5hmC immunohistochemistry: a predictor of TERT promoter mutational status in follicular thyroid carcinoma?

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Martin Hysek, Samuel Hellgren, Vincenzo Condello, Catharina Larsson, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2642987/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Purpose TERT promoter mutations and TERT gene expression correlate to adverse prognosis in follicular thyroid carcinoma (FTC), identifying cases at risk of poor outcome. As loss of 5-hydroxymethylcytosine (5hmC) immunoreactivity has been associated with TERT promoter mutations in papillary thyroid carcinoma, this study sought to analyze the levels of 5hmC in a well-characterized cohort of follicular thyroid tumors with available TERT data. Methods 29 tumors (26 FTCs, 2 follicular thyroid tumors of uncertain malignant potential (FT-UMPs), and one oncocytic thyroid carcinoma) with known TERT promoter mutational status and TERT gene expression levels were assessed for 5hmC immunoreactivity using two monoclonal antibodies (clones RM236 and 4D9.) Slides were analyzed using a semiquantitative scoring system. Results Of the 10 tumor cases with a TERT promoter mutation and TERT expression, only one was scored as negative with both antibodies (1/10; 10%), while the remaining 9 cases (9/10; 90%) exhibited various degrees of positivity for at least one antibody. Of the 19 TERT wild-type tumors, no case (0/19; 0%) was scored as negative using the RM236 clone, and two cases (2/19; 11%) using the 4D9 clone. The differences between TERT promoter mutated and wildtype groups were non-significant (Fisher’s Exact test P = 0.35 and 0.59 respectively). The sensitivity and specificity for 5hmC IHC to detect mutated cases were 10% and 100% for RM236 and 20% and 89% for 4D9 respectively. Conclusion 5hmC IHC is not a highly sensitive marker for the detection of TERT promoter mutations in follicular thyroid tumors. Further analyses in larger cohorts are warranted. TERT promoter mutation 5hmC immunohistochemistry thyroid Figures Figure 1 Introduction Follicular thyroid tumors pose a diagnostic difficulty as the differentiation between follicular thyroid adenoma (FTA) and follicular thyroid carcinoma (FTC) is based on the identification of invasive properties using routine histology. To establish a correct diagnosis, the material often needs to be scrutinized thoroughly to identify areas of invasion. In the past years, activating hotspot mutations in the Telomerase reverse transcriptase ( TERT ) gene promoter (denoted C228T and C250T) have been intimately coupled to FTC [ 1 ] and subsets of follicular thyroid tumors of uncertain malignant potential (FT-UMPs), but are rarely observed in FTA. This genotype-phenotype correlation could therefore help in evaluating the true malignant potential of follicular thyroid tumors [ 2 ]. Moreover, TERT promoter mutations and TERT gene expression are prognostic factors in thyroid cancer, as these events are closely associated with adverse histopathological factors and poor patient outcomes [ 3 – 7 ]. As of this, the 2022 WHO classification of endocrine and neuroendocrine tumors acknowledges the role of TERT promoter mutations in highlighting cases at risk of disease dissemination [ 8 ], and more focus is currently diverted to developing robust screening tools to detect TERT -expressing tumors in clinical routine. Specifically, while TERT immunohistochemistry (IHC) is not yet considered a reliable method, studies using TERT mRNA in situ hybridization (ISH) have shown promising results [ 9 , 10 ]. 5-hydroxymethylcytosine (5hmC) immunoreactivity is decreased in various malignant tumors, such as melanoma [ 11 ] and glioma [ 12 ] as well as in papillary thyroid carcinoma (PTC) [ 13 , 14 ]. 5hmC is produced when 5-methylcytosine (5mc) is demethylated via ten-eleven translocation (TET) enzymes, and 5hmC may thus serve as an epigenetic marker of global de-methylation events across the genome. Oishi et al. [ 14 ] recently showed an association between the loss of 5hmC and TERT promoter mutations in PTC, but to our knowledge, no efforts to predict TERT promoter mutational status by 5hmC IHC in FTCs have been published. The aim of this study was therefore to evaluate whether loss of immunohistochemical 5hmC expression can predict TERT promotor mutations in a cohort of FTCs with established TERT promoter genotypes and TERT mRNA expression by quantitative real-time PCR (qRT-PCR) and ISH. Material And Methods Cohort A cohort of differentiated follicular cell-derived and oncocytic thyroid tumors which has previously been evaluated for TERT promoter mutations and TERT expression using qRT-PCR and ISH [ 10 ] was chosen for 5hmC IHC analyses. The cohort consisted of 26 FTCs, 2 FT-UMPs, and one oncocytic thyroid carcinoma (OTC), of which 10 (7 FTCs, 2 FT-UMPs, and the single OTC) showed TERT promoter mutations and TERT gene expression, while the remaining cases were TERT promoter wildtype and displayed absent TERT mRNA levels. A de-identified case with thyroid follicular nodular disease (TFND) was included as a non-tumorous control for both antibodies, and a de-identified sample of normal testis tissue as a positive control of 5hmC immunoreactivity, which was used for identifying the best dilution of the 4D9 antibody. To test the validity of the antibody used in clinical routine, three de-identified malignant thyroid tumors with concurrent BRAF and TERT promoter mutations were included as well. 5mhC immunohistochemistry (clone RM236) Formalin-fixed paraffin-embedded (FFPE) tissues from all cases were sectioned in 4-micrometer thick sections. The chosen sections all contained tumor tissue as well as adjacent normal thyroid tissue. After de-paraffinization in xylene and rehydration in alcohol, sections were subjected to heat-induced antigen retrieval using the Ventana Ultra CC1, pH 8.5 for 32 min. The staining was automated using the Ventana Ultra Benchmark methodology and a primary antibody (5hmC clone RM236, Abcam, Cambridge, UK) was applied at a dilution of 1:1,000 for 32 min. Visualization was performed