SIX1 Expression and its Clinicopathological Significance: Difference between Classic and Follicular Variant Papillary Thyroid Carcinoma

SIX1 Expression and its Clinicopathological Significance: Difference between Classic and Follicular Variant Papillary Thyroid Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article SIX1 Expression and its Clinicopathological Significance: Difference between Classic and Follicular Variant Papillary Thyroid Carcinoma Elzahraa Ibrahim Khalil, Ahmed S. Issa, Rehab M. Kamal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4664320/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Dec, 2024 Read the published version in Thyroid Research → Version 1 posted 11 You are reading this latest preprint version Abstract Background Papillary thyroid carcinoma (PTC) is the most common type of thyroid carcinoma, representing the majority of thyroid cancer cases. Most patients with PTC have an excellent prognosis following treatment, yet approximately 10% face mortality within ten years, primarily due to lymph node metastasis (LNM) or local recurrence. The SIX1 gene, a member of the SIX gene superfamily, encodes a transcription factor integral to the development of certain tissues during embryogenesis. The impact of SIX1 in different subtypes of PTC ha s not been studied previously. Objective The purpose of this study was to investigate the expression of SIX1 protein in PTC and to explore its relationship with clinical behavior in two subtypes of PTC: classic PTC (C-PTC) and follicular variant PTC (FV-PTC). Material and Methods Using immunohistochemistry, the study analyzed 125 primary PTC cases, including 85 cases of C-PTC and 40 cases of FV-PTC. Results The study found significant positive associations between high SIX1 expression and several adverse clinical features across the PTC samples. High SIX1 expression was linked with increased tumor size, multifocal tumors, LNM, high-grade tumor features, advanced tumor stage, lymphovascular invasion, perineural invasion, and extrathyroidal extension (ETE). Within the classic PTC subgroup, high SIX1 expression showed significant positive correlations with Tumor size (P = 0.04), Multifocality (P = 0.02) and High-grade features (P = 0.03). In the follicular variant subgroup, high SIX1 expression was significantly associated with Lymph node metastasis (LNM) (P = 0.001), Lymphovascular invasion (P = 0.03), ETE (P = 0.003) and Tumor stage (P = 0.007). Conclusions The findings of this study indicate that SIX1 expression is a marker of poor prognosis in PTC, suggesting that its high expression is linked with more aggressive tumor characteristics and advanced disease stages. Importantly, the impact of SIX1 expression varies between C-PTC and FV-PTC, predicting distinct prognostic factors in each subtype. This suggests that SIX1 could be utilized not only as a prognostic biomarker but also in developing subtype-specific therapeutic strategies for PTC patients. SIX1 Papillary thyroid carcinoma classic PTC follicular variant PTC Immunohistochemistry Figures Figure 1 Figure 2 1. Background Thyroid cancer (TC), the predominant endocrine malignancy globally, ranks as the 10th most frequently diagnosed cancer worldwide [ 1 ], and the 6th most prevalent among women [ 2 ]. Incidence of thyroid cancer is nearly three times higher in women than in men [ 3 ]. In Egypt, TC stands as the 5th most prevalent cancer among women [ 4 ]. The rise in TC incidence can be attributed to advancements in diagnostic techniques, facilitating early detection [ 5 ]. To date, childhood exposure to ionizing radiation remains the most established modifiable risk factor for TC [ 6 ]. Thyroid cancers originate from either follicular cells or parafollicular cells (C cells) [ 7 ]. Among the various histopathologic types of TC, approximately 85% are papillary thyroid carcinomas (PTC), 4% are follicular thyroid carcinomas (FTC), 2% are Hurtle-cell carcinomas, 2% are medullary thyroid carcinomas (MTC), and 1% are anaplastic thyroid carcinomas (ATC) [ 3 ]. The majority of PTC cases comprise classic PTC (C-PTC) and follicular variant PTC (FV-PTC) histologic subtypes. Historically, clinical equivalence was assumed; however, subsequent studies revealed that C-PTC may exhibit more aggressive features [ 8 , 9 ]. BRAF mutations are less common in FV-PTC compared to C-PTC [ 10 ], which could account for the different roles in patient outcomes between C-PTC and FV-PTC. Cancers originating from follicular cells are typically classified as differentiated thyroid carcinomas (DTC), constituting 85% of tumors with generally favorable treatment outcomes. However, the remaining 10%-15% of tumors may progress into more aggressive forms of thyroid cancer [ 7 ]. These aggressive variants often necessitate more extensive surgical intervention and additional adjuvant therapies. For PTC, conventional surgical thyroidectomy combined with radioactive iodine ablation and thyrotropin hormone-suppressive levothyroxine administration can achieve excellent prognoses for the majority of patients [ 11 ]. Unfortunately, 10% of PTC patients present with locally advanced or metastatic disease at diagnosis, representing the primary causes of thyroid cancer-related mortality [ 12 ]. Consequently, comprehending the molecular basis of thyroid cancer is crucial for the development of novel and effective treatment strategies for advanced PTC patients. Transcription factors (TFs), regulatory proteins coexist in genomic regions, interact with one another to facilitate the regulation of gene expression. TFs exert their regulatory influence by binding to specific DNA elements and forming protein complexes [ 13 ]. The development of cancer can be attributed to alterations in TFs through various direct mechanisms, including gene amplification or deletion, chromosomal translocations, point mutations, or changes in expression levels [ 14 ]. SIX1, a member of the transcription factor family, plays critical roles in organ development and differentiation by regulating the expression of various proteins implicated in embryogenesis. Meanwhile, abnormal reactivation of embryological genes, including SIX1 and the EYA family, can contribute to tumorigenesis. The aberrant activation of SIX1 and EYA has been implicated in the development of several cancers [ 15 ]. Studies have revealed that SIX1 can function as an oncogene by inducing epithelial-mesenchymal transition (EMT) and activating the transforming growth factor (TGF) signal transduction pathway, thereby promoting tumor progression [ 16 , 17 ]. Upregulation of SIX1 has been observed in multiple tumor types, such as breast cancer [ 18 ], colorectal cancer [ 19 ], and ovarian carcinoma [ 20 ], exerting influence on their clinical prognosis. Few studies have investigated the role of SIX1 in TC, particularly PTC, and its possible relation with tumor behavior. Most of these studies have been conducted using cell lines, while others have employed functional assays or tissue microarray (TMA) analyses rather than whole-tissue section examinations. Additionally, there is a lack of research characterizing SIX1 expression and its relationship with clinical behavior specifically in C-PTC and FV-PTC. We hypothesize that SIX1 is involved in the development of PTC. Therefore, utilizing immunohistochemistry, our study aims to investigate the expression levels of SIX1 in PTC and its association with clinicopathological data. Furthermore, comparing the immunohistochemical expression of SIX1 between C-PTC and FV-PTC patients and examining its association with the clinicopathological features of these patients are investigated. 2. Material and Methods 2.1. Tissue specimens The present study comprised 125 patients of primary PTC (85 cases C-PTC and 40 FV-PTC) that were collected from Pathology Department, Minia University Hospital and Minia Oncology Center, Egypt during the period from 2018 to 2022. Patients were referred from Radiology Department initially for cytological examination of their proven solitary or multinodular goiter based on Thyroid Imaging Reporting and Data Systems (TIRADS) staging. Pathologically proven papillary thyroid carcinomas underwent surgical resection and tissue specimen histoplathological assessment. The corresponding clinical data were obtained from patients’ medical records after approval of the corresponding hospital districts and the study protocol was approved by Local Ethics Research Committee of Faculty of Medicine, Minia University. Hematoxylin and Eosin (H&E) stained slides for all cases were reviewed to confirm the diagnosis. Tumor type and grade were evaluated according to WHO classification of thyroid neoplasms, 5th edition [ 21 ]. Tumor size and stage was estimated by AJCC 8th Edition/TNM classification system for DTC [ 22 ]. 2.2. Immunohistochemistry Five µm sections were prepared on positive charged slides for immunohistochemistry of SIX1 primary antibodies utilizing the avidin biotin-peroxidase complex method with diaminobenzidine (DAB) chromogen detection system. Initially tissue sections on the positive charged slides were deparaffinized and rehydrated. Then the endogenous peroxidase was blocked by immersion in a 3% solution of hydrogen peroxide and incubated for 30 minutes. Antigen retrieval was performed by immersing the slides in citrate buffer solution (pH 6) for 2 times (10 minutes each) at 750-W. To block nonspecific background staining, the slides were treated by UV block. Primary antibody SIX1 was then added, and tissue sections were incubated for 1 hour at room temperature (dilution 1:500). Excess reagent was thrown off and the slides were then rinsed gently with buffer solution for 5 minutes. After that Secondary biotinylated antibody was added for each slide for 30 minutes. DAB substrate and chromogen. The Positive control for SIX1 was normal humanendocervicalglands. 2.3. Scoring of Immunostaining Cytoplasmic or nuclear SIX1 expression is considered positive. Immunostaining was scored by two independent pathologists. The immunohistochemical scores were obtained as the staining intensity (scored from 0–3) multiplied by the percentage of positive cells (scored from 1–4). The intensity of SIX1 protein expression was scored as; 0 (no staining), 1 (weak), 2 (moderate) and 3 (strong). The percentage of positive cells was scored as; 1 (0–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%). The cut-off value for high versus low expression of the SIX1 protein, defining a final immunostaining score of > 6 as high SIX1 protein expression and score ≤ 6 as low SIX1 expression [ 17 ]. 