1. Introduction

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Nodular Lesions of the thyroid gland comprise benign and malignant tumors, as well as some forms of hyperplasia [1,2].Clinically obvious nodule develops in about 5 % of general population. With introduction of ultrasound, the detection of non-palpable nodules reached 20-67 % of non-palpable nodules [3].

Follicularthyroid lesions either originate from follicular cells or have follicular pattern of growth. According to the presence or absence of some features like presence of capsule, vascular and/or capsularinvasion, papillary- carcinoma type nuclei, follicular lesions are classified into: adenomatoid nodules, follicular adenomas (FA), papillarythyroid carcinoma (PTC), follicular thyroid carcinoma(FTC), well differentiated tumours of uncertain malignant potential (WDT-UMP), follicular tumour of uncertain malignant potential (FT-UMP) and Hurthle cell adenoma/carcinoma [45]. Follicular nodular thyroid lesions have many common morphological features, with an inter-observer and intra-observer variability in diagnosis of routinely stained slides [6].

Thyroid cancer constitutes about 1 % of all epithelial malignancies worldwide and is the commonest inendocrine system. It is the 5thmost common malignancy in women and is three times more common than in men [7,8].In Egypt, it represents about 1.5% of all cancers and about 30% of endocrine malignancies. Its rate among Egyptian females is 3.28 % ranking as the 6th most common malignancy [9,10].This low incidence of thyroid cancer has led to little improvements in therapy and survival rates over last two decades[11].

PTC is the most common type of thyroid cancer, accounting for 80-85% of the cases followed by FTC that accounts for 10-15% [12].The prognosis is more favourable in females and most well differentiated carcinomas occur during reproductive age. The use of oral contraceptives, estrogen therapy and pregnancy during this period increase the risk of thyroid cancer [13].This supports the role of female hormones especially estrogen in thyroid carcinogenesis and hypothesised the use of anti-estrogen hormone therapy for multidrug resistant thyroid cancer treatment.

The G protein-coupled estrogen receptor 1 (GPER1), previously known G protein receptor 30 (GPR30), is a transmembrane receptor. GPER1 mediates a rapid response to estrogensvia binding with it. Also, it regulates variable cellular physiological functions of estrogen. GPER1 activation leads to intracellular mechanisms controlling growth, proliferation, differentiation, invasion and migration [14,15].

Expression ofGPER1 was detected in various tissues throughout the body[14], and it had been found to be a significant prognostic parameter in breast, endometrial and ovarian malignant tumors. Whereas, its high expression was associated with metastases and poor survival [16,17].

CXCR1 is one of the 2 high affinity receptors for the CXC chemokine interleukin-8 (IL-8). It is involved in immune and inflammatory responses in many disorders. CXCR1 is mainly expressed in neutrophils and induces chemotaxis. Also, over expression of CXCR1 was related to drug-resistance, invasion, and metastasis in several solid tumors [18,19].

Human epidermal growth factor receptor 2 (HER-2) is a proto-oncogene, that belongs to epidermal growth factor receptor (EGFR) family. HER-2 contributes to physiological mechanisms of cell proliferation and differentiation by intrinsic tyrosine-kinase activity. In malignant tumours, HER-2 over expression is associated with increased disease recurrence and poor prognosis [20,21]. The importance of HER-2 in cancer has emerged by the effectiveness of the anti-HER-2 humanized monoclonal antibody trastuzumab (Herceptin), especially when combined with chemotherapy, for the treatment of HER2-overexpressing breast cancers. Previous studies have proposed that combining CXCR1 inhibitors with HER-2 targeted therapies can play a role in better survival during the treatment of HER-2 over expressing breast cancers [22,23]. In thyroid cancer, previous studies showed conflicting results with HER-2 positivity rates varying from 0% to 70% [24-26]. Moreover, HER-2 expression was linked to estrogen receptors expression in thyroid tumours[13].

In the setting of breast cancer; that is more common also in females, anti-estrogen hormone therapy as well as Herceptin are already well established among protocols for treatment of breast cancer. This study aims to identify a similar role in treatment of thyroid cancer by examining the expression and significance of one of oestrogen receptors (GPER1), HER-2 and their potential therapeutic target CXCR1. Also, to determine the association between their expression progression in different follicular thyroid lesions and clinicopathological characters. And to determine their role for prognosis and their expected value as targets for treatment.

