Determining the Prevalence of RET/PTC Mutation in Cases Where Thyroid Nodules in American Thyroid Association (ATA) Ultrasonography (USG) Guidance According to Risk Category is Determined and investigating the Relation of Malignancy

OBJECTIVE


Introduction
More than 20% of the general population has a palpable thyroid nodule and the percentage rises to 70% based on ultrasound identi cations. In 95 % of the cases, the nodule is simply a hyperplastic or benign lesion [1]. Thyroid cancer is the most common malignancy of the endocrine system [2]. The best reliable diagnostic test for thyroid nodules is ne needle aspiration (FNA). Cytological analysis is now, almost routinely, being combined with molecular genetics to enable the pathologist to make a more objective diagnosis [1,3]. The molecular mechanism of thyroid cancers is now properly well cognised. Molecular research might be used as a rule-in test for analysis of cancer in thyroid nodules [4,5]. Nowadays there is a better identi cation of the process implicated in the start and progression of thyroid cancer. It is actually now recognised that BRAF and RAS genetic abnormality and RET/PTC and PAX8/PPARγ rearrangements account for the majority of molecular alterations detected in differentiated thyroid cancers [6]. The most common genetic mutations in papillary and follicular thyroid cancers are point mutations of the BRAF or RAS genes, while the well known chromosomal alterations are RET/PTC and PAX8/PPARγ rearrangements [2,7]. The RET/PTC rearrangement and the BRAF(V600E) mutation are the two prevalent molecular alterations associated with papillary thyroid carcinoma (PTC), and their recognition is gradually being used as an assistant to cytology in diagnosing PTC. On the other hand, there are warnings related with the use of the molecular method in ne-needle aspiration (FNA), particularly for RET/PTC, that should be taken into consideration. It has been declared that a clonal or sporadic existence of this pathology in follicular cells can distinguish between malignant and benign nodules [8]. There are many factors for thyroid cancer etiology one of these is rearranged during transfection (RET) mutations. The RET receptor is fundamentally a tirosinkinase membran receptor that is controlled at the 10th chromosome [9]. Rearrangement of the RET gene, also known as RET/PTC rearrangement, is the most common genetic alteration identi ed to date in thyroid papillary carcinomas [10]. There are now at least 15 types of RET/PTC rearrangements involving RET and 10 different genes [11]. Con icting results on the frequency and type of RET/PTC rearrangements have been reported in relation to age, radiation exposure, and histological tumor variant [12].
This study aims to determine the prevalence of RET/PTC mutation in cases where thyroid nodules in ATA USG according to risk category is determined and investigating the relation of the malignancy.

Patients
Medical records of patients were prospectively evaluated. 102 patients met inclusion criteria and were assessed for eligibility. 42 patients were excluded. We exclude; 2 patients who had previously been exposed to high levels of radiation, 13 patients who had Hashimoto's thyroiditis and Graves' disease, 18 patients who do not want to undergo surgery and 9 patients who had disappeared on follow-up. Sixty patients with thyroid nodules were included in this study. The study protocol was approved by the ethics committee of the Ege University Medical School. Informed consent had been obtained before the study in all patients. The study was carried out in the department of endocrinology and metabolism diseases at the Ege University Faculty of Medicine.
Physical examination, thyroid pro les, thyroid ultrasonography and FNA (Ret/PTC) were performed before surgical procedure. Sonographic images were classi ed according to the American Thyroid Association guidelines [13]. 3 USG groups: A-high suspicion, B-intermediate suspicion and C-low suspicion were included in the study according to ATA nodule sonographic patterns and risk of malignancy.
In the sonographic pattern; High suspicion (groups A) features are solid hypoechoic nodule or solid hypoechoic component of a partially cystic nodule with one or more of the following features: irregular margins (in ltrative, microlobulated), microcalci cations, taller than wide shape, rim calci cations with small extrusive soft tissue component, evidence of extrathyroidal extension (ETE). Intermediate suspicion (groups B) features are hypoechoic solid nodule with smooth margins without microcalci cations, ETE, or taller than wide shape. Low suspicion (groups C) features are isoechoic or hyperechoic solid nodule, or partially cystic nodule with eccentric solid areas, without microcalci cation, irregular margin or ETE, or taller than wide shape.
In the thyroid nodules, indications for surgical treatment included suspicious lesions detected by ne needle aspiration biopsy and growing nodules. Primary surgical treatment was Total Tyroidectomy (TT) or neartotal tyroidectomy when preoperative diagnosis was available.
Thyroid FNA samples were provided from the Department of Endocrinology for molecular examinations of the patients with thyroid nodules. RET/PTC analyses from FNA samples were carried out at Molecular Medicine Research Laboratory of Ege University.
RET-PTC genes rearrangement method from ne needle aspiration samples.