2.4. Statistical analysis Statistical analysis was conducted using the IBM SPSS 20.0 statistical package software. Chi-square and Fisher’s exact tests were used to compare categorical variables. Comparison between the expression of SIX1 in the primary tumor and their corresponding lymph node metastasis was evaluated using spearman’s test. Results were considered statistically significant when P value ≤ 0.05. Data were analyzed using the Statistical Package for Social Sciences (SPSS) version 17 software. 3. Results 3.1. Patient and tumor characteristics Overall, the mean age ± standard deviation (SD) of the studied patients was 42.02 ± 14.6, ranging from 18–80 years. Patients were classified into < 55 years and ≥ 55 years according to the median age of patients included in this study. Most patients 84% (105/125) were 2 and ≤ 4 cm and > 4cm, the mean size ± SD was 2.31 ± 1.46, ranging from 0.5- 6cm at the maximum dimension, most tumors were ≤ 2cm. Of patients, 68% were C-PTC and 32% were FV-PTC. Most tumors have low grade features 100/125 (80%). Lympho-vascular invasion (LVI) and perineural invasion (PNI) were detected in 28% and 16% respectively. Positive lymph node metastasis (LNM) was diagnosed in 24% of patients. Twenty-three tumors (18.4%) showed extrathyroidal extension (ETE). Most tumors (42.4%) were stage I. Patients’ characteristics listed in Table (1). 3.2. Expression of SIX1 in PTC and adjacent non-cancerous tissue SIX1 was expressed in the cytoplasm of cancer cells. SIX1 was expressed diffusely without heterogeneity throughout the tumor. Overall, the positive expression ratio of SIX1 was 36% (45/125), while 80/125 tumors (64%) were negative/low expression. SIX1 positive staining was detected in 6 cases (3.2%) of adjacent non-cancerous thyroid tissue (ANCT) (4/80). On comparing SIX1 expression between non-tumor and PTC; an obvious significant increase in PTC was found (p < 0.001) (Fig. 1). 3.3. Association of SIX1 with clinicopathological features in PTC The statistical analyses revealed significant positive associations between high SIX1 expression and increased tumor size, multifocal tumor, LNM, tumor with high grade features, advanced tumor stage, LVI, PNI and ETE,( P = 0.04, P = 0.003, P = 0.002, P = 0.01, P = 0.002, P = 0.002, P = 0.01and P = 0.001, respectively) (Fig. 2). No significant associations were observed between SIX1 expression and patients’ age, gender, or histological subtype. (Table 1) 3.4. SIX1 expression in classic PTC and follicular variant PTC High SIX1 expression was observed in 38.8% (33/85) of C-PTC versus 30% (12/40) in FV-PTC tumors. On comparing SIX1 expression in C-PTC and FV-PTC in relation to their different clinicopathological parameters, the statistical analyses revealed significant positive associations between high SIX1 expression and tumor size, multifocality and tumors with high grade features in C-PTC cases (P = 0.04, P = 0.02 and P = 0.03, respectively), and significant positive associations between high SIX1 expression and LNM, LVI, ETE and stage in FV-PTC tumors (P = 0.001, P = 0.03, P = 0.003 and P = 0.007, respectively). (Table 2) 4. Discussion As mentioned previously, mortality due to local recurrence or lymph node metastasis occurs in approximately 10% of papillary thyroid carcinoma (PTC) patients within 10 years [ 7 ]. Therefore, there is an urgent need to clarify the pathogenesis of PTC to develop new treatment strategies. The present study found that SIX1 was upregulated in PTC cases overall, with particularly notable upregulation observed in PTC patients exhibiting adverse prognostic features. This highlights the potential significance of SIX1 as a biomarker for identifying high-risk PTC cases and underscores the importance of further research into its role in PTC pathogenesis and potential therapeutic targeting. SIX1, recognized as a developmental transcription factor crucial in embryonic myogenesis, has been identified to also play a significant role in tumorigenesis. For instance, in cervical intraepithelial or cervical cancer, the expression of SIX1 was markedly higher compared to that in normal cervical tissues [ 23 ]. In the current study, SIX1 expression was obviously elevated in PTC compared to ANCT, where almost non-cancerous tissues showed no expression of SIX1. These findings align with previous research indicating the upregulation of SIX1 in PTC while being absent in non-cancerous tissue [ 16 , 17 , 24 ]. Additionally, our findings are consistent with analyses of The Cancer Genome Atlas (TCGA) dataset, which demonstrated higher Six1 mRNA levels in thyroid cancers compared to non-cancerous thyroid tissues [ 24 ]. In this regard, functional assays conducted on two thyroid cancer cell lines to examine cell viability and colony formation revealed that SIX1 enhanced PTC proliferation and provided significant protection against apoptosis, whereas knockdown of SIX1 using siRNA inhibited growth rate and colony formation [ 16 , 17 , 24 ]. These results confirm the role of SIX1 in promoting growth in thyroid cancer cells, suggesting its potential as an oncogene in PTC tumorigenesis. The precise mechanism by which SIX1 mediates tumorigenesis remains unclear [ 25 ]. Earlier studies have shown that overexpression of SIX1 facilitates tumorigenesis and proliferation of breast cancer cells by directly activating cyclin A1 transcription [ 26 ]. In colorectal and cervical cancers, SIX1 has been implicated in EMT and is described as a mediator of ZEB1 transcription and regulation of TGF-β signaling [ 23 , 27 ]. More recently, the proliferation of PTC has been linked to the activation of classical STAT3 signaling and the TGF-β/Smad2/3 signaling pathways [ 16 , 17 ]. In our study, SIX1 was primarily found to be localized in the cytoplasm, a result consistent with the findings of Min and Wei as well as Kong et al. whereas Yang et al. observed nuclear localization of SIX1 expression in their series [ 16 , 17 , 20 ]. High SIX1 expression was detected in 36% of tumors, whereas previous studies have reported a higher positivity ranging from 52–63% in PTC tumors [ 16 , 17 , 24 ]. This variance could be attributed to differences in scoring systems, variations in the clone used, or differences in the methodology employed for the study, such as the use of tissue microarrays instead of larger surgical specimens. Previous literature has consistently associated the upregulation of SIX1 with aggressive tumor characteristics and unfavorable patient prognosis across various cancers [ 18 , 19 , 20 ]. A recent study linked SIX1's role in tumor invasiveness to its promotion of glucose metabolism and invasion through the regulation of GLUT3, MMP2, and snail in thyroid cancer [ 24 ]. In our study, we found a significant association between high SIX1 expression and aggressive features in PTC, including larger tumor size, multifocality, LNM, tumors with high-grade features, advanced tumor stage, LVI, PNI, and ETE. These results are consistent with other studies [ 16 , 17 , 24 ] that have reported a positive relation between invasiveness and high SIX1 expression. Additionally, our findings align with previous studies utilizing Matrigel invasion assays, which demonstrated that SIX1 upregulation increased invasion in thyroid cancer, while knockdown of SIX1 using siRNA reduced invasive ability in both cell lines. Furthermore, our results are supported by an analysis of TCGA data, which showed positive relations between SIX1 mRNA expression and nodal metastasis [ 24 ]. Collectively, these findings suggest that upregulation of SIX1 expression could serve as a biomarker for predicting PTC behavior and clinical outcomes. Historically, C-PTC and FV-PTC were often regarded as having comparable prognosis and treatment approaches. However, recent research by the TCGA group has revealed distinct differences in clinical outcomes and genetic profiles between these variants. Studies by Henke et al. and Shi et al. [ 8 , 9 ] examined large series of C-PTC and FV-PTC and found that C-PTC exhibits a higher prevalence of ETE, LNM, disease recurrence, and mortality compared to FV-PTC. They concluded that C-PTC and FV-PTC represent biologically distinct disease entities with different oncogenic drivers and tumor behaviors. In the current study, the clinicopathological parameters appeared comparable between C-PTC and FV-PTC cases. However, this may be attributed to the small sample size of our study. Little is currently understood about SIX1 expression across various histologic subtypes of PTC. To our knowledge, our study represents the first investigation into the association between immunohistochemical expression of SIX1 and clinicopathological features among both C-PTC and FV-PTC patients. We observed no statistically significant difference in high SIX1 expression between C-PTC and FV-PTC, although there was a slight increase in C-PTC (38.2% versus 30%). This finding contrasts with a recent study that reported a significant difference [ 16 ]. The lack of significance in our study may be attributed to the disproportionate number of each histological subtype and differences in the scoring system used. In the present study, we observed a significant upregulation of SIX1 in C-PTC tumors with larger tumor size, multifocality and high-grade features. Conversely, SIX1 was significantly upregulated in FV-PTC tumors with LVI, LNM, ETE, and advanced tumor stage. Particularly noteworthy in the FV-PTC group, unlike in C-PTC, was the clear association between high SIX1 expression and LNM and ETE, with all LNM patients and nearly all ETE tumors showing high SIX1 expression. It has been suggested that patients with LNM have a higher mortality rate, with incomplete surgical excision being a significant contributing factor to increased mortality in stage I PTC patients [ 28 ]. Our findings indicate that SIX1 expression may serve as a useful marker for predicting which patients with clinically node-negative (cN0) FV-PTC may benefit from prophylactic central lymph node dissection. Previous studies have indicated that locoregional extension to perithyroidal soft tissues and lymph nodes is less common in FV-PTC [ 8 , 9 ]. In this context, our study may aid in selecting FV-PTC patients who require aggressive management while avoiding overtreatment of the predominantly non-aggressive FV-PTC tumors. In C-PTC group, the notable association of high SIX1 expression with multifocality holds significant clinical value. This finding suggests that total thyroidectomy, rather than lobectomy or hemithyroidectomy, might be more appropriate for C-PTC patients. This approach could be justified by the potential presence of small foci of disease in the contralateral thyroid lobe, thereby reducing the likelihood of locoregional recurrence. Moreover, the relation between high SIX1 with tumor size, a key factor in the tumor (T) classification of the AJCC staging system, implies that SIX1 could serve as a predictor of post-treatment recurrence. This suggests that patients with high SIX1 expression may require closer monitoring and more aggressive management strategies to mitigate the risk of recurrence following initial treatment. 