Expression of EGFR and FAK proteins was evaluated by immunohistochemistry in 104 formalin‐fixed, paraffin‐embedded tissue sections from patients diagnosed with PTC. Representative microphotographs are given in Figure 1. When present, non‐malignant (normal or hyperplastic) tissue did not exhibit immunoreactivity for EGFR or FAK. The analysed PTC cases were divided into two categories: low‐expressing (staining scores 0 and 1) and high‐expressing cases (staining scores 2 and 3), as detailed in the Materials and Methods section. High (moderate to strong) cytoplasmic/membranous staining for EGFR was found in 58 of 104 cases (55.77%), and high (moderate to strong) cytoplasmic staining for FAK was evident in 60 of 104 cases (57.69%). We observed an apparent correlation between EGFR and FAK expression profiles, which was confirmed by the high level of statistical significance (r = 0.844, P < 0.0001) given by the Fisher exact and Spearman correlation tests (Table 1).

Figure 1.

Immunohistochemical expression of epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) in papillary thyroid carcinoma (PTC). Weak staining (scored as 1, low‐expressing group) for EGFR (a) and FAK (d). Moderate staining (scored as 2, high‐expressing group) for EGFR (b) and FAK (e). Inserts located in the upper‐right corner of (b) and (e) are displays of 40× magnification of the same cases showing moderate membranous and cytoplasmic staining for EGFR and moderate cytoplasmic staining for FAK. Strong staining (scored as 3, high‐expressing group) for EGFR (c) and FAK (f). ABC immunotechnique, haematoxylin–diaminobenzidine, original magnification: ×20. Scale bars correspond to 50 μm. [Colour figure can be viewed at http://wileyonlinelibrary.com]

Table 1.

Correlation between epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) protein expressions in papillary thyroid carcinoma (n = 104)

		FAK
		Low (n = 44)	High (n = 60)	P valuea	Correlation coefficientb
EGFR	Low (n = 46)	41	5	<0.0001	0.844
	High (n = 58)	3	55

^aFisher exact test, P < 0.05 – statistically significant.

^bSpearman correlation test.

Correlation of EGFR and FAK immunohistochemical expression with clinicopathological PTC

We further evaluated possible association between high expression of EGFR and FAK proteins and clinicopathological data for the analysed PTC cases (Table 2). Statistical analysis revealed no significant differences concerning age and sex, but significant positive relationships were found between high level of expression of each molecule and the presence of lymph node metastasis (LNM), extrathyroid invasion (EI), degree of neoplastic infiltration and advanced pT status (P < 0.05). High levels of expression of EGFR or FAK were not significantly associated with advanced pTNM stages. It should be noted that in our series of PTCs, almost the half of the highly positive EGFR and FAK cases, having LNM or EI, were young patients, classified as stage I according to the TNM classification proposed by the American Joint Committee on Cancer. Interestingly, high expression of both EGFR and FAK showed statistically significant differences regarding the histomorphological growth pattern of PTC, being higher in the classical than in the follicular variant of PTC (P < 0.001).

Table 2.

Correlation of epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) protein expressions with clinicopathological parameters in papillary thyroid carcinoma (n = 104)

		EGFR			FAK
Parameter	Total (n = 104)	Low (n = 46)	High (n = 58)	P valuea	Low (n = 44)	High (n = 60)	P valuea
Age
<45	49	19	30	0.290	20	29	0.771
≥45	55	27	28		24	31
Gender
F	80	36	44	0.773	32	48	0.384
M	24	10	14		12	12
Tumour size
≤2 cm	34	11	23	0.089	9	25	0.023
>2 cm	70	35	35		35	35
LNM
Absent	55	38	17	<0.0001	34	21	<0.0001
Present	49	8	41		10	39
EI
Absent	60	35	25	0.0007	33	27	0.002
Present	44	11	33		11	33
pT status
T1/T2	48	28	20	0.007	26	22	0.023
T3/T4	56	18	38		18	38
TNM stage
I/II	68	33	35	0.225	32	36	0.178
III/IV	36	13	23		12	24
Degree of infiltration
A/B	56	35	21	<0.0001	33	23	0.0002
C/D	48	11	37		11	37
Histotype
Classical	59	13	46	<0.0001	11	48	<0.0001
Follicular	27	16	11		17	10

TNM stage and the degree of infiltration – as detailed in the Materials and Methods section; LNM, lymph node metastasis; EI, extrathyroid invasion.

^aFisher exact test, P < 0.05 – statistically significant (bolded).