using OptiView and slides were then counterstained using Mayer´s hematoxylin for 3 min. 5hmC immunohistochemistry (clone 4D9) Following de-paraffinization and rehydration of FFPE sections, heat-induced antigen retrieval was performed using a decloaking chamber (Biocare Medical, CA) set for 5 min at 110°C in citrate buffer pH 6 (article C-9999, Sigma-Aldrich, MA). For quenching of endogenous peroxidase, a 30 min incubation in 0.15% hydrogen peroxidase was performed at room temperature, followed by a 30 min blocking step using 1% bovine serum albumin (article A-4503, Sigma-Aldrich, MA). The primary 5hmC monoclonal antibody (HMC/4D9, Epigentek, NY) was diluted 1:2,000 in Renior Red diluent (article #PD9004M, Biocare Medical, CA), before incubation at 4°C overnight in a humidified chamber. For detection, the Mach-1 Universal HRP-Polymer Kit was used (article MIU539L10, Biocare Medical, CA) according to the protocol provided by the manufacturer. The sections were counterstained in Mayer´s hematoxylin for 1 min, followed by dehydration with graded alcohols, xylene treatment, and coverslipped with Pertex (Histolab, Askim, Sweden). Scoring methodology The stained slides were evaluated by two pathologists independently following the same semi-quantitative scoring system as Oishi et al. for their study on PTC [ 14 ]: 0 = complete lack of nuclear staining in tumor cells i.e. negative; 1 = positive staining only in scarce tumor cells (1–9%); 2 = positive staining in 10–24% of tumor cells; 3 = positive staining in 25–74% of tumor cells; and 4 = diffuse and strong nuclear staining in > = 75% of tumor cells. Examples of different staining patterns are shown in Fig. 1 Statistical analyses Fisher’s exact tests were used to compare 5hmC expression and TERT promoter mutational status. Chi-square and Mann-Whitney-U tests were used to compare clinicopathological features and TERT promoter mutational status. P values < 0.05 were considered significant. Statistical computations were performed using RStudio v12.0 (Posit Software PBC, MA). Results Clinicopathological features of the study cohort A total of 29 tumors from 29 patients were included in this study. The mean age at diagnosis was 53.2 years and 72% of the patients were female. The tumors were between 15 and 100 mm in size (mean 44.8 mm) and we calculated an average proliferation index (Ki-67) of 5.9%. In 5 cases, distant metastases were identified. The comparisons between TERT promoter mutated and wild-type cases are summarized in Table 1 . Table 1 Clinicopathological features of the follicular and oncocytic thyroid tumors included in the study cohort. Clinicopathological Parameters TERT promoter mutated (n = 10) TERT promoter wildtype (n = 19) Total p-value Gender Female (n, %) 6 (60%) 15 (79%) 21 (72%) p = 0.51 Age at diagnosis, years 65.4 46.7 53.2 p = 0.49 Size, mm 47.2 43.6 44.8 p = 0.73 Ki-67, % 7.3 5.1 5.86 p = 0.06 Distant Metastasis (n, %) 4 (40%) 1 (5%) 5 (17%) p = 0.06 Evaluation of 5hmC immunoreactivity across the tumor cohort For both antibodies, positive and negative controls were evaluated. The IHC for 5hmC yielded varying results regarding staining intensity in tumor tissues, requiring us to implement the semi-quantitative scoring system described in the Material and Methods section based on nuclear staining in an estimated percentage of tumor cells. The staining outcomes and TERT gene status are detailed together with the histopathological diagnosis for each case in Table 2 . The control case exhibiting TFND and the normal testicular tissue sample used as positive controls displayed a score of 4 for both antibody clones, as did all cases of adjacent normal thyroid tissue whenever present on the same slide as the tumor tissue (data not shown). The three de-identified malignant thyroid tumors with concurrent BRAF and TERT promoter mutations all exhibited complete nuclear absence of 5hmC staining (tier 0). Table 2 Summarized genetic and expressional cohort data. Sample no. Diagnosis TERT mutation TERT Expression 5hmC IHC (RM236) score 5hmC IHC (4D9) score 1 FTC Mut 0.65 4 1 2 FTC Mut 1.00 3 3 3 FTC Mut 5.28 3 3 4 FTC Mut 14.93 3 3 5 FTC Mut 1.01 3 2 6 FTC Mut 1.23 3 0 7 FTC Mut 2.57 3 2 8 FT-UMP Mut 2.69 0 0 9 FT-UMP Mut 10.86 3 3 10 OTC Mut 5.37 4 3 11 FTC WT 0 4 4 12 FTC WT 0 3 2 13 FTC WT 0 3 2 14 FTC WT 0 3 2 15 FTC WT 0 4 4 16 FTC WT 0 4 3 17 FTC WT 0 3 3 18 FTC WT 0 3 3 19 FTC WT 0 3 1 20 FTC WT 0 2 2 21 FTC WT 0 4 3 22 FTC WT 0 3 2 23 FTC WT 0 4 4 24 FTC WT 0 4 3 25 FTC WT 0 4 4 26 FTC WT 0 4 4 27 FTC WT 0 4 0 28 FTC WT 0 4 0 29 FTC WT 0 4 1 30 TFND Not known Not known 4 4 TERT promoter mutational status (mut = mutated, WT = wildtype), normalized TERT expression value (qRT-PCR), 5hmC IHC semiquantitative (0 74%). FTC; Follicular thyroid carcinoma. FT-UMP; follicular thyroid tumor of uncertain malignant potential. OTC; Oncocytic thyroid carcinoma. TFND; thyroid follicular nodular disease Comparing 5hmC IHC to TERT promotor mutational status and TERT expression For the 4D9 clone, of the 10 tumors with an established TERT promoter mutation and TERT gene expression, 2 (20%) stained negative (tier 0) while 8 cases exhibited positive staining in more than 1% of tumor cells (tier 1 or above). Of the 19 TERT promoter wild-type cases, 2 (10.5%) demonstrated a negative 5hmC immunoreactivity, while 17 cases (89.5%) were seen with more than 1% positive tumor cells (Fisher’s Exact test P = 0.59). The sensitivity and specificity for global loss of 5hmC (clone 4D9) to detect TERT mutated cases were 20% and 89% respectively (Table 3 ). For the RM236 clone, one of the TERT promoter mutated tumors stained negative (tier 0), whereas 9 cases demonstrated a tier 1–4 stain, compared to TERT promoter wild-type cases in which the corresponding numbers were 0 and 19 respectively (Fisher's Exact test P = 0.35). The sensitivity and specificity for the RM236 clone to detect TERT promoter mutations were 10% and 100% respectively (Table 3 ). Table 3 Sensitivity, specificity, positive and negative predictive values (PPV/NPV) of the semiquantitative scoring. 