5. Conclusions This study indicates that SIX1 could serve as a hallmark of malignancy in PTC. High SIX1 expression was closely associated with adverse prognostic factors in PTC. Furthermore, SIX1 expression may predict distinct prognostic factors in C-PTC and FV-PTC. This suggests that SIX1 could be utilized not only as a prognostic biomarker but also in developing subtype-specific therapeutic strategies for PTC patients. However, these findings require validation in independent large cohorts, and the potential implications for management and staging warrant further investigation. Further research is needed to fully understand the clinical significance of SIX1 expression in PTC and its implications for patient outcomes and treatment strategies. Abbreviations TC thyroid cancer PTC papillary thyroid carcinoma DTC differentiated thyroid carcinoma C-PTC classic PTC FV-PTC follicular variant ETE extrathyroidal extension LNM lymph node metastasis PNI perineural infiltration LVI lymphovascular infiltration Declarations Author Contribution Study design, data acquisition, analysis and drafting of manuscript: Khalil EI, Issa AS and Kamal RM. Statistical analysis: Khalil EI. References Shank JB, Are C, Wenos CD. "Thyroid Cancer: Global Burden and Trends." Indian Journal of Surgical Oncology 2022; 13, no. 1 40-45. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2021. Ca Cancer J Clin 2021; 71(1), 7-33. ‏ Kitahara CM, Schneider AB. Cancer Epidemiol Biomarkers Prev. 2022 Jul 1; 31(7):1284-1297. Gohar M, Mohamed A, Al-Azzouny H. 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Int J Endocrinol. 2014; 2014: 385787. ‏ Tables Table (1): Association between SIX1 expression and clinicopathological features (n=125): Clinicopathological features Number of cases SIX1 Expression P value Low Expression (N=80) High expression (N=45) Age <55 y ≥ 55 y 105 20 70 (66.7%) 10 (50%) 35 (33.3%) 10 (50%) 0.3 Gender Male Female 15 110 5 (33.3%) 75 (68.2 %) 10 (66.7%) 35 (31.8%) 0.1 Histological subtypes C-PTC FV- PTC 85 40 21 (61.8%) 11 (68.8%) 13 (38.2%) 5 (31.2%) 0.4 Tumor size ≤ 2 >2 - ≤ 4 > 4 70 42 13 48 (68.5%) 30 (71.4%) 2 (15.3%) 22 (31.4%) 12 (29.6 %) 11 (84.7%) 0.04 Tumor Focality Unifocal Multifocal 82 43 64 (78.1%) 15 (34.8%) 18 (21.9%) 28 (65.2%) 0.003 Tumor grade Low grade features High grade features 100 25 75 (75%) 5 (20%) 25 (25%) 20 (80%) 0.002 LVI Absent Present 90 35 74 (82.2%) 6 (17.1%) 16 (27.8%) 29 (82.9%) 0.001 PNI Absent Present 105 20 76 (72.3%) 4 (20%) 29 (27.7%) 16 (80%) 0.001 ETE Absent Present 102 23 77 (75.4%) 3 (13.1%) 25 (24.5%) 20 (86.9%) 0.001 LNM Absent Present 95 30 73 (76.8%) 7 (23.4%) 22 (23.2%) 23 (76.6%) 0.002 Tumor Stage pT1 pT2 pT3 pT4 52 40 25 8 43(82.7%) 29(72.5%) 8(32%) 0 9(17.3%) 11(22.5%) 17(68%) 8(100%) 0.01 * P - value > 0.05 are considered statistically significant. Chi-Square test. Table (2): Comparison between the expression of SIX1 in classic and follicular variant PTC with clinicopathological data: Clinico-pathological Variables N. 85 C-PTC N. 40 FV-PTC SIX1 Expression p-value SIX1 Expression p-value Low (N=52) High (N=33) Low (N=28) High (N=12) Age <55 ≥55 72 13 47(65.2%) 5(38.4%) 25(34.8%) 8(61.6%) 0.3 33 7 23(69.6%) 5(71.4%) 10(30.4%) 2(28.6%) 0.7 Gender Male Female 11 74 2(18.1%) 50(67.6%) 9(81.9%) 24(32.4%) 0.09 4 36 4(100%) 24(66.6%) 0 12(100%) 0.6 Tumor Size ≤ 2 >2 - ≤ 4 >4 50 23 12 35(70%) 15(65.2%) 2(1.6%) 15(30%) 8(34.8%) 10(83.4%) 0.04* 20 19 1 15(75%) 12(63.2%) 1(100%) 5(25%) 7(36.8%) 0 0.5 Tumor Focality Unifocal Multifocal 54 31 43(78.1%) 9(29.1%) 11(21.9%) 22(71.9%) 0.02* 28 12 23(82.1%) 5(41.6%) 5(17.9%) 7(59.4%) 0.2 Tumor Grade Low grade features High grade features 64 21 48(73.8%) 4(19.1%) 16(26.2%) 17(80.9%) 0.03* 36 4 26(72.3%) 2(50%) 10(27.7%) 2(50%) 0.5 LVI Absent Present 62 23 44(70.9%) 8(34,7%) 18(2914%) 15(65.3%) 0.05 30 10 26(86.6%) 2(20%) 4(13.4%) 8(80%) 0.03* PNI Absent Present 66 19 44(66.6%) 8(42.2%) 22(33.4%) 11(57.8%) 0.2 39 1 29(74.3%) 2(50%) 10(25.7%) 2(50%) 0.7 ETE Absent Present 73 12 49(67.1%) 3(25%) 24(32.9%) 9 (75%) 0.2 29 11 27(93.1%) 1(9%) 2(6.9%) 10(91%) 0.003* LNM Absent Present 63 22 43(68.2%) 9(40.9%) 20(31.8%) 13(59.1%) 0.1 32 8 28(87.5%) 0 4(12.5%) 8(100%) 0.001* Stage pT1 pT2 pT3 pT4 40 20 19 6 29 (72.5%) 14(70%) 8(42.1%) 1(16.7%) 11(27.5%) 6(30%) 11(57.9%) 5(83.3%) 0.1 12 20 6 2 12(100%) 16(80) 0 0 0 4(20%) 6(100%) 2(100%) 0.007* * P - value > 0.05 are considered statistically significant. Chi-Square test. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 Dec, 2024 Read the published version in Thyroid Research → Version 1 posted Editorial decision: Revision requested 24 Jul, 2024 Reviews received at journal 24 Jul, 2024 Reviews received at journal 23 Jul, 2024 Reviews received at journal 12 Jul, 2024 Reviewers agreed at journal 08 Jul, 2024 Reviewers agreed at journal 07 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers invited by journal 02 Jul, 2024 Editor assigned by journal 02 Jul, 2024 Submission checks completed at journal 02 Jul, 2024 First submitted to journal 30 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4664320","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":330742180,"identity":"e4ef1d68-552f-4432-be03-f87630b2967c","order_by":0,"name":"Elzahraa Ibrahim Khalil","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIie3RMUsDMRTA8VcC6fLg1hyRfoaAIEhL+kFcTgLt0oO6FTpYuKGbc0U/hO4OTwJ207VwSw/ByUEQRHDQd4XSQeLhJpj/khD48R4EIBb7i7UBRH0qBS3Kxr3No/qRiB0ByszgtwSMbyaJaFevJzcW0otiTWvzcGRI3JYIPTsLkLQApxdPDvTeneHFyvyKpOsiDFyI8CakkQR0VLYleKARvAst1vet4gPplMnwhck9k+SNyWeQGCEkT/Gg1aieQvUUfgGyIaK8lF2kJaaL0ZiJy8+93D+8NC4LkWQ+fyyRph21Gl5X7xObny2LavU8sf0Q2Ya76+abDBzPmsy3GqfEYrHYv+kLm95S4UA2y9cAAAAASUVORK5CYII=","orcid":"","institution":"Pathology Department, Minia University","correspondingAuthor":true,"prefix":"","firstName":"Elzahraa","middleName":"Ibrahim","lastName":"Khalil","suffix":""},{"id":330742181,"identity":"f2d8e8c5-f498-4fd5-abc4-6776f66d9af8","order_by":1,"name":"Ahmed S. Issa","email":"","orcid":"","institution":"Radiology Department, Minia University","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"S.","lastName":"Issa","suffix":""},{"id":330742182,"identity":"aec5eb96-8e08-49a3-a12a-bbb569c912f3","order_by":2,"name":"Rehab M. Kamal","email":"","orcid":"","institution":"Pathology Department, Minia University","correspondingAuthor":false,"prefix":"","firstName":"Rehab","middleName":"M.","lastName":"Kamal","suffix":""}],"badges":[],"createdAt":"2024-06-30 21:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4664320/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4664320/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13044-024-00212-9","type":"published","date":"2024-12-09T15:57:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61177604,"identity":"573f372e-05f7-4f31-856c-32e07a1e3f9a","added_by":"auto","created_at":"2024-07-26 15:55:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":689882,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figurespage0001.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4664320/v1/762554a8f2f73d8c3544a84a.jpg"},{"id":61176589,"identity":"3c0a85f6-1e95-4f74-b88c-5d1058f28053","added_by":"auto","created_at":"2024-07-26 15:47:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1107663,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figurespage0002.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4664320/v1/630e107f804b56710b71e222.jpg"},{"id":71552647,"identity":"0e90c52d-09c2-4104-aa6e-ca315a18de51","added_by":"auto","created_at":"2024-12-16 16:07:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2598006,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4664320/v1/513e77bd-a373-4311-af57-3365f0118219.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"SIX1 Expression and its Clinicopathological Significance: Difference between Classic and Follicular Variant Papillary Thyroid Carcinoma","fulltext":[{"header":"1. Background","content":"\u003cp\u003eThyroid cancer (TC), the predominant endocrine malignancy globally, ranks as the 10th most frequently diagnosed cancer worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], and the 6th most prevalent among women [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Incidence of thyroid cancer is nearly three times higher in women than in men [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In Egypt, TC stands as the 5th most prevalent cancer among women [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The rise in TC incidence can be attributed to advancements in diagnostic techniques, facilitating early detection [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To date, childhood exposure to ionizing radiation remains the most established modifiable risk factor for TC [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThyroid cancers originate from either follicular cells or parafollicular cells (C cells) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Among the various histopathologic types of TC, approximately 85% are papillary thyroid carcinomas (PTC), 4% are follicular thyroid carcinomas (FTC), 2% are Hurtle-cell carcinomas, 2% are medullary thyroid carcinomas (MTC), and 1% are anaplastic thyroid carcinomas (ATC) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The majority of PTC cases comprise classic PTC (C-PTC) and follicular variant PTC (FV-PTC) histologic subtypes. Historically, clinical equivalence was assumed; however, subsequent studies revealed that C-PTC may exhibit more aggressive features [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. BRAF mutations are less common in FV-PTC compared to C-PTC [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], which could account for the different roles in patient outcomes between C-PTC and FV-PTC.