manufacturer's instructi ne
The Fine Needle biopsy sample is placed in the tube containing Ambion RNAlater Stabilization solution (Thermo Fisher Scienti c Waltham, MA USA) and the needle is washed several times.
The RNA Stabilization Solution is an aqueous tissue storage reagent that rapidly permeates most tissues to stabilize and protect RNA in fresh specimens. It eliminates the need to immediately process or freeze samples; the specimen can simply be submerged in RNAlater Solution and stored for analysis at a later date. Samples stored in RNAlater Solution have been used successfully with TRI Reagent® Solution kit according to the manufacturer's instructions.
Dissolved samples at room temperature were rotated for 5 minutes at a high speed of 14,000 rpm in a refrigerated centrifuge and the top was thrown. The Pellet is washed with PBS and centrifuged again.
Isotaion of mRNA from 200 mkl PBS suspended sample is performed according to prospectus using the QIAmp RNAlater mini kit.
The nal elution product is kept in 50µl RNAse-free Water. RNA concentration is measured in NanoDrop The mixture obtained is added in Veriti Thermal Cycler (Applied Biosystems) by adjusting the following temperature and time: + 25 0 C 10 minute a cycle + 48 0 C 60 minute a cycle + 4 0 C ∞ cDNA material can be kept at + 4 0 C or -20 0 C for a long time.
The integrity of the RNA and e ciency of the RT reaction in each sample was con rmed for the housekeeping genes GAPDH or PGK1 whereas the presence of follicular epithelial cells was assessed by the presence of thyroglobulin (Tg) mRNA on RT-PCR as previously described by Rebelo S et al (Rebelo S Domingues R, Catarino AL et al. Immunostaining and RT-PCR: different approaches to search for RET rearrangements in patients with papillary thyroid carcinoma [14]. PCR for ret/PTC-1, -2, and − 3 was performed as described previously. The analysis of ret/PTC gene rearrangements re nes the ne needle aspiration diagnosis of thyroid cancer [15]. Negative controls performed with each RT-PCR reaction omitted either template or reverse transcriptase. The products were resolved on a 2 % agarose gel containing ethidium bromide and visualized under UV light.

Statistical Analyses
Statistical tests were performed using the IBM SPSS Statistics for Windows, Version 25.0. (Released 2017, Armonk, NY: IBM Corp.) Whether the numerical variables showed a normal distribution or was not examined by Shapiro Wilk Test in each subgroup. An independent Two-Group Student-t Test was used for two-group comparisons, and One-Way Analysis of variance (One-Way ANOVA) was used for multiplegroup comparisons. After variance analysis, Bonferroni Test was used as Post HOC method.
Relationships between categorical variables were examined using the Chi-Square Test. All statistical hypothesis checks were carried out at the 0.05 signi cance level.
There was a statistical difference related to in the USG groups frequency between Classic type PTC (70%) and Follicular type PTC (30%)(p = 0.019)( Table 4). There was statistical difference related to between RET/PTC and PTC types in the USG groups (p = 0.012)( Table 4). There was no statistical difference related to between RET/PTC and PTC subtypes (P = 0.613) ( Table 5). There was no statistical difference related to between PTC type (Classic type and Follicular type) and TSH, age and tumor diameter (respectively p = 0.740, p = 0.138, p = 0.875) ( Table 6). There was no statistical difference related to between RET PTC type (1,3) and TSH, age and tumor diameter (respectively p = 0.626, p = 0.241, p = 0.752) ( Table 7).   There was no statistical difference related to between RET/PTC and PTC subtypes (P = 0.613) There was no statistical difference related to between sex and usg groups and PTC and RET/PTC (respectively p = 1, p = 1, p = 0.935) ( Table 8). There was no statistical difference related to between age and usg groups and PTC and RET/PTC (respectively p = 0.274, p = 0.185, p = 0.092) ( Table 8). There was no statistical difference related to between TSH and usg groups and PTC and RET/PTC (respectively p = 0.512, p = 0.675, p = 0.870) ( Table 8). There was no statistical difference related to between RET/PTC positive (12.46 ± 3.71mm) and RET/PTC negative (13.80 ± 3.27mm) tumor diameter (p = 0.485). There was statistical difference related to between usg groups tumor diameter A and C (P = 0.010) (Table 8).