Figure 1

2. Material And Methods

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2.1. Case Selection And Tissue Sample Preparation
In this retrospective study, all specimens that are presented with thyroid nodules at pathology lab of Minia university hospital and Minia oncology center between 2014 to 2016 were collected. There were 217 specimens divided as 98 fine needle aspiration cytology, 15 true cut biopsies and 104 surgically removed nodules. Thirty two [32] cases with fine needle aspiration were followed by excision biopsies and three cases with micropapillary carcinoma were missed and diagnosed only with biopsies. This discrepancy does not occur for true cut biopsies. Patient data were retrospectively collected and summarized.

The specimens for immuno histochemistry were obtained from paraffin blocks of 104 follicular thyroid lesions divided into, Nodular hyperplasia (n= 53), Thyroiditis (n=13), FTAs (n=17), PTC (n=15) and FTC (n=6) patients who underwent thyroidectomy in the Department of Surgery, between January 2014 and January 2016. No other hisopathological variants of thyroid carcinoma was detected. Staging was done according to AJCC classifica-tion system [27]. Lymph node metastasis was positive in 16 of the 21 malignant cases.

Five μm sections placed on positively charged slides (CITOGLAS) were prepared. Sections were de-paraffinized with xylene, hydrated through 99.9%, 95%, and 70% ethanol, and then treated with 3% hydrogen peroxide for 30 minutes to inactivate endogenous peroxides, washed in phosphate-buffered saline (PBS) solution. For antigen retrieval, sections were treated for 20 minutes in 0.01 M citrate buffer (pH 6.0)by microwave, and then allow cooling.

After rinse in PBS, primary antibodies were incubated overnight in a humidity chamber using GPER1 antibody (rabbit polyclonal antibody; 1:100 dilution, ab39742; Abcam, USA), anti-CXCR1 antibody (rabbit polyclonal antibody; 1:100 dilution, ab150548; Abcam, USA) and c-erbB-2/HER-2antibody (Monoclonal mouse antibody clone e2-4001 + 3B5, Lab Vision Laboratories, Ready to use). Then slides were washed with PBS before applying the biotinylated secondary antibody (Lab Vision Laboratories) for 30 minutes. Sections washed in PBS, incubated with the streptavidin-biotin complex reagent (Lab Vision Laboratories) for 30 minutes.

A brown color was developed with 3, 3-diaminobenzidinetetra hydrochloride (DAB, Lab Vision Laboratories) for 5 minutes, then washed in distilled water, counterstained with Mayer's haematoxylin, dehydrated, cleared in xylene, mounted and covered slipped. Each staining batch included positive and negative control sections.

2.2. Scoring Of Immunostaining
A semiquantitative assessment of immunohis-tochemical (IHC) scoring for GPER1 and CXCR1 was performed by two observers blinded to the diagnosis. The IHC score was assigned based on staining intensity and percentage of positive cells. The intensity score was assigned as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). The proportion score was assigned as 0 (< 5% positive cells), 1 (6-25% positive cells), 2 (26-50% positive cells), 3 (51-75% positive cells), and 4 (> 75% positive cells). Multi-plication of the intensity and percentage scores gave rise to the final staining score: 0 (negative), + (1-4), ++ (5-8), and +++ (9-12). For statistical analysis, a final staining score of negative or + or ++ without gene amplification was combined into the low expression group, and a final staining score of ++ with gene amplification or +++ was combined into the high expression group [28].

HER-2 expression was evaluated using immunohistochemistry (IHC) and scored according to the updated ASCO-CAP system[29]. Fluorescent in situ hybridization (FISH) was done if HER-2 expression was classed as +2 in IHC. HER-2 was considered to be over expressed if it was classed as +3 by IHC or if showed gene amplification in FISH test. Fluorescent in situ hybridization was performed using Vysis DNA probe kit (PathVysion, USA). Gene amplification was recorded when the ratio HER-2/centromeric probe for chromosome 17 (CEP17) signal was >2.0. For statistical analysis, a final staining score of negative or +1 was combined into the low expression group, and a final staining score of +2 or +3 was combined into the high expression group.