\u003c/p\u003e \u003cp\u003eCancers originating from follicular cells are typically classified as differentiated thyroid carcinomas (DTC), constituting 85% of tumors with generally favorable treatment outcomes. However, the remaining 10%-15% of tumors may progress into more aggressive forms of thyroid cancer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These aggressive variants often necessitate more extensive surgical intervention and additional adjuvant therapies. For PTC, conventional surgical thyroidectomy combined with radioactive iodine ablation and thyrotropin hormone-suppressive levothyroxine administration can achieve excellent prognoses for the majority of patients [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Unfortunately, 10% of PTC patients present with locally advanced or metastatic disease at diagnosis, representing the primary causes of thyroid cancer-related mortality [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consequently, comprehending the molecular basis of thyroid cancer is crucial for the development of novel and effective treatment strategies for advanced PTC patients.\u003c/p\u003e \u003cp\u003eTranscription factors (TFs), regulatory proteins coexist in genomic regions, interact with one another to facilitate the regulation of gene expression. TFs exert their regulatory influence by binding to specific DNA elements and forming protein complexes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The development of cancer can be attributed to alterations in TFs through various direct mechanisms, including gene amplification or deletion, chromosomal translocations, point mutations, or changes in expression levels [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSIX1, a member of the transcription factor family, plays critical roles in organ development and differentiation by regulating the expression of various proteins implicated in embryogenesis. Meanwhile, abnormal reactivation of embryological genes, including SIX1 and the EYA family, can contribute to tumorigenesis. The aberrant activation of SIX1 and EYA has been implicated in the development of several cancers [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Studies have revealed that SIX1 can function as an oncogene by inducing epithelial-mesenchymal transition (EMT) and activating the transforming growth factor (TGF) signal transduction pathway, thereby promoting tumor progression [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Upregulation of SIX1 has been observed in multiple tumor types, such as breast cancer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], colorectal cancer [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and ovarian carcinoma [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], exerting influence on their clinical prognosis.\u003c/p\u003e \u003cp\u003eFew studies have investigated the role of SIX1 in TC, particularly PTC, and its possible relation with tumor behavior. Most of these studies have been conducted using cell lines, while others have employed functional assays or tissue microarray (TMA) analyses rather than whole-tissue section examinations. Additionally, there is a lack of research characterizing SIX1 expression and its relationship with clinical behavior specifically in C-PTC and FV-PTC.\u003c/p\u003e \u003cp\u003eWe hypothesize that SIX1 is involved in the development of PTC. Therefore, utilizing immunohistochemistry, our study aims to investigate the expression levels of SIX1 in PTC and its association with clinicopathological data. Furthermore, comparing the immunohistochemical expression of SIX1 between C-PTC and FV-PTC patients and examining its association with the clinicopathological features of these patients are investigated.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Tissue specimens\u003c/h2\u003e \u003cp\u003eThe present study comprised 125 patients of primary PTC (85 cases C-PTC and 40 FV-PTC) that were collected from Pathology Department, Minia University Hospital and Minia Oncology Center, Egypt during the period from 2018 to 2022. Patients were referred from Radiology Department initially for cytological examination of their proven solitary or multinodular goiter based on Thyroid Imaging Reporting and Data Systems (TIRADS) staging. Pathologically proven papillary thyroid carcinomas underwent surgical resection and tissue specimen histoplathological assessment. The corresponding clinical data were obtained from patients\u0026rsquo; medical records after approval of the corresponding hospital districts and the study protocol was approved by Local Ethics Research Committee of Faculty of Medicine, Minia University. Hematoxylin and Eosin (H\u0026amp;E) stained slides for all cases were reviewed to confirm the diagnosis. Tumor type and grade were evaluated according to WHO classification of thyroid neoplasms, 5th edition [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Tumor size and stage was estimated by AJCC 8th Edition/TNM classification system for DTC [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Immunohistochemistry\u003c/h2\u003e \u003cp\u003eFive \u0026micro;m sections were prepared on positive charged slides for immunohistochemistry of SIX1 primary antibodies utilizing the avidin biotin-peroxidase complex method with diaminobenzidine (DAB) chromogen detection system. Initially tissue sections on the positive charged slides were deparaffinized and rehydrated. Then the endogenous peroxidase was blocked by immersion in a 3% solution of hydrogen peroxide and incubated for 30 minutes. Antigen retrieval was performed by immersing the slides in citrate buffer solution (pH 6) for 2 times (10 minutes each) at 750-W. To block nonspecific background staining, the slides were treated by UV block. Primary antibody SIX1 was then added, and tissue sections were incubated for 1 hour at room temperature (dilution 1:500). Excess reagent was thrown off and the slides were then rinsed gently with buffer solution for 5 minutes. After that Secondary biotinylated antibody was added for each slide for 30 minutes. DAB substrate and chromogen. The Positive control for SIX1 was normal humanendocervicalglands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Scoring of Immunostaining\u003c/h2\u003e \u003cp\u003eCytoplasmic or nuclear SIX1 expression is considered positive. Immunostaining was scored by two independent pathologists. The immunohistochemical scores were obtained as the staining intensity (scored from 0\u0026ndash;3) multiplied by the percentage of positive cells (scored from 1\u0026ndash;4). The intensity of SIX1 protein expression was scored as; 0 (no staining), 1 (weak), 2 (moderate) and 3 (strong). The percentage of positive cells was scored as; 1 (0\u0026ndash;25%), 2 (26\u0026ndash;50%), 3 (51\u0026ndash;75%) and 4 (76\u0026ndash;100%). The cut-off value for high versus low expression of the SIX1 protein, defining a final immunostaining score of \u0026gt;\u0026thinsp;6 as high SIX1 protein expression and score\u0026thinsp;\u0026le;\u0026thinsp;6 as low SIX1 expression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted using the IBM SPSS 20.0 statistical package software. Chi-square and Fisher\u0026rsquo;s exact tests were used to compare categorical variables. Comparison between the expression of SIX1 in the primary tumor and their corresponding lymph node metastasis was evaluated using spearman\u0026rsquo;s test. Results were considered statistically significant when P value\u0026thinsp;\u0026le;\u0026thinsp;0.05. Data were analyzed using the Statistical Package for Social Sciences (SPSS) version 17 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Patient and tumor characteristics\u003c/h2\u003e \u003cp\u003eOverall, the mean age\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of the studied patients was 42.02\u0026thinsp;\u0026plusmn;\u0026thinsp;14.6, ranging from 18\u0026ndash;80 years. Patients were classified into \u0026lt;\u0026thinsp;55 years and \u0026ge;\u0026thinsp;55 years according to the median age of patients included in this study. Most patients 84% (105/125) were \u0026lt;\u0026thinsp;55 years of age. Forty-four patients (88%) were females. Bilaterality was detected in 37/125 (29.6%) of tumors and multifocality was seen in 43/125(34.4%). Tumor size was dichotomized into \u0026le;\u0026thinsp;2cm, \u0026gt; 2 and \u0026le;\u0026thinsp;4 cm and \u0026gt;\u0026thinsp;4cm, the mean size\u0026thinsp;\u0026plusmn;\u0026thinsp;SD was 2.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46, ranging from 0.5- 6cm at the maximum dimension, most tumors were \u0026le;\u0026thinsp;2cm. Of patients, 68% were C-PTC and 32% were FV-PTC. Most tumors have low grade features 100/125 (80%). Lympho-vascular invasion (LVI) and perineural invasion (PNI) were detected in 28% and 16% respectively. Positive lymph node metastasis (LNM) was diagnosed in 24% of patients. Twenty-three tumors (18.4%) showed extrathyroidal extension (ETE). Most tumors (42.4%) were stage I. Patients\u0026rsquo; characteristics listed in Table\u0026nbsp;(1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Expression of SIX1 in PTC and adjacent non-cancerous tissue\u003c/h2\u003e \u003cp\u003eSIX1 was expressed in the cytoplasm of cancer cells. SIX1 was expressed diffusely without heterogeneity throughout the tumor. Overall, the positive expression ratio of SIX1 was 36% (45/125), while 80/125 tumors (64%) were negative/low expression. SIX1 positive staining was detected in 6 cases (3.2%) of adjacent non-cancerous thyroid tissue (ANCT) (4/80). On comparing SIX1 expression between non-tumor and PTC; an obvious significant increase in PTC was found (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Association of SIX1 with clinicopathological features in PTC\u003c/h2\u003e \u003cp\u003eThe statistical analyses revealed significant positive associations between high SIX1 expression and increased tumor size, multifocal tumor, LNM, tumor with high grade features, advanced tumor stage, LVI, PNI and ETE,( P\u0026thinsp;=\u0026thinsp;0.04, P\u0026thinsp;=\u0026thinsp;0.003, P\u0026thinsp;=\u0026thinsp;0.002, P\u0026thinsp;=\u0026thinsp;0.01, P\u0026thinsp;=\u0026thinsp;0.002, P\u0026thinsp;=\u0026thinsp;0.002, P\u0026thinsp;=\u0026thinsp;0.01and P\u0026thinsp;=\u0026thinsp;0.001, respectively) (Fig.