Discussion
Recently, RET/PTC rearrangements have been shown not only in PTC but also in benign thyroid lesions. In recent years, thyroid cancer has been at the forefront of molecular pathology as a result of the consequences of the Chernobyl disaster and the recognition of the role of RET/PTC rearrangements in papillary thyroid carcinomas (PTCs). The evolution of molecular tests for thyroid nodules followed the discovery of various diagnostic and prognostic molecular markers of thyroid cancer that can be applied to thyroid FNA [16].
The most frequent initial manifestation of thyroid cancer is the appearance of a nodüle, most of which are benign nodules. Estimating, less than 5% are malignant nodules. In reality, some cases are misdiagnosed, and many patients undergo unnecessary surgery. Because of this, an accurate pre-surgery evaluation is required. In most cases, the most reliable diagnostic test for thyroid nodules is ne needle aspiration (FNA) cytology.
In our study, we performed the molecular analysis using a procedure that involves RET/PTC gene mutation in easily obtainable FNA samples. We aimed to attempt to improve the e cacy of the FNA diagnosis of thyroid nodules and thus patient management.
In a multicenter study in Italy, the rate of mutation-positive FNAs were found related to the risk of malignancy of in each Bethesda diagnostic categories class [21]. On the other hand, in a study in Germany, their data suggest that the application of the current seven-gene panel in a routine primary referral setting does not improve the presurgical diagnosis of thyroid FNAs [22].  Table 2). Similarly with the literature, in our study signi cant correlation was found between USG groups and PTC (respectively group A,B,C: 60%, 30%, 10%)(p = 0.003)( Table 3) [13]. When we considered patients with PTC according to histological types, they were composed of classical type and follicular variant (FV). The FV is the most common type of PTC after classic PTC [23]. In our study, classical type and FV of PTC prevalence was similar with literature [24]. In our study, in frequency between Classic type and Follicular varient PTC signi cant correlation was found with USG groups (p = 0.019) ( Table 4).
The prevalence of RET rearrangements have been examined in cohorts of papillary carcinomas in many countries. The prevalence has shown a marked geographic discordance [17,[25][26][27][28]. RET/PTC1 and RET/PTC3 are the most encountered types [29]. Similarly, RET/PTC1 and RET/PTC3 the most type of RET/PTC was found in our study.
The relationship between RET/PTC prevalence and types of PTC were investigated. No correlation difference was found between RET/PTC and types of PTC (Table 5).
In a study Basolo F at al. investigated the prognostic meaning of RET/PTC rearrangement on the long term outcome of PTC. No correlation was found between RET expression and other parameters such as age at diagnosis, sex, histological variant and tumor class [30].
The relationship between prognostic factors and RET/PTC prevalence and types in patients with PTC were investigated in our study. There was no statistical difference related to TSH, age and tumor diameter in both PTC type (Classic and Follicular) and RET PTC type (1,3) (Table 6 and Table 7). No correlation was found with RET/PTC releated to multifocality, bilaterality, soft tissue invasion and lymph node metastases.
In the beginning of the literature rstly, our study investigated the prevalence of RET/PTC mutation in thyroid nodules according to risk category ATA USG. On the other hand, we were determined the relation of the between RET/PTC and malignancy. No differant was found between USG groups and TSH, sex, age at diagnosis and tumor diameter (Table 8). In addition, No difference was found with sex in USG groups neither RET/PTC nor PTC. There was a statistical difference related tumor to diameter with groups A and C (Table 8).
The weaknesses of our study were the absence of other molecular markers and the number of patients. Strengths of the study were the cytological evaluation and histopathological con rmation of RET/PTC according to the ATA USG risk category for the rst time.
As a result, Cytological analysis is now, practically routinely, being joined with molecular analysis to enable the patholog to make a more actual diagnosis. Molecular testing of FNA samples improves presurgical diagnosis. RET/PTC frequency in FNA signi cant difference was found in ATA USG risk category. RET/PTC was found to be signi cantly higher in the diagnoses of the PTC with 75% sensitivity and 65% speci city. However, a larger clinical study will be required to verify this resuls.

Declarations
Compliance with ethical standards Con ict of interest The authors declare no con ict of interest.
Ethical approval Consent forms and protocols were approved by the ethics committee of the Ege University Medical School. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/ or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.
Informed consent Informed consent was obtained from all individual participants included in the study.