Correlation of concomitant high expression of EGFR and FAK with lymph node metastasis and extrathyroid invasion

As we found a statistically significant association between immunohistochemical expression of each of the examined proteins (EGFR, FAK) and clinicopathological parameters of the patients, we further analysed the concomitant high expression of EGFR and FAK in relation to LNM and EI, which are important parameters of tumour progression (Table 3).

Table 3.

Correlation of concomitant high expression of epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) with lymph node metastasis and extrathyroid invasion in papillary thyroid carcinoma

	EGFR/FAK
Parameter	Low/Low (n = 41)	High/High (n = 55)	P valuea
Lymph node metastasis
Absent (n = 49)	33	16	<0.0001
Present (n = 47)	8	39
Extrathyroid invasion
Absent (n = 56)	32	24	0.0007
Present (n = 40)	9	31

^aFisher exact test, P < 0.05 – statistically significant.

Concomitant high expression of EGFR and FAK (i.e. coexpression of high levels) closely correlated with the presence of LNM or EI.

Since in the analysed series of PTCs (n = 104) there were 49 patients with LNM and 44 patients with EI (Table 2), the coexpression of high levels of both EGFR and FAK found in 39 of 49 metastatic PTCs and in 31 of 44 patients with extrathyroid invasion, gave positive rates of 79.59% and 70.45% respectively.

Thus, high expression of a single protein (EGFR or FAK) or coexpression of high levels of both (due to their strong correlation with each other) was significantly associated with positive lymph node metastatic status and the presence of the extrathyroid invasion in patients with PTC.

Western blot analysis of EGFR and FAK protein expression

In the next step, we analysed the protein expression of EGFR and FAK by Western blot using snap‐frozen tissue extracts to confirm the specificity of antibodies and to extend immunohistochemical findings. Western blot analysis of a representative panel of PTC tissues illustrating the coexpression of high EGFR and FAK protein levels in association with adverse clinicopathological parameters is shown in Figure 2.

Figure 2.

Western blot analysis of epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) in a representative panel of papillary thyroid carcinoma (PTC) tissues. Concomitant high expression of EGFR and FAK in cases 1 and 2 (lymph node metastasis and extrathyroid extension present), and low EGFR and FAK expression in cases 3 and 4 (lymph node metastasis and extrathyroid extension absent). [Colour figure can be viewed at http://wileyonlinelibrary.com]

Immunohistochemical analysis of EGFR and FAK expression in primary tumours and matched lymph node metastases

To determine whether expression profiles of EGFR and FAK change during tumour progression via metastatic spreading, in addition we analysed 20 PTC tissues paired with their metastatic tissue in lymph nodes (Figures 3 and 4). An increase in staining intensity compared to the primary tumour or maintenance of high expression levels in metastatic tissues was found for both proteins, which reached 18 (90%) high‐EGFR‐expressing and 15 (75%) high‐FAK‐expressing metastatic tissues (Table 4), demonstrating their concordant high expression during lymphatic spreading of PTC.

Figure 3.

Immunohistochemical expression of epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) in two cases of primary papillary thyroid carcinoma (PTC) and corresponding metastatic tissue in lymph nodes. Immunostaining for EGFR (a) and FAK (c) in the primary tumours and their paired lymph node metastases (b and d). ABC immunotechnique, original magnification: ×10. Scale bars correspond to 50 μm. [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 4.

Coexpressed high levels of epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) in the papillary thyroid carcinoma (PTC) case (left) and its matched lymph node metastasis (right), showing maintenance of concomitant high expression levels during tumour progression via lymphogenic metastatic spreading. ABC immunotechnique, original magnifications: a and c: ×10; b and d: ×20. Scale bars correspond to 50 μm. [Colour figure can be viewed at http://wileyonlinelibrary.com]

Table 4.

Immunohistochemical expressions of epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) in primary papillary thyroid carcinoma (n = 20) and matched lymph node metastases

	Immunohistochemical staining
	Low	High
EGFR
Primary tumour	5	15	(75%)
Lymph node metastasis	2	18	(90%)
FAK
Primary tumour	6	14	(70%)
Lymph node metastasis	5	15	(75%)

Discussion

Papillary thyroid carcinoma is usually not life‐threatening, but may recur or progress to aggressive forms resistant to conventional therapies. Therefore, better understanding of molecular alterations in PTC signalling pathways in relation to clinical phenotype may help in risk stratification of the patients by predicting aggressive tumour behaviour and possibly identify novel therapeutic approaches against these tumours.

To contribute to this issue, we have analysed the expression profiles of EGFR and FAK in clinical PTC samples: (i) in correlation with clinicopathological parameters of the patients and (ii) in comparison with matched metastatic tissue in lymph nodes to detect changes during tumour progression via metastatic spreading. For the first time, our results demonstrated concomitant high levels of EGFR and FAK in primary tumours and corresponding metastatic tissue suggesting synchronous activity during tumour progression. Furthermore, these results support roles for EGFR and its downstream effector, FAK, as a part of the mechanism for invasion and metastasis of PTC.