\u0026nbsp;2). No significant associations were observed between SIX1 expression and patients\u0026rsquo; age, gender, or histological subtype. (Table\u0026nbsp;1)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4. SIX1 expression in classic PTC and follicular variant PTC\u003c/h2\u003e \u003cp\u003eHigh SIX1 expression was observed in 38.8% (33/85) of C-PTC versus 30% (12/40) in FV-PTC tumors. On comparing SIX1 expression in C-PTC and FV-PTC in relation to their different clinicopathological parameters, the statistical analyses revealed significant positive associations between high SIX1 expression and tumor size, multifocality and tumors with high grade features in C-PTC cases (P\u0026thinsp;=\u0026thinsp;0.04, P\u0026thinsp;=\u0026thinsp;0.02 and P\u0026thinsp;=\u0026thinsp;0.03, respectively), and significant positive associations between high SIX1 expression and LNM, LVI, ETE and stage in FV-PTC tumors (P\u0026thinsp;=\u0026thinsp;0.001, P\u0026thinsp;=\u0026thinsp;0.03, P\u0026thinsp;=\u0026thinsp;0.003 and P\u0026thinsp;=\u0026thinsp;0.007, respectively). (Table\u0026nbsp;2)\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAs mentioned previously, mortality due to local recurrence or lymph node metastasis occurs in approximately 10% of papillary thyroid carcinoma (PTC) patients within 10 years [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, there is an urgent need to clarify the pathogenesis of PTC to develop new treatment strategies. The present study found that SIX1 was upregulated in PTC cases overall, with particularly notable upregulation observed in PTC patients exhibiting adverse prognostic features. This highlights the potential significance of SIX1 as a biomarker for identifying high-risk PTC cases and underscores the importance of further research into its role in PTC pathogenesis and potential therapeutic targeting.\u003c/p\u003e \u003cp\u003eSIX1, recognized as a developmental transcription factor crucial in embryonic myogenesis, has been identified to also play a significant role in tumorigenesis. For instance, in cervical intraepithelial or cervical cancer, the expression of SIX1 was markedly higher compared to that in normal cervical tissues [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the current study, SIX1 expression was obviously elevated in PTC compared to ANCT, where almost non-cancerous tissues showed no expression of SIX1. These findings align with previous research indicating the upregulation of SIX1 in PTC while being absent in non-cancerous tissue [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, our findings are consistent with analyses of The Cancer Genome Atlas (TCGA) dataset, which demonstrated higher Six1 mRNA levels in thyroid cancers compared to non-cancerous thyroid tissues [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this regard, functional assays conducted on two thyroid cancer cell lines to examine cell viability and colony formation revealed that SIX1 enhanced PTC proliferation and provided significant protection against apoptosis, whereas knockdown of SIX1 using siRNA inhibited growth rate and colony formation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These results confirm the role of SIX1 in promoting growth in thyroid cancer cells, suggesting its potential as an oncogene in PTC tumorigenesis.\u003c/p\u003e \u003cp\u003eThe precise mechanism by which SIX1 mediates tumorigenesis remains unclear [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Earlier studies have shown that overexpression of SIX1 facilitates tumorigenesis and proliferation of breast cancer cells by directly activating cyclin A1 transcription [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In colorectal and cervical cancers, SIX1 has been implicated in EMT and is described as a mediator of ZEB1 transcription and regulation of TGF-β signaling [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. More recently, the proliferation of PTC has been linked to the activation of classical STAT3 signaling and the TGF-β/Smad2/3 signaling pathways [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, SIX1 was primarily found to be localized in the cytoplasm, a result consistent with the findings of \u003cb\u003eMin\u003c/b\u003e and \u003cb\u003eWei\u003c/b\u003e as well as \u003cb\u003eKong et al.\u003c/b\u003e whereas \u003cb\u003eYang et al.\u003c/b\u003e observed nuclear localization of SIX1 expression in their series [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. High SIX1 expression was detected in 36% of tumors, whereas previous studies have reported a higher positivity ranging from 52\u0026ndash;63% in PTC tumors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This variance could be attributed to differences in scoring systems, variations in the clone used, or differences in the methodology employed for the study, such as the use of tissue microarrays instead of larger surgical specimens.\u003c/p\u003e \u003cp\u003ePrevious literature has consistently associated the upregulation of SIX1 with aggressive tumor characteristics and unfavorable patient prognosis across various cancers [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. A recent study linked SIX1's role in tumor invasiveness to its promotion of glucose metabolism and invasion through the regulation of GLUT3, MMP2, and snail in thyroid cancer [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In our study, we found a significant association between high SIX1 expression and aggressive features in PTC, including larger tumor size, multifocality, LNM, tumors with high-grade features, advanced tumor stage, LVI, PNI, and ETE. These results are consistent with other studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] that have reported a positive relation between invasiveness and high SIX1 expression. Additionally, our findings align with previous studies utilizing Matrigel invasion assays, which demonstrated that SIX1 upregulation increased invasion in thyroid cancer, while knockdown of SIX1 using siRNA reduced invasive ability in both cell lines. Furthermore, our results are supported by an analysis of TCGA data, which showed positive relations between SIX1 mRNA expression and nodal metastasis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Collectively, these findings suggest that upregulation of SIX1 expression could serve as a biomarker for predicting PTC behavior and clinical outcomes.\u003c/p\u003e \u003cp\u003eHistorically, C-PTC and FV-PTC were often regarded as having comparable prognosis and treatment approaches. However, recent research by the TCGA group has revealed distinct differences in clinical outcomes and genetic profiles between these variants. Studies by \u003cb\u003eHenke et al.\u003c/b\u003e and \u003cb\u003eShi et al.\u003c/b\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] examined large series of C-PTC and FV-PTC and found that C-PTC exhibits a higher prevalence of ETE, LNM, disease recurrence, and mortality compared to FV-PTC. They concluded that C-PTC and FV-PTC represent biologically distinct disease entities with different oncogenic drivers and tumor behaviors. In the current study, the clinicopathological parameters appeared comparable between C-PTC and FV-PTC cases. However, this may be attributed to the small sample size of our study.\u003c/p\u003e \u003cp\u003eLittle is currently understood about SIX1 expression across various histologic subtypes of PTC. To our knowledge, our study represents the first investigation into the association between immunohistochemical expression of SIX1 and clinicopathological features among both C-PTC and FV-PTC patients. We observed no statistically significant difference in high SIX1 expression between C-PTC and FV-PTC, although there was a slight increase in C-PTC (38.2% versus 30%). This finding contrasts with a recent study that reported a significant difference [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The lack of significance in our study may be attributed to the disproportionate number of each histological subtype and differences in the scoring system used.\u003c/p\u003e \u003cp\u003eIn the present study, we observed a significant upregulation of SIX1 in C-PTC tumors with larger tumor size, multifocality and high-grade features. Conversely, SIX1 was significantly upregulated in FV-PTC tumors with LVI, LNM, ETE, and advanced tumor stage. Particularly noteworthy in the FV-PTC group, unlike in C-PTC, was the clear association between high SIX1 expression and LNM and ETE, with all LNM patients and nearly all ETE tumors showing high SIX1 expression. It has been suggested that patients with LNM have a higher mortality rate, with incomplete surgical excision being a significant contributing factor to increased mortality in stage I PTC patients [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our findings indicate that SIX1 expression may serve as a useful marker for predicting which patients with clinically node-negative (cN0) FV-PTC may benefit from prophylactic central lymph node dissection.\u003c/p\u003e \u003cp\u003ePrevious studies have indicated that locoregional extension to perithyroidal soft tissues and lymph nodes is less common in FV-PTC [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In this context, our study may aid in selecting FV-PTC patients who require aggressive management while avoiding overtreatment of the predominantly non-aggressive FV-PTC tumors.\u003c/p\u003e \u003cp\u003eIn C-PTC group, the notable association of high SIX1 expression with multifocality holds significant clinical value. This finding suggests that total thyroidectomy, rather than lobectomy or hemithyroidectomy, might be more appropriate for C-PTC patients. This approach could be justified by the potential presence of small foci of disease in the contralateral thyroid lobe, thereby reducing the likelihood of locoregional recurrence.\u003c/p\u003e \u003cp\u003eMoreover, the relation between high SIX1 with tumor size, a key factor in the tumor (T) classification of the AJCC staging system, implies that SIX1 could serve as a predictor of post-treatment recurrence. This suggests that patients with high SIX1 expression may require closer monitoring and more aggressive management strategies to mitigate the risk of recurrence following initial treatment.