5hmC IHC (4D9 clone) semiquantitative score = 0 5hmC IHC (4D9 clone) semiquantitative score = 1–4 5hmC IHC (RM236 clone) semiquantitative score = 0 5hmC IHC (RM236 clone) semiquantitative score = 1–4 Tumors with TERT mutation/ expression 2 8 1 9 Tumors without TERT mutation/ expression 2 17 0 19 Sensitivity, % 20 10 Specificity, % 89 100 PPV, % 50 100 NPV, % 68 68 Fisher’s Exact p = 0.59 p = 0.35 IHC; immunohistochemistry. NPV; negative predictive value. PPV; positive predictive value. Discussion TERT promoter mutations and TERT expression are harbingers of worse clinical outcomes in patients with FTC, constituting important prognostic markers. While it is certainly feasible to interrogate both DNA and RNA acquired from FFPE material in the clinical setting, the advent of an IHC marker with the ability to triage cases for further testing would of course be a valuable tool in the clinical setting, given the low cost and general availability across pathology laboratories. In this cohort of follicular thyroid tumors, we could not verify earlier observations made in PTC suggesting that loss of 5hmC would reliably signify an underlying TERT promoter mutation. If accepting global loss (score 0) as a pathogenic staining pattern, the sensitivity of the method reached 20% and 10% for the 4D9 and RM236 clones respectively in our series, while the specificity was rather low. Given the rather low percentages of follicular thyroid tumors exhibiting TERT promoter mutations in unselected series (around 10%), the method would require high specificity to minimize the risk of false positive cases. In this cohort of follicular thyroid tumors and OTCs, we could not fully reproduce the observations of global loss of 5hmC immunoreactivity in PTCs with TERT promoter mutations [ 13 , 14 ] The discrepancies may in part be due to the different thyroid tumor types studied. Indeed, the inheritably different biology of a BRAF mutation compared to predominantly RAS -driven FTCs may affect the global epigenetic landscape differently in TERT promoter mutated cases. A similar result was recently presented by Seok et al., showing a correlation between BRAF -mutated PTC and reduced 5hmC levels, but no such correlation in other groups of follicular patterned thyroid tumors [ 15 ]. The pathway linking BRAF mutations with lower levels of TET proteins and 5hmC is well established [ 16 ], whereas to our knowledge no such linkage has been examined for the more heterogeneous group of non- BRAF -driven thyroid neoplasms. Whereas Oishi et al. used a single, polyclonal antibody, we incorporated two different monoclonal anti-5hmC antibodies. However, our staining of small subsets of de-identified BRAF- driven thyroid tumors with TERT promoter mutations gave rise to similar results as Oishi and colleagues observed, namely a global loss of 5hmC (Fig. 1 ). Therefore, the discrepancies in the staining outcomes in association with underlying genotypes are most likely due to intrinsic differences in tumor biology rather than methodological diversities. Furthermore, we saw no apparent differences in terms of sensitivity and specificity regarding the two antibody clones used in this study. Moreover, since we employed two different staining techniques (automated vs manual), this parameter is not believed to affect the outcome either. TERT promoter mutations and TERT gene expression are not unequivocally needed for the global loss of 5hmC in thyroid cancer. There is much to be revealed regarding the biology of TERT promoter mutations in follicular thyroid tumors, as TERT protein expression itself may not be correlated to TERT promoter mutations and TERT mRNA expression [ 9 ] as well as the recent observation of nuclear-specific TERT mRNA expression [ 10 ]. TERT aberrancies may exhibit spatial heterogeneity in follicular thyroid tumors, which has been shown for TERT promoter mutations [ 17 , 18 ] as well as for TERT mRNA expression visualized by ISH [ 10 ]. It was therefore expected that some heterogeneity could be observed regarding the expression of 5hmC; it is however interesting that as many as half of all cases showed heterogeneous expression patterns - a finding that lacks a credible explanation. Evaluating immunohistochemical staining where the required result is a negative staining pattern may not be without problems on its own. It is not always evident whether negative staining is due to technical mishaps or relates to a true biological reason. However, in the examined material in this study, the surrounding normal thyroid as well as stromal elements within the tumor tissue itself were available for internal control purposes, suggesting preserved antigenicity for all cases Conclusion In conclusion, even though the loss of 5hmC immunoreactivity may signify TERT promoter mutations in subsets of FTCs, we could not prove its clinical value to predict the TERT promoter mutational status in this tumor entity. Further studies are therefore warranted. Declarations Funding: This study was financially supported by grants generously provided by the Swedish Cancer Society, the Swedish Society for Medical Research, the Cancer Research Funds of Radiumhemmet, and the Stockholm City Council. Competing interests: The authors have no relevant financial or non-financial interests to disclose. Author Contributions: All authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by Martin Hysek, Samuel Hellgren, Vincenzo Condello, and C. Christofer Juhlin. Funding was provided by Jan Zedenius, Catharina Larsson, and C. Christofer Juhlin. The first draft of the manuscript was written by Martin Hysek and C. Christofer Juhlin and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethics approval: The study was approved by the Swedish Ethical Review Authority (Dnr 2015.959.31) Consent to participate: Informed consent was obtained from all individual participants included in the study. Consent to publish: The authors affirm that human research participants provided informed consent for publication. References C. Bournaud, F. Descotes, M. Decaussin-Petrucci, J. Berthiller, C. de la Fouchardière, A.-L. Giraudet, M. Bertholon-Gregoire, P. Robinson, J.-C. 