2.3. Statistical Analysis
Statistical analysis was performed using SPSS 18.0(SPSS Inc., Chicago, IL, USA) statistical software. Chi-square test was used to determine the significance of any differences between the IHC scores for groups of tissue specimens. A P- value< 0.05 was considered statistically significant.

3. Results

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3.1. Clinicopathological Data Of Patients
The examined cases were 82 females and 22 males. The mean age of cases was 40.17±1.47 years with a median of 40 years (range 14–85 years).

3.2. Expression Of GPER1, CXCR1 And HER- 2 In Different Nodular Thyroid Tissues And Their Association With Clinico Pathological Features
GPER1, CXCR1 and HER-2 protein expressions were detected in the cytoplasm and cell membrane. Their expression levels were significantly higher with advanced age ≥ 40 years (p=0.045, 0.005 and 0.015 respectively), and with female gender only with HER-2 expression (p=0.005). The expression was low in all cases of nodular hyperplasia and most cases of thyroiditis. While, the expression was increased in thyroid carcinomas compared to FA for all three molecules as shown in Table 1. No significant association was detected between protein expression of the three molecules with size in FA. The same previous association was significant only with GPER1 in PTC (p=0.017) and only for CXCR1 in FC (p=0.050).Lymph node metastasis and advanced tumor stage were significantly associated with GPER1 and CXCR1 expression. Whereas, Her-2 expression was significantly higher with only tumor stage (p=0.023).

The concomitant low expression of the three molecules was noted in 76.9% in all thyroid cases studied, in 70.6% of FA and in 33.3% of thyroid carcinomas (Table 4). While only one case (5.9%) of FA and 5 cases (23.8%) of thyroid carcinomas showed concomitant high expression and they were stratified as 2cases of PTC and 3 cases of FC.

3.3. Correlation Of GPER1, CXCR1 And HER-2 Protein Expression With One Another In Different Nodular Thyroid Tissues
As shown in Table 2, the correlation of the studied molecules was highly significant in all cases studied (104 cases). On studying this correlation in malignant thyroid tumors (Table 3), a significant correlation was noted between GPER1 and CXCR1. HER-2 expression was significant only with CXCR1 (p=0.013) and not with GPER1 (p= 0.76).

4. Discussion

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Many studies suggested estrogen role in thyroid carcinogenesis [30]. GPER1 is a new member of the estrogen receptor family that mediates response to estrogen through activation of epidermal growth factor receptor (EGFR). This leads to production of several downstream signaling molecules that are associated with cancer cell proliferation, invasion, metastasis and resistance to therapy [31]. CXCR1 was found to be involved in EGFR activation through interaction with GPER1 especially in estrogen negative cancer [32]. It was reported that GPER1 protein expression was higher in endometrial carcinoma, ovarian carcinomas and lung tumors, when compared with their normal or benign tissues [33-35].On the contrary; GPER1was positive in all samples of normal breast, while in carcinoma cases only 42% were positive [36].

In the present study, upregulation of GPER1, CXCR1 and HER-2 expression was found from FA to PTC and FC cases. The expression of the three molecules was higher in FC (83.3%, 66.7%, 50% respectively) when compared to either FA (29.4%, 23.5%, 5.9% respectively) or PC (60%, 46.7%, 13.3% respectively). Similar results were observed in previous studies [1415,26].However, concomitant expression of GPER1, CXCR1 and HER-2 was not investigated in previous studies. In the present study, the differences in GPER1, CXCR1, and HER-2 protein expression between non neoplastic thyroid tissues and thyroid tumors were statistically significant (P < 0.001).On the contrary,CXCR1 expressionwas lower in thyroid normal cells compared to transformed cells[37].

When the correlation of GPER1, CXCR1and HER-2 protein expression with clinicopathological parameters was assessed, it was found that GPER and CXCR1 protein expression were associated with age, histopathological diagnosis, nodal metastasis and TNM stage. Previous study found significant correlation between both protein expressions and lymph node metastasis in PC [28].Another study on follicular adenomas and carcinomas, detected positive correlation only with histopathological diagnosis and both proteins [15].