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study indicates that SIX1 could serve as a hallmark of malignancy in PTC. High SIX1 expression was closely associated with adverse prognostic factors in PTC. Furthermore, SIX1 expression may predict distinct prognostic factors in C-PTC and FV-PTC. This suggests that SIX1 could be utilized not only as a prognostic biomarker but also in developing subtype-specific therapeutic strategies for PTC patients. However, these findings require validation in independent large cohorts, and the potential implications for management and staging warrant further investigation. Further research is needed to fully understand the clinical significance of SIX1 expression in PTC and its implications for patient outcomes and treatment strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethyroid cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePTC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epapillary thyroid carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDTC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edifferentiated thyroid carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eC-PTC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eclassic PTC\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFV-PTC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efollicular variant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eETE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextrathyroidal extension\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLNM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elymph node metastasis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePNI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eperineural infiltration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLVI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elymphovascular infiltration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eStudy design, data acquisition, analysis and drafting of manuscript: Khalil EI, Issa AS and Kamal RM. Statistical analysis: Khalil EI.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShank JB, Are C, Wenos CD. \u0026quot;Thyroid Cancer: Global Burden and Trends.\u0026quot; Indian Journal of Surgical Oncology 2022; 13, no. 1 40-45. \u003c/li\u003e\n\u003cli\u003eSiegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2021. Ca Cancer J Clin 2021; 71(1), 7-33.\u003cspan dir=\"RTL\"\u003e\u0026rlm;\u003c/span\u003e \u003c/li\u003e\n\u003cli\u003eKitahara CM, Schneider AB. Cancer Epidemiol Biomarkers Prev. 2022 Jul 1; 31(7):1284-1297. \u003c/li\u003e\n\u003cli\u003eGohar M, Mohamed A, Al-Azzouny H. Risk of hypocalcemia after total thyroidectomy and bilateral central neck dissection in patients with well differentiated thyroid carcinoma. Al-Azhar International Medical Journal, 2020; 1(11): 139-143. doi: 10.21608/aimj.2021.44026.1327\u003c/li\u003e\n\u003cli\u003eMalaguarnera R, Ledda C, Filippello A, Frasca F, Francavilla VC, Ramaci T, Piro S. Thyroid cancer and circadian clock disruption. Cancers (Basel). 2020 Nov; 12(11): 3109. \u003c/li\u003e\n\u003cli\u003eBJ Duffy Jr, Fitzgerald PJ. Thyroid cancer in childhood and adolescence. A report on 28 cases. Cancer. 1950 Nov; 3(6):1018-32. \u003c/li\u003e\n\u003cli\u003eShah JP. Thyroid carcinoma: epidemiology, histology, and diagnosis. Clin Adv Hematol Oncol. 2015 Apr; 13(4 Suppl 4): 3\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eLauren E Henke, John D Pfeifer, Thomas J Baranski, Todd DeWees, Perry W Grigsby; Long-term outcomes of follicular variant vs classic papillary thyroid carcinoma. Endocr Connect. 2018 Dec; 7(12): 1226\u0026ndash;1235. \u003c/li\u003e\n\u003cli\u003eShi X, Liu R, Basolo F, Giannini R, Shen X, Teng D et al. Differential Clinicopathological Risk and Prognosis of Major Papillary Thyroid Cancer Variants. J Clin Endocrinol Metab. 2016 Jan; 101(1):264-74. \u003c/li\u003e\n\u003cli\u003eAgrawal N, Akbani R, Aksoy BA, Ally A, Arachchi H, Asa SL, Protopopov A. Integrated genomic characterization of papillary thyroid carcinoma. Cell. 2014 Oct 23; 159 (3):676-90. \u003c/li\u003e\n\u003cli\u003eHaugen BR, AlexanderErik K, BibleKeith C, DohertyGerard M, MandelSusan J, NikiforovYuri E, Michael T. 2015 American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: the American Thyroid Association guidelines task force on thyroid nodules and differentiated thyroid cancer. \u003cbr\u003e Thyroid. 2016 Jan; 26(1):1-133.\u003c/li\u003e\n\u003cli\u003eValerio L, Pieruzzi L, Giani C, Agate L, Bottici V, Lorusso L, Elisei R. Targeted therapy in thyroid cancer: state of the art. Clinical oncology. 2017 May; 29(5):316-324.\u003c/li\u003e\n\u003cli\u003eCeddia G, Martino LN, Parodi A, Secchi P, Campaner S, Masseroli M. Association rule mining to identify transcription factor interactions in genomic regions. \u003cem\u003eBioinformatics\u003c/em\u003e, Volume 36, Issue 4, February 2020, Pages 1007\u0026ndash;1013. \u003c/li\u003e\n\u003cli\u003eBushweller JH. Targeting transcription factors in cancer\u0026mdash;from undruggable to reality. \u003cbr\u003e Nat Rev Cancer. 2019 Nov;19 (11):611-624. \u003c/li\u003e\n\u003cli\u003eRafiq A, Aashaq S, Jan I, Beigh MA. SIX1 transcription factor: A review of cellular functions and regulatory dynamics. Int J Biol Macromol. 2021 Dec 15;193(Pt B):1151-1164. doi: 10.1016/j.ijbiomac.2021.10.133. \u003c/li\u003e\n\u003cli\u003eMin WP, Wei XF. Silencing SIX1 inhibits epithelial mesenchymal transition through regulating TGF-\u0026beta;/Smad2/3 signaling pathway in papillary thyroid carcinoma. Auris Nasus Larynx. 2021 Jun; 48(3):487-495. \u003c/li\u003e\n\u003cli\u003eKong D, Li A, Liu Y, Cui Q, Wang K, Zhang D, Wu K. SIX1 activates STAT3 signaling to promote the proliferation of thyroid carcinoma via EYA1. Front Oncol. 2019 Dec 20:9:1450.\u003c/li\u003e\n\u003cli\u003eXu HX, Wu K J, Tian YJ, Liu Q, Han N, He X L, Wu K M. Expression profile of SIX family members correlates with clinic-pathological features and prognosis of breast cancer: A systematic review and meta-analysis. Medicine. 2016 Jul;95(27):e4085. \u003c/li\u003e\n\u003cli\u003eSong W, Ma J, Lei B, Yuan X, Cheng B, Yang H, Wang, L. Sine oculis homeobox 1 promotes proliferation and migration of human colorectal cancer cells through activation of Wnt/\u0026beta;‐catenin signaling. Cancer Sci. 2019 Feb;110(2):608-616.\u003cspan dir=\"RTL\"\u003e\u0026rlm;\u003c/span\u003e \u003c/li\u003e\n\u003cli\u003eYang Z, Feng Z, Gu J, Li X, Dong Q, Liu K, OuYang, L. microRNA-488 inhibits chemoresistance of ovarian cancer cells by targeting Six1 and mitochondrial function. Oncotarget. 2017 Oct 6; 8(46): 80981\u0026ndash;80993. \u003c/li\u003e\n\u003cli\u003eBaloch ZW, Asa SL, Barletta JA, Ghossein RA, Juhlin CC, Jung, CK, Mete O. Overview of the 2022 who classification of thyroid neoplasms. Endocr Pathol. 2022 Mar; 33(1):27-63.\u003c/li\u003e\n\u003cli\u003ePerrier, N. D., Brierley, J. D., \u0026amp; Tuttle, R. M. Differentiated and anaplastic thyroid carcinoma: major changes in the American Joint Committee on Cancer eighth edition cancer staging manual. CA Cancer J Clin. 2018 Jan;68(1):55-63. doi: 10.3322/caac.21439. \u003c/li\u003e\n\u003cli\u003eTan J, Zhang C, Qian J. Expression and significance of Six1 and Ezrin in cervical cancer tissue. Tumour Biol. 2011 Dec; 32(6):1241-7.\u003cspan dir=\"RTL\"\u003e\u0026rlm;\u003c/span\u003e \u003c/li\u003e\n\u003cli\u003eYang C, Xu W, Gong J, Chai F, Cui D, Liu Z. Six1 overexpression promotes glucose metabolism and invasion through regulation of GLUT3, MMP2 and snail in thyroid cancer cells. Onco Targets Ther. 2020; 13: 4855\u0026ndash;4863. \u003c/li\u003e\n\u003cli\u003eWu W, Ren Z, Li P, Yu D, Chen J, Huang R, Liu H. Six1: a critical transcription factor in tumorigenesis. Int J Cancer. 2015 Mar 15;136(6):1245-53. \u003c/li\u003e\n\u003cli\u003eTerunuma A, Putluri N, Mishra P, Math\u0026eacute; EA, Dorsey TH, Yi M, Ambs S. MYC-driven accumulation of 2-hydroxyglutarate is associated with breast cancer prognosis. J Clin Invest. 2014 Jan; 124(1):398-412. \u003c/li\u003e\n\u003cli\u003eChristensen KL, Brennan JDG, Aldridge CS, Ford HL. Cell cycle regulation of the human Six1 homeoprotein is mediated by APCCdh1. Oncogene. 2007 May 17; 26(23):3406-14. \u003c/li\u003e\n\u003cli\u003eJiang LH, Chen C, Tan Z, Lu XX, Hu SS, Wang QL, Ge MH. Clinical characteristics related to central lymph node metastasis in cN0 papillary thyroid carcinoma: a retrospective study of 916 patients. Int J Endocrinol. 2014; 2014: 385787.\u003cspan dir=\"RTL\"\u003e \u0026rlm;\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable (1):\u003cem\u003e\u0026nbsp;Association between SIX1 expression and clinicopathological features (n=125):\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"606\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinicopathological features\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.871287128712872%\" rowspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of cases\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.51485148514851%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eSIX1 Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.910891089108912%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"51.02040816326531%\" style=\"width: 29.5122%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow Expression (N=80)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.97959183673469%\" style=\"width: 24.2832%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;High expression (N=45)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026lt;55 y\u003c/p\u003e\n \u003cp\u003e\u0026ge; 55 y\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.871287128712872%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e105\u003c/p\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.752475247524753%\" style=\"width: 29.5122%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e70 (66.7%)\u003c/p\u003e\n \u003cp\u003e10 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.762376237623762%\" style=\"width: 24.2832%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e35 (33.3%)\u003c/p\u003e\n \u003cp\u003e10 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.910891089108912%\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eGender\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.871287128712872%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003cp\u003e110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.752475247524753%\" style=\"width: 29.5122%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5 (33.3%)\u003c/p\u003e\n \u003cp\u003e75 (68.2 %)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.762376237623762%\" style=\"width: 24.2832%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10 (66.7%)\u003c/p\u003e\n \u003cp\u003e35 (31.