Epidermal growth factor receptor signalling is involved in human cancer cell progression and is responsible for aggressive biological behaviour and poor clinical outcome in several human malignancies. With respect to thyroid carcinomas, EGFR upregulation occurs in anaplastic thyroid cancer and is associated with dedifferentiation, an aggressive phenotype and poor outcome (Landriscina et al. 2011). In PTC, high expression of EGFR has been associated with adverse clinicopathological features, that is local aggressiveness (Fisher et al. 2013) and metastatic spreading (Tang et al. 2014).

In thyroid cancer cells, ligand binding to EGFR leads to activation of both the ERK and Akt pathways, driving uncontrolled proliferation of tumour cells, conferring the ability to evade programmed cell death, enhancing migration and facilitating metastasis. Indeed, EGF has been shown to stimulate proliferation, migration and invasiveness of follicular and PTC cells in vitro (Hoelting et al. 1994). Furthermore, it has been demonstrated that thyroid cancer cell invasion is regulated by activation of matrix metalloproteinases (MMPs) downstream of EGFR (Yeh et al. 2006). Several molecular studies have proposed a functional link between EGFR, MMPs and FAK, impacting the migratory potential and invasiveness of malignant cells.

Focal adhesion kinase is a non‐receptor tyrosine kinase implicated in cancer progression and plays a pivotal role in integrating signals from growth factors and the extracellular matrix, acting as both a mediator of integrin‐dependent cell motility and an effector molecule in growth factor‐triggered signalling pathways (Parsons et al. 1994; Schlaepfer et al. 1999). In cancer cells, an increased FAK/Src complex confirms the activation of both the MAPK and PI3K signalling pathways already activated by EGFR, enhancing in that way their proliferation, survival and ability to migrate and metastasize (Hauck et al. 2002; Zhao & Guan 2011; Sulzmaier et al. 2014; Tai et al. 2015). The FAK/Src complex empowers tyrosine phosphorylation cascades to modulate signalling pathways relevant for malignant cell behaviour.

Elevated levels of FAK have been correlated with invasive properties in a variety of human tumours (Chatzizacharias et al. 2008; Golubovskaya et al. 2009; Tai et al. 2015; Yoon et al. 2015). Regarding thyroid cancer, high FAK expression levels were found in thyroid carcinomas, but not in non‐malignant tissue (Kim et al. 2004), and were associated with aggressive phenotypes, such as anaplastic thyroid cancer (Owens et al. 1996; Michailidi et al. 2010).

In vitro studies have indicated that matrix metalloproteinases (MMPs) and FAK are associated with the EGFR activation and that they promote tumour cell motility and invasion. Thus, inhibition of FAK expression or activity was shown to disrupt EGF‐stimulated invasion and decrease MMP‐9 activity in human adenoma cells (A549), suggesting that FAK promotes motility of cancer cells through regulation of MMP‐9 (Hauck et al. 2001). In breast cancer cells, pomolic acid inhibited EGF‐induced invasion, migration and cell motility by reducing FAK phosphorylation and MMP‐9 expression (Park et al. 2016). Similarly, it has been reported (Rothhut et al. 2007) that EGFR stimulation of thyroid malignant cells in vitro leads to FAK phosphorylation and MMP‐9 expression. Inhibition of the EGFR signalling pathway suppressed MMP‐9 expression and FAK phosphorylation, while FAK antisense treatment inhibited MMP‐9 secretion and subsequent cell invasion, thus proving its direct relation to the process of invasion.

Our results obtained in a clinical setting support a role for FAK in EGF‐induced cell migration and invasion, as indicated from in vitro functional studies on malignant cells.

In conclusion, the results of the current study have demonstrated high levels of EGFR and its downstream effector, FAK, in association with lymphatic spread and tumour infiltration. This indicates their involvement in PTC progression and suggests that both molecules may predict aggressive behaviour. The potential impact of our results is worth of consideration as a preliminary evidence to be confirmed in a larger series of clinical samples. Furthermore, studies evaluating cytological assessment of EGFR and FAK expressions in fine‐needle aspiration biopsy samples could possibly contribute to preoperative risk stratification of patients with PTC requiring more extensive surgery, careful follow‐up and therapeutic strategy.

Funding source

This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, project 173050: ‘Molecular characterization of thyroid gland tumours: biological and clinical aspects’.

Conflict of interest

The authors declare that they have no conflict of interests.