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Also discoverable on Platform About In Review Editorial Policies 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-2642987","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":183730187,"identity":"507d5c6e-f785-4c8c-9aeb-95f6a3207fe2","order_by":0,"name":"Martin Hysek","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIie3OsQqCQBzH8b+4+gA3ZS8QKII4+DAegi3S0h436RLNNvUKjY0HB00Xt95oBLYaLbUlpqt3Y9B9ufH34X8AJtOvlvQPbGgp0gIWGYlVaRMYiO1Qjf2ivNzr6wlWnmAsiHkEbkmmSciXPsEc1p7M0jSXCDyuOBXSzCK4AHyUTsDytiMoURDRDESIJ4s64h5qBZHjFZrbKXQfAzktOtJYFS4Q3sss8LccOR5XfiyDx7uI8U6wG3qdNzO3pIoz3xDMh6Gjte9zif7WZDKZ/qwPGC9KBWGEG7UAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-6544-5399","institution":"Karolinska Institutet","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Hysek","suffix":""},{"id":183730188,"identity":"43902d37-19b6-4acf-b642-ca8518fe0316","order_by":1,"name":"Samuel Hellgren","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Hellgren","suffix":""},{"id":183730189,"identity":"4a7ab3ef-c88e-4de4-ab7e-9c9f90bfa1ec","order_by":2,"name":"Vincenzo Condello","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Vincenzo","middleName":"","lastName":"Condello","suffix":""},{"id":183730190,"identity":"ff2c8084-96f8-41a5-994b-828d6f9acb9f","order_by":3,"name":"Catharina Larsson","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Catharina","middleName":"","lastName":"Larsson","suffix":""},{"id":183730191,"identity":"6e0d6b7f-f29d-4e60-b769-1346580c3f22","order_by":4,"name":"Jan Zedenius","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Zedenius","suffix":""},{"id":183730192,"identity":"d27c1b54-69bd-4b10-8e25-f8371d0befb4","order_by":5,"name":"C. Christofer Juhlin","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"C.","middleName":"Christofer","lastName":"Juhlin","suffix":""}],"badges":[],"createdAt":"2023-03-01 12:17:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2642987/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2642987/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":34587408,"identity":"f6722799-e805-4038-b59d-abefe7fce960","added_by":"auto","created_at":"2023-03-21 14:20:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3059497,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferent 5hmC staining patterns.\u003c/strong\u003e \u003cstrong\u003e(A-C) Control tissues:\u003c/strong\u003e A – normal testicular tissue (200x magnification), manually stained with clone 4D6; B – Negative stain in a \u003cem\u003eBRAF\u003c/em\u003e+ \u003cem\u003eTERT\u003c/em\u003e promoter mutated oncocytic papillary thyroid carcinoma (PTC), Ventana clone RM236 (200x); C – Negative stain in a BRAF + \u003cem\u003eTERT\u003c/em\u003e promoter mutated tall cell PTC, Ventana clone RM236 (200x); \u003cstrong\u003e(D-F) Clone RM236:\u003c/strong\u003e D – Score 4 (Sample #28, a \u003cem\u003eTERT\u003c/em\u003e promoter wildtype follicular thyroid carcinoma (FTC) without \u003cem\u003eTERT\u003c/em\u003e mRNA expression, 200x); E – Score 3 (Sample #4, a \u003cem\u003eTERT\u003c/em\u003epromoter mutated FTC with established \u003cem\u003eTERT\u003c/em\u003e mRNA expression, 200x); F – Score 0 (Sample #8, a \u003cem\u003eTERT\u003c/em\u003e promoter mutated follicular thyroid tumor of uncertain malignant potential (FT-UMP) with established \u003cem\u003eTERT\u003c/em\u003e mRNA expression, 200x). \u003cstrong\u003e(G-I) Clone 4D6:\u003c/strong\u003e G – Score 4 (Sample #2, 200x) H – Score 3 (Sample #4, 200x); I – Score 1 (Sample #8, 200x).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2642987/v1/321363d29e56648c03fb5015.png"},{"id":34588822,"identity":"8637a4e9-aef8-42a7-9551-b44c6b95cc90","added_by":"auto","created_at":"2023-03-21 14:28:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2535933,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2642987/v1/c366653a-14c7-4387-b5e1-53c7c2e13da9.pdf"}],"financialInterests":"","formattedTitle":"5hmC immunohistochemistry: a predictor of TERT promoter mutational status in follicular thyroid carcinoma?","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFollicular thyroid tumors pose a diagnostic difficulty as the differentiation between follicular thyroid adenoma (FTA) and follicular thyroid carcinoma (FTC) is based on the identification of invasive properties using routine histology. To establish a correct diagnosis, the material often needs to be scrutinized thoroughly to identify areas of invasion. In the past years, activating hotspot mutations in the \u003cem\u003eTelomerase reverse transcriptase\u003c/em\u003e (\u003cem\u003eTERT\u003c/em\u003e) gene promoter (denoted C228T and C250T) have been intimately coupled to FTC [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and subsets of follicular thyroid tumors of uncertain malignant potential (FT-UMPs), but are rarely observed in FTA. This genotype-phenotype correlation could therefore help in evaluating the true malignant potential of follicular thyroid tumors [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Moreover, \u003cem\u003eTERT\u003c/em\u003e promoter mutations and \u003cem\u003eTERT\u003c/em\u003e gene expression are prognostic factors in thyroid cancer, as these events are closely associated with adverse histopathological factors and poor patient outcomes [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As of this, the 2022 WHO classification of endocrine and neuroendocrine tumors acknowledges the role of \u003cem\u003eTERT\u003c/em\u003e promoter mutations in highlighting cases at risk of disease dissemination [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and more focus is currently diverted to developing robust screening tools to detect \u003cem\u003eTERT\u003c/em\u003e-expressing tumors in clinical routine. Specifically, while \u003cem\u003eTERT\u003c/em\u003e immunohistochemistry (IHC) is not yet considered a reliable method, studies using \u003cem\u003eTERT\u003c/em\u003e mRNA \u003cem\u003ein situ\u003c/em\u003e hybridization (ISH) have shown promising results [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e5-hydroxymethylcytosine (5hmC) immunoreactivity is decreased in various malignant tumors, such as melanoma [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and glioma [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] as well as in papillary thyroid carcinoma (PTC) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. 