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.910891089108912%\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eHistological subtypes\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eC-PTC\u003c/p\u003e\n \u003cp\u003eFV- PTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.871287128712872%\" valign=\"bottom\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.752475247524753%\" valign=\"bottom\" style=\"width: 29.5122%;\"\u003e\n \u003cp\u003e21 (61.8%)\u003c/p\u003e\n \u003cp\u003e11 (68.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.762376237623762%\" valign=\"bottom\" style=\"width: 24.2832%;\"\u003e\n \u003cp\u003e13 (38.2%)\u003c/p\u003e\n \u003cp\u003e5 (31.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.910891089108912%\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor size\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026le; 2\u003c/p\u003e\n \u003cp\u003e\u0026gt;2 - \u0026le; 4\u003c/p\u003e\n \u003cp\u003e\u0026gt; 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.871287128712872%\" valign=\"bottom\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.752475247524753%\" valign=\"bottom\" style=\"width: 29.5122%;\"\u003e\n \u003cp\u003e48 (68.5%)\u003c/p\u003e\n \u003cp\u003e30 (71.4%)\u003c/p\u003e\n \u003cp\u003e2 (15.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.762376237623762%\" valign=\"bottom\" style=\"width: 24.2832%;\"\u003e\n \u003cp\u003e22 (31.4%)\u003c/p\u003e\n \u003cp\u003e12 (29.6 %)\u003c/p\u003e\n \u003cp\u003e11 (84.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.910891089108912%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor Focality\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eUnifocal\u003c/p\u003e\n \u003cp\u003eMultifocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.871287128712872%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.752475247524753%\" style=\"width: 29.5122%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e64 (78.1%)\u003c/p\u003e\n \u003cp\u003e15 (34.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.762376237623762%\" style=\"width: 24.2832%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e18 (21.9%)\u003c/p\u003e\n \u003cp\u003e28 (65.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.910891089108912%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.003\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor grade\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eLow grade features\u003c/p\u003e\n \u003cp\u003eHigh grade features\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.871287128712872%\" valign=\"bottom\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.752475247524753%\" valign=\"bottom\" style=\"width: 29.5122%;\"\u003e\n \u003cp\u003e75 (75%)\u003c/p\u003e\n \u003cp\u003e5 (20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.762376237623762%\" valign=\"bottom\" style=\"width: 24.2832%;\"\u003e\n \u003cp\u003e25 (25%)\u003c/p\u003e\n \u003cp\u003e20 (80%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.910891089108912%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLVI\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.871287128712872%\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.752475247524753%\" style=\"width: 29.5122%;\"\u003e\n \u003cp\u003e74 (82.2%)\u003c/p\u003e\n \u003cp\u003e6 (17.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.762376237623762%\" style=\"width: 24.2832%;\"\u003e\n \u003cp\u003e16 (27.8%)\u003c/p\u003e\n \u003cp\u003e29 (82.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.910891089108912%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003ePNI\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.871287128712872%\" valign=\"bottom\"\u003e\n \u003cp\u003e105\u003c/p\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.752475247524753%\" valign=\"bottom\" style=\"width: 29.5122%;\"\u003e\n \u003cp\u003e76 (72.3%)\u003c/p\u003e\n \u003cp\u003e4 (20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.762376237623762%\" valign=\"bottom\" style=\"width: 24.2832%;\"\u003e\n \u003cp\u003e29 (27.7%)\u003c/p\u003e\n \u003cp\u003e16 (80%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.910891089108912%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eETE\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.871287128712872%\" valign=\"bottom\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.752475247524753%\" valign=\"bottom\" style=\"width: 29.5122%;\"\u003e\n \u003cp\u003e77 (75.4%)\u003c/p\u003e\n \u003cp\u003e3 (13.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.762376237623762%\" valign=\"bottom\" style=\"width: 24.2832%;\"\u003e\n \u003cp\u003e25 (24.5%)\u003c/p\u003e\n \u003cp\u003e20 (86.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.910891089108912%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eLNM\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.871287128712872%\" valign=\"bottom\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.752475247524753%\" valign=\"bottom\" style=\"width: 29.5122%;\"\u003e\n \u003cp\u003e73 (76.8%)\u003c/p\u003e\n \u003cp\u003e7 (23.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.762376237623762%\" valign=\"bottom\" style=\"width: 24.2832%;\"\u003e\n \u003cp\u003e22 (23.2%)\u003c/p\u003e\n \u003cp\u003e23 (76.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.910891089108912%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.702970297029704%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor Stage\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003epT1\u003c/p\u003e\n \u003cp\u003epT2\u003c/p\u003e\n \u003cp\u003epT3\u003c/p\u003e\n \u003cp\u003epT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.871287128712872%\" valign=\"bottom\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.752475247524753%\" valign=\"bottom\" style=\"width: 29.5122%;\"\u003e\n \u003cp\u003e43(82.7%)\u003c/p\u003e\n \u003cp\u003e29(72.5%)\u003c/p\u003e\n \u003cp\u003e8(32%)\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.762376237623762%\" valign=\"bottom\" style=\"width: 24.2832%;\"\u003e\n \u003cp\u003e9(17.3%)\u003c/p\u003e\n \u003cp\u003e11(22.5%)\u003c/p\u003e\n \u003cp\u003e17(68%)\u003c/p\u003e\n \u003cp\u003e8(100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.910891089108912%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.01\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e* P - value \u003cspan dir=\"RTL\"\u003e\u0026gt;\u0026nbsp;\u003c/span\u003e0.05 are considered statistically significant. \u0026nbsp;Chi-Square test.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable (2): \u003cem\u003eComparison between the expression of SIX1 in classic and follicular variant PTC with clinicopathological data:\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"684\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.42105263157895%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eClinico-pathological\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eVariables\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.2631578947368425%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eN.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e85\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eC-PTC\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.140350877192983%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eN.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e40\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.8421052631579%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFV-PTC\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSIX1 Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSIX1 Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.666666666666668%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow (N=52)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh (N=33)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow (N=28)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.333333333333332%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh (N=12)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.42105263157895%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026lt;55\u003c/p\u003e\n \u003cp\u003e\u0026ge;55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.2631578947368425%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.403508771929825%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e47(65.2%)\u003c/p\u003e\n \u003cp\u003e5(38.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e25(34.8%)\u003c/p\u003e\n \u003cp\u003e8(61.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.140350877192983%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e23(69.6%)\u003c/p\u003e\n \u003cp\u003e5(71.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.912280701754385%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10(30.4%)\u003c/p\u003e\n \u003cp\u003e2(28.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.42105263157895%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGender\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.2631578947368425%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.403508771929825%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2(18.1%)\u003c/p\u003e\n \u003cp\u003e50(67.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9(81.9%) \u0026nbsp;24(32.4%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.140350877192983%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4(100%)\u003c/p\u003e\n \u003cp\u003e24(66.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.912280701754385%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003cp\u003e12(100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.42105263157895%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor Size\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026le; 2\u003c/p\u003e\n \u003cp\u003e\u0026gt;2 - \u0026le; 4\u003c/p\u003e\n \u003cp\u003e\u0026gt;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.2631578947368425%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.403508771929825%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e35(70%) 15(65.2%) \u0026nbsp; \u0026nbsp; \u0026nbsp;2(1.