HER-2 protein expression was associated only with age, gender, histopathological diagnosis and TNM stage. But, neither of molecules studies was associated with the size. These results suggested that GPER1, EGFR and HER-2 may play an important role in invasion and metastasis of malignant thyroid tumors.

Additionally, our study, for the first time, demonstrated a significantly positive correlation between GPER1, CXCR1 and HER-2 expression in different thyroid lesions. These findings could be supported by the high protein expression of the three molecules especially in neoplastic thyroid tissues in previous studies [15,26]. However, it is necessary to further elucidate the molecular mechanisms underlying these correlations.

To our knowledge, this is the first study that demonstrated a positive correlation of GPER1, CXCR1 and HER-2 expression in FA, PTC and FC of the thyroid. High expression of GPER1, CXCR1 and HER-2 was associated with tumor stage. So studying the protein expression level of the three molecules can predict the tumor stage and subsequent response to targeted therapy and patients' prognosis. Future studies on larger number of cases will be necessary to determine the utility of these molecules as molecular markers for therapy in thyroid tumors.

Reparixin, a clinical CXCR1/2 inhibitor. When it was combined with 5-Fluorouracil, it prevented malignant gastric cells proliferation, survival and migration both in vitro and in vivo [38]. Recent studies had provided evidence that using Reparixin affects various phenotypes of thyroid cancer cells. Also it did not exert any effect on non-neoplastic thyroid cell growth and survival even at the high concentrations [37,39].So, detecting CXCR1 in thyroid carcinoma is important to select those who could be targets for Reparixin.

HER-2 expression have been previously reported in thyroid cancer with controversial results. Either due to variation in number of cases, different methodology or variable scoring criteria [40-42]. In the present study; the updated ASCO-CAP system was used for assessment of HER-2 expression. High expression of HER-2 was not detected in non neoplastic thyroid tissue but was obvious in 5.9% FA, 13.3% PTC and in 50% FC. A previous study has detected high expression of HER-2 protein in 44% of FC and in 18% of PTC with no expression in normal thyroid tissue . As HER-2 expression was more evident in metastatic thyroid carcinomas. Therefore, They suggest a role of HER-2 expression towards worse prognosis and eligibility for targeted therapy, such as trastuzumab especially in iodine refractory cancers . Also, HER-2 expression was reported to be associated with disease recurrence [40]. On the contrary, others reported no significant variation in HER-2 expression in benign or malignant thyroid tumors [41,42]. On the other hand, Low rate of HER-2 over expression was reportedin 6.9% of PTC and not detected in FC cases [43].

The occurrence of cytoplasmic expression of HER-2 with membranous expression was considered non significant and should be ignored because trastuzumab only target membranous HER-2 . In colon cancer; cytoplasmic HER-2 was associated with survival and prognosis. Whereas, Lapatinib that targets cytoplasmic HER-2 can be used for treatment[44]. Several mechanisms were suggested to explain the cytoplasmic expression of HER-2. One of them is by binding to p155 that presents in the mitochondria leading to instability of the HER-2 protein and its mRNA. Also when the incubation time is more than 30 minutes; the cytoplasmic stain appears. Another theory is due to upregulation of promoter-binding proteins leading to increase in HER-2 production [45]. Moreover, cytoplasmic expression of HER-2 was not associated with gene amplification [46].

On molecular basis, HER-2 expression was detected in 38.5% of familial PTC and restricted to less than 10% of tumor cells. On comparing IHC with FISH, inconsistent results were obtained [47]. In another series, No DNA amplification of HER-2 was detected in either PTC or FC. They found that cytoplasmic immunoreactivity of HER-2 was correlated with tumor differentiation [48].

Due to contradictory results between many studies, HER-2 expression profile still needs further evaluation regarding its role in prognosis of thyroid tumors.

5. Conclusion

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The rates of GPER1, CXCR1 and HER-2 high expression were significantly higher in FTC than in FTA. However, their expression should not be alone suggested as a diagnostic marker for discriminating between FTC and FTA diagnosis. Concomitant high expression of any two or all of the three molecules had stronger correlation with the occurrence of FTC than did each alone. Consequently, concomitant high expression of GPER1, CXCR1 and HER-2 should be suggested as a valuable tool in the differential diagnosis between FTC and FTA.

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