5hmC is produced when 5-methylcytosine (5mc) is demethylated via ten-eleven translocation (TET) enzymes, and 5hmC may thus serve as an epigenetic marker of global de-methylation events across the genome. Oishi \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] recently showed an association between the loss of 5hmC and \u003cem\u003eTERT\u003c/em\u003e promoter mutations in PTC, but to our knowledge, no efforts to predict \u003cem\u003eTERT\u003c/em\u003e promoter mutational status by 5hmC IHC in FTCs have been published.\u003c/p\u003e \u003cp\u003eThe aim of this study was therefore to evaluate whether loss of immunohistochemical 5hmC expression can predict \u003cem\u003eTERT\u003c/em\u003e promotor mutations in a cohort of FTCs with established \u003cem\u003eTERT\u003c/em\u003e promoter genotypes and \u003cem\u003eTERT\u003c/em\u003e mRNA expression by quantitative real-time PCR (qRT-PCR) and ISH.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCohort\u003c/h2\u003e \u003cp\u003eA cohort of differentiated follicular cell-derived and oncocytic thyroid tumors which has previously been evaluated for \u003cem\u003eTERT\u003c/em\u003e promoter mutations and \u003cem\u003eTERT\u003c/em\u003e expression using qRT-PCR and ISH [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] was chosen for 5hmC IHC analyses. The cohort consisted of 26 FTCs, 2 FT-UMPs, and one oncocytic thyroid carcinoma (OTC), of which 10 (7 FTCs, 2 FT-UMPs, and the single OTC) showed \u003cem\u003eTERT\u003c/em\u003e promoter mutations and \u003cem\u003eTERT\u003c/em\u003e gene expression, while the remaining cases were \u003cem\u003eTERT\u003c/em\u003e promoter wildtype and displayed absent \u003cem\u003eTERT\u003c/em\u003e mRNA levels. A de-identified case with thyroid follicular nodular disease (TFND) was included as a non-tumorous control for both antibodies, and a de-identified sample of normal testis tissue as a positive control of 5hmC immunoreactivity, which was used for identifying the best dilution of the 4D9 antibody. To test the validity of the antibody used in clinical routine, three de-identified malignant thyroid tumors with concurrent \u003cem\u003eBRAF\u003c/em\u003e and \u003cem\u003eTERT\u003c/em\u003e promoter mutations were included as well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e5mhC immunohistochemistry (clone RM236)\u003c/h2\u003e \u003cp\u003eFormalin-fixed paraffin-embedded (FFPE) tissues from all cases were sectioned in 4-micrometer thick sections. The chosen sections all contained tumor tissue as well as adjacent normal thyroid tissue. After de-paraffinization in xylene and rehydration in alcohol, sections were subjected to heat-induced antigen retrieval using the Ventana Ultra CC1, pH 8.5 for 32 min. The staining was automated using the Ventana Ultra Benchmark methodology and a primary antibody (5hmC clone RM236, Abcam, Cambridge, UK) was applied at a dilution of 1:1,000 for 32 min. Visualization was performed using OptiView and slides were then counterstained using Mayer\u0026acute;s hematoxylin for 3 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e5hmC immunohistochemistry (clone 4D9)\u003c/h2\u003e \u003cp\u003eFollowing de-paraffinization and rehydration of FFPE sections, heat-induced antigen retrieval was performed using a decloaking chamber (Biocare Medical, CA) set for 5 min at 110\u0026deg;C in citrate buffer pH 6 (article C-9999, Sigma-Aldrich, MA). For quenching of endogenous peroxidase, a 30 min incubation in 0.15% hydrogen peroxidase was performed at room temperature, followed by a 30 min blocking step using 1% bovine serum albumin (article A-4503, Sigma-Aldrich, MA). The primary 5hmC monoclonal antibody (HMC/4D9, Epigentek, NY) was diluted 1:2,000 in Renior Red diluent (article #PD9004M, Biocare Medical, CA), before incubation at 4\u0026deg;C overnight in a humidified chamber. For detection, the Mach-1 Universal HRP-Polymer Kit was used (article MIU539L10, Biocare Medical, CA) according to the protocol provided by the manufacturer. The sections were counterstained in Mayer\u0026acute;s hematoxylin for 1 min, followed by dehydration with graded alcohols, xylene treatment, and coverslipped with Pertex (Histolab, Askim, Sweden).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eScoring methodology\u003c/h2\u003e \u003cp\u003eThe stained slides were evaluated by two pathologists independently following the same semi-quantitative scoring system as Oishi \u003cem\u003eet al.\u003c/em\u003e for their study on PTC [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]: 0\u0026thinsp;=\u0026thinsp;complete lack of nuclear staining in tumor cells i.e. negative; 1\u0026thinsp;=\u0026thinsp;positive staining only in scarce tumor cells (1\u0026ndash;9%); 2\u0026thinsp;=\u0026thinsp;positive staining in 10\u0026ndash;24% of tumor cells; 3\u0026thinsp;=\u0026thinsp;positive staining in 25\u0026ndash;74% of tumor cells; and 4\u0026thinsp;=\u0026thinsp;diffuse and strong nuclear staining in \u0026gt;\u0026thinsp;=\u0026thinsp;75% of tumor cells.\u003c/p\u003e \u003cp\u003eExamples of different staining patterns are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eFisher\u0026rsquo;s exact tests were used to compare 5hmC expression and \u003cem\u003eTERT\u003c/em\u003e promoter mutational status. Chi-square and Mann-Whitney-U tests were used to compare clinicopathological features and \u003cem\u003eTERT\u003c/em\u003e promoter mutational status. P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant. Statistical computations were performed using RStudio v12.0 (Posit Software PBC, MA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eClinicopathological features of the study cohort\u003c/h2\u003e \u003cp\u003eA total of 29 tumors from 29 patients were included in this study. The mean age at diagnosis was 53.2 years and 72% of the patients were female. The tumors were between 15 and 100 mm in size (mean 44.8 mm) and we calculated an average proliferation index (Ki-67) of 5.9%. In 5 cases, distant metastases were identified. The comparisons between \u003cem\u003eTERT\u003c/em\u003e promoter mutated and wild-type cases are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinicopathological features of the follicular and oncocytic thyroid tumors included in the study cohort.