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e15(30%) \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8(34.8%)\u003c/p\u003e\n \u003cp\u003e10(83.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e0.04*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.140350877192983%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e15(75%)\u003c/p\u003e\n \u003cp\u003e12(63.2%)\u003c/p\u003e\n \u003cp\u003e1(100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.912280701754385%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5(25%)\u003c/p\u003e\n \u003cp\u003e7(36.8%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;0 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.42105263157895%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor Focality\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eUnifocal\u003c/p\u003e\n \u003cp\u003eMultifocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.2631578947368425%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.403508771929825%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e43(78.1%)\u003c/p\u003e\n \u003cp\u003e9(29.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11(21.9%)\u003c/p\u003e\n \u003cp\u003e22(71.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e0.02*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.140350877192983%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e23(82.1%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;5(41.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.912280701754385%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5(17.9%)\u003c/p\u003e\n \u003cp\u003e7(59.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.42105263157895%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor Grade\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eLow grade features\u003c/p\u003e\n \u003cp\u003eHigh grade features\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.2631578947368425%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.403508771929825%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e48(73.8%)\u003c/p\u003e\n \u003cp\u003e4(19.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e16(26.2%)\u003c/p\u003e\n \u003cp\u003e17(80.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e0.03*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.140350877192983%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e26(72.3%)\u003c/p\u003e\n \u003cp\u003e2(50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.912280701754385%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10(27.7%)\u003c/p\u003e\n \u003cp\u003e2(50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.42105263157895%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLVI\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAbsent\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.2631578947368425%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.403508771929825%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e44(70.9%)\u003c/p\u003e\n \u003cp\u003e8(34,7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e18(2914%)\u003c/p\u003e\n \u003cp\u003e15(65.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.140350877192983%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e26(86.6%)\u003c/p\u003e\n \u003cp\u003e2(20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.912280701754385%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4(13.4%)\u003c/p\u003e\n \u003cp\u003e8(80%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e0.03*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.42105263157895%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePNI\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.2631578947368425%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.403508771929825%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e44(66.6%)\u003c/p\u003e\n \u003cp\u003e8(42.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e22(33.4%)\u003c/p\u003e\n \u003cp\u003e11(57.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.140350877192983%\" 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width=\"13.157894736842104%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e12(100%)\u003c/p\u003e\n \u003cp\u003e16(80)\u003c/p\u003e\n \u003cp\u003e0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.912280701754385%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;0\u003c/p\u003e\n \u003cp\u003e4(20%)\u003c/p\u003e\n \u003cp\u003e6(100%)\u003c/p\u003e\n \u003cp\u003e2(100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.771929824561404%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e0.007*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e* P - value \u003cspan dir=\"RTL\"\u003e\u0026gt;\u0026nbsp;\u003c/span\u003e0.05 are considered statistically significant. Chi-Square test.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"thyroid-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"thyr","sideBox":"Learn more about [Thyroid Research](http://thyroidresearchjournal.biomedcentral.com/)","snPcode":"13044","submissionUrl":"https://submission.nature.com/new-submission/13044/3","title":"Thyroid Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"SIX1, Papillary thyroid carcinoma, classic PTC, follicular variant PTC, Immunohistochemistry","lastPublishedDoi":"10.21203/rs.3.rs-4664320/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4664320/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePapillary thyroid carcinoma (PTC) is the most common type of thyroid carcinoma, representing the majority of thyroid cancer cases. Most patients with PTC have an excellent prognosis following treatment, yet approximately 10% face mortality within ten years, primarily due to lymph node metastasis (LNM) or local recurrence. The SIX1 gene, a member of the SIX gene superfamily, encodes a transcription factor integral to the development of certain tissues during embryogenesis. The impact of SIX1 in different subtypes of PTC ha\u003cem\u003es\u003c/em\u003e not been studied previously.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThe purpose of this study was to investigate the expression of SIX1 protein in PTC and to explore its relationship with clinical behavior in two subtypes of PTC: classic PTC (C-PTC) and follicular variant PTC (FV-PTC).\u003c/p\u003e\u003ch2\u003eMaterial and Methods\u003c/h2\u003e \u003cp\u003eUsing immunohistochemistry, the study analyzed 125 primary PTC cases, including 85 cases of C-PTC and 40 cases of FV-PTC.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe study found significant positive associations between high SIX1 expression and several adverse clinical features across the PTC samples. High SIX1 expression was linked with increased tumor size, multifocal tumors, LNM, high-grade tumor features, advanced tumor stage, lymphovascular invasion, perineural invasion, and extrathyroidal extension (ETE). Within the classic PTC subgroup, high SIX1 expression showed significant positive correlations with Tumor size (P\u0026thinsp;=\u0026thinsp;0.04), Multifocality (P\u0026thinsp;=\u0026thinsp;0.02) and High-grade features (P\u0026thinsp;=\u0026thinsp;0.03). In the follicular variant subgroup, high SIX1 expression was significantly associated with Lymph node metastasis (LNM) (P\u0026thinsp;=\u0026thinsp;0.001), Lymphovascular invasion (P\u0026thinsp;=\u0026thinsp;0.03), ETE (P\u0026thinsp;=\u0026thinsp;0.003) and Tumor stage (P\u0026thinsp;=\u0026thinsp;0.007).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe findings of this study indicate that SIX1 expression is a marker of poor prognosis in PTC, suggesting that its high expression is linked with more aggressive tumor characteristics and advanced disease stages. Importantly, the impact of SIX1 expression varies between C-PTC and FV-PTC, predicting distinct prognostic factors in each subtype. This suggests that SIX1 could be utilized not only as a prognostic biomarker but also in developing subtype-specific therapeutic strategies for PTC patients.\u003c/p\u003e","manuscriptTitle":"SIX1 Expression and its Clinicopathological Significance: Difference between Classic and Follicular Variant Papillary Thyroid Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-26 15:47:04","doi":"10.21203/rs.3.rs-4664320/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-24T13:34:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-24T12:19:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-23T13:09:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-12T07:55:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232012458919458665215408110819360525148","date":"2024-07-08T17:12:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"140826826031424828771362738005304325249","date":"2024-07-07T23:40:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39908897927500194749580401485820471663","date":"2024-07-02T09:47:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-02T08:40:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-02T05:34:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-02T05:34:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Thyroid Research","date":"2024-06-30T21:31:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"thyroid-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"thyr","sideBox":"Learn more about [Thyroid Research](http://thyroidresearchjournal.biomedcentral.com/)","snPcode":"13044","submissionUrl":"https://submission.nature.com/new-submission/13044/3","title":"Thyroid Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"761074f3-6e6a-4ea3-94bd-6a52bfdd8b98","owner":[],"postedDate":"July 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-16T16:06:58+00:00","versionOfRecord":{"articleIdentity":"rs-4664320","link":"https://doi.org/10.1186/s13044-024-00212-9","journal":{"identity":"thyroid-research","isVorOnly":false,"title":"Thyroid Research"},"publishedOn":"2024-12-09 15:57:04","publishedOnDateReadable":"December 9th, 2024"},"versionCreatedAt":"2024-07-26 15:47:04","video":"","vorDoi":"10.1186/s13044-024-00212-9","vorDoiUrl":"https://doi.org/10.1186/s13044-024-00212-9","workflowStages":[]},"version":"v1","identity":"rs-4664320","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4664320","identity":"rs-4664320","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}