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinicopathological Parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTERT promoter mutated (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTERT promoter wildtype (n\u0026thinsp;=\u0026thinsp;19)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender Female (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (60%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (79%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21 (72%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge at diagnosis, years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSize, mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKi-67, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistant Metastasis (n, %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (40%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (17%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of 5hmC immunoreactivity across the tumor cohort\u003c/h2\u003e \u003cp\u003eFor both antibodies, positive and negative controls were evaluated. The IHC for 5hmC yielded varying results regarding staining intensity in tumor tissues, requiring us to implement the semi-quantitative scoring system described in the \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003eMaterial and Methods\u003c/span\u003e section based on nuclear staining in an estimated percentage of tumor cells. The staining outcomes and \u003cem\u003eTERT\u003c/em\u003e gene status are detailed together with the histopathological diagnosis for each case in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The control case exhibiting TFND and the normal testicular tissue sample used as positive controls displayed a score of 4 for both antibody clones, as did all cases of adjacent normal thyroid tissue whenever present on the same slide as the tumor tissue (data not shown). The three de-identified malignant thyroid tumors with concurrent \u003cem\u003eBRAF\u003c/em\u003e and \u003cem\u003eTERT\u003c/em\u003e promoter mutations all exhibited complete nuclear absence of 5hmC staining (tier 0).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummarized genetic and expressional cohort data.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiagnosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTERT\u003c/em\u003e mutation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eTERT\u003c/em\u003e Expression\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5hmC IHC\u003c/p\u003e \u003cp\u003e(RM236) score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5hmC IHC (4D9) score\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMut\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.65\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMut\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e1.00\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMut\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e5.28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMut\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e14.93\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMut\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e1.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMut\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e1.23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMut\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e2.57\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFT-UMP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMut\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e2.69\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFT-UMP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMut\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e10.86\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMut\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e5.37\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTFND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNot known\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNot known\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTERT\u003c/em\u003e promoter mutational status (mut\u0026thinsp;=\u0026thinsp;mutated, WT\u0026thinsp;=\u0026thinsp;wildtype), normalized \u003cem\u003eTERT\u003c/em\u003e expression value (qRT-PCR), 5hmC IHC semiquantitative (0\u0026thinsp;\u0026lt;\u0026thinsp;1%; 1\u0026thinsp;=\u0026thinsp;1\u0026ndash;9%; 2\u0026thinsp;=\u0026thinsp;10\u0026ndash;24%; 3\u0026thinsp;=\u0026thinsp;25\u0026ndash;74%; 4\u0026thinsp;\u0026gt;\u0026thinsp;74%). FTC; Follicular thyroid carcinoma. FT-UMP; follicular thyroid tumor of uncertain malignant potential. OTC; Oncocytic thyroid carcinoma. TFND; thyroid follicular nodular disease\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eComparing 5hmC IHC to TERT promotor mutational status and TERT expression\u003c/h2\u003e \u003cp\u003eFor the 4D9 clone, of the 10 tumors with an established \u003cem\u003eTERT\u003c/em\u003e promoter mutation and \u003cem\u003eTERT\u003c/em\u003e gene expression, 2 (20%) stained negative (tier 0) while 8 cases exhibited positive staining in more than 1% of tumor cells (tier 1 or above). Of the 19 \u003cem\u003eTERT\u003c/em\u003e promoter wild-type cases, 2 (10.5%) demonstrated a negative 5hmC immunoreactivity, while 17 cases (89.5%) were seen with more than 1% positive tumor cells (Fisher\u0026rsquo;s Exact test P\u0026thinsp;=\u0026thinsp;0.59). The sensitivity and specificity for global loss of 5hmC (clone 4D9) to detect \u003cem\u003eTERT\u003c/em\u003e mutated cases were 20% and 89% respectively (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For the RM236 clone, one of the \u003cem\u003eTERT\u003c/em\u003e promoter mutated tumors stained negative (tier 0), whereas 9 cases demonstrated a tier 1\u0026ndash;4 stain, compared to \u003cem\u003eTERT\u003c/em\u003e promoter wild-type cases in which the corresponding numbers were 0 and 19 respectively (Fisher's Exact test P\u0026thinsp;=\u0026thinsp;0.35). The sensitivity and specificity for the RM236 clone to detect \u003cem\u003eTERT\u003c/em\u003e promoter mutations were 10% and 100% respectively (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSensitivity, specificity, positive and negative predictive values (PPV/NPV) of the semiquantitative scoring.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5hmC IHC\u003c/p\u003e \u003cp\u003e(4D9 clone) semiquantitative score\u0026thinsp;=\u0026thinsp;0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5hmC IHC\u003c/p\u003e \u003cp\u003e(4D9 clone) semiquantitative score\u0026thinsp;=\u0026thinsp;1\u0026ndash;4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5hmC IHC\u003c/p\u003e \u003cp\u003e(RM236 clone)\u003c/p\u003e \u003cp\u003esemiquantitative score\u0026thinsp;=\u0026thinsp;0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5hmC IHC\u003c/p\u003e \u003cp\u003e(RM236 clone)\u003c/p\u003e \u003cp\u003esemiquantitative score\u0026thinsp;=\u0026thinsp;1\u0026ndash;4\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumors with \u003cem\u003eTERT\u003c/em\u003e mutation/ expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumors without \u003cem\u003eTERT\u003c/em\u003e mutation/ expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSensitivity, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecificity, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePPV, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNPV, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFisher\u0026rsquo;s Exact\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIHC; immunohistochemistry. NPV; negative predictive value. PPV; positive predictive value.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eTERT\u003c/em\u003e promoter mutations and \u003cem\u003eTERT\u003c/em\u003e expression are harbingers of worse clinical outcomes in patients with FTC, constituting important prognostic markers. While it is certainly feasible to interrogate both DNA and RNA acquired from FFPE material in the clinical setting, the advent of an IHC marker with the ability to triage cases for further testing would of course be a valuable tool in the clinical setting, given the low cost and general availability across pathology laboratories. In this cohort of follicular thyroid tumors, we could not verify earlier observations made in PTC suggesting that loss of 5hmC would reliably signify an underlying \u003cem\u003eTERT\u003c/em\u003e promoter mutation. If accepting global loss (score 0) as a pathogenic staining pattern, the sensitivity of the method reached 20% and 10% for the 4D9 and RM236 clones respectively in our series, while the specificity was rather low. Given the rather low percentages of follicular thyroid tumors exhibiting \u003cem\u003eTERT\u003c/em\u003e promoter mutations in unselected series (around 10%), the method would require high specificity to minimize the risk of false positive cases.\u003c/p\u003e \u003cp\u003eIn this cohort of follicular thyroid tumors and OTCs, we could not fully reproduce the observations of global loss of 5hmC immunoreactivity in PTCs with \u003cem\u003eTERT\u003c/em\u003e promoter mutations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] The discrepancies may in part be due to the different thyroid tumor types studied. Indeed, the inheritably different biology of a \u003cem\u003eBRAF\u003c/em\u003e mutation compared to predominantly \u003cem\u003eRAS\u003c/em\u003e-driven FTCs may affect the global epigenetic landscape differently in \u003cem\u003eTERT\u003c/em\u003e promoter mutated cases. A similar result was recently presented by Seok et al., showing a correlation between \u003cem\u003eBRAF\u003c/em\u003e-mutated PTC and reduced 5hmC levels, but no such correlation in other groups of follicular patterned thyroid tumors [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The pathway linking \u003cem\u003eBRAF\u003c/em\u003e mutations with lower levels of TET proteins and 5hmC is well established [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], whereas to our knowledge no such linkage has been examined for the more heterogeneous group of non-\u003cem\u003eBRAF\u003c/em\u003e-driven thyroid neoplasms.\u003c/p\u003e \u003cp\u003eWhereas Oishi \u003cem\u003eet al.\u003c/em\u003e used a single, polyclonal antibody, we incorporated two different monoclonal anti-5hmC antibodies. However, our staining of small subsets of de-identified \u003cem\u003eBRAF-\u003c/em\u003edriven thyroid tumors with \u003cem\u003eTERT\u003c/em\u003e promoter mutations gave rise to similar results as Oishi and colleagues observed, namely a global loss of 5hmC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, the discrepancies in the staining outcomes in association with underlying genotypes are most likely due to intrinsic differences in tumor biology rather than methodological diversities. Furthermore, we saw no apparent differences in terms of sensitivity and specificity regarding the two antibody clones used in this study. Moreover, since we employed two different staining techniques (automated vs manual), this parameter is not believed to affect the outcome either.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTERT\u003c/em\u003e promoter mutations and \u003cem\u003eTERT\u003c/em\u003e gene expression are not unequivocally needed for the global loss of 5hmC in thyroid cancer. There is much to be revealed regarding the biology of \u003cem\u003eTERT\u003c/em\u003e promoter mutations in follicular thyroid tumors, as \u003cem\u003eTERT\u003c/em\u003e protein expression itself may not be correlated to \u003cem\u003eTERT\u003c/em\u003e promoter mutations and \u003cem\u003eTERT\u003c/em\u003e mRNA expression [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] as well as the recent observation of nuclear-specific \u003cem\u003eTERT\u003c/em\u003e mRNA expression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eTERT\u003c/em\u003e aberrancies may exhibit spatial heterogeneity in follicular thyroid tumors, which has been shown for \u003cem\u003eTERT\u003c/em\u003e promoter mutations [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] as well as for \u003cem\u003eTERT\u003c/em\u003e mRNA expression visualized by ISH [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It was therefore expected that some heterogeneity could be observed regarding the expression of 5hmC; it is however interesting that as many as half of all cases showed heterogeneous expression patterns - a finding that lacks a credible explanation.\u003c/p\u003e \u003cp\u003eEvaluating immunohistochemical staining where the required result is a negative staining pattern may not be without problems on its own. It is not always evident whether negative staining is due to technical mishaps or relates to a true biological reason. However, in the examined material in this study, the surrounding normal thyroid as well as stromal elements within the tumor tissue itself were available for internal control purposes, suggesting preserved antigenicity for all cases\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, even though the loss of 5hmC immunoreactivity may signify \u003cem\u003eTERT\u003c/em\u003e promoter mutations in subsets of FTCs, we could not prove its clinical value to predict the \u003cem\u003eTERT\u003c/em\u003e promoter mutational status in this tumor entity. Further studies are therefore warranted.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was financially supported by grants generously provided by the Swedish Cancer Society, the Swedish Society for Medical Research, the Cancer Research Funds of Radiumhemmet, and the Stockholm City Council.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eAll authors contributed to the study\u0026apos;s conception and design. Material preparation, data collection, and analysis were performed by Martin Hysek, Samuel Hellgren, Vincenzo Condello, and C. Christofer Juhlin. Funding was provided by Jan Zedenius, Catharina Larsson, and C. Christofer Juhlin. The first draft of the manuscript was written by Martin Hysek and C. Christofer Juhlin and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eThe study was approved by the Swedish Ethical Review Authority (Dnr 2015.959.31)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConsent to participate:\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent to publish:\u0026nbsp;\u003c/strong\u003eThe authors affirm that human research participants provided informed consent for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eC. Bournaud, F. Descotes, M. Decaussin-Petrucci, J. 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Paulsson, N. Mu, I. Shabo, N. Wang, J. Zedenius, C. Larsson, and C. C. Juhlin, Endocrine-Related Cancer \u003cstrong\u003e25\u003c/strong\u003e, 723 (2018).\u003c/li\u003e\n\u003cli\u003eT. Liu, N. Wang, J. Cao, A. Sofiadis, A. Dinets, J. Zedenius, C. Larsson, and D. Xu, Oncogene \u003cstrong\u003e33\u003c/strong\u003e, 4978 (2014).\u003c/li\u003e\n\u003cli\u003eG. Melo, J. Da Rocha, M. Vinagre, M. Batista, M. Peixoto, M. Tavares, M. Celestino, M. Almeida, M. Salgado, M. Eloy, M. Castro, M. Prazeres, M. Lima, M. Amaro, M. Lobo, M. Martins, M. Moura, M. Cavaco, M. Leite, M. Cameselle-Teijeiro, M. Carrilho, M. Carvalheiro, M. M\u0026aacute;ximo, M. Sobrinho-Sim\u0026otilde;es, and M. Soares, The Journal of Clinical Endocrinology \u0026amp; Metabolism \u003cstrong\u003e99\u003c/strong\u003e, E754 (2014).\u003c/li\u003e\n\u003cli\u003eZ. W. Baloch, S. L. Asa, J. A. Barletta, R. A. Ghossein, C. C. Juhlin, C. K. Jung, V. A. LiVolsi, M. G. Papotti, M. Sobrinho-Sim\u0026otilde;es, G. 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Br\u0026auml;nstr\u0026ouml;m, E. Darai-Ramqvist, J. O. Paulsson, N. Wang, C. Larsson, J. Zedenius, and C. C. Juhlin, Endocr Pathol \u003cstrong\u003e30\u003c/strong\u003e, 246 (2019).\u003c/li\u003e\n\u003cli\u003eM. Hysek, K. Jatta, L. S. Hellgren, A. Stenman, C. Larsson, J. Zedenius, and C. C. Juhlin, The Journal of Molecular Diagnostics \u003cstrong\u003e23\u003c/strong\u003e, 212 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"endocrine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"endo","sideBox":"Learn more about [Endocrine](https://www.springer.com/journal/12020)","snPcode":"12020","submissionUrl":"https://submission.nature.com/new-submission/12020/3","title":"Endocrine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"TERT promoter mutation, 5hmC, immunohistochemistry, thyroid","lastPublishedDoi":"10.21203/rs.3.rs-2642987/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2642987/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003e \u003cem\u003eTERT\u003c/em\u003e promoter mutations and \u003cem\u003eTERT\u003c/em\u003e gene expression correlate to adverse prognosis in follicular thyroid carcinoma (FTC), identifying cases at risk of poor outcome. As loss of 5-hydroxymethylcytosine (5hmC) immunoreactivity has been associated with \u003cem\u003eTERT\u003c/em\u003e promoter mutations in papillary thyroid carcinoma, this study sought to analyze the levels of 5hmC in a well-characterized cohort of follicular thyroid tumors with available \u003cem\u003eTERT\u003c/em\u003e data.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e29 tumors (26 FTCs, 2 follicular thyroid tumors of uncertain malignant potential (FT-UMPs), and one oncocytic thyroid carcinoma) with known \u003cem\u003eTERT\u003c/em\u003e promoter mutational status and \u003cem\u003eTERT\u003c/em\u003e gene expression levels were assessed for 5hmC immunoreactivity using two monoclonal antibodies (clones RM236 and 4D9.) Slides were analyzed using a semiquantitative scoring system.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOf the 10 tumor cases with a \u003cem\u003eTERT\u003c/em\u003e promoter mutation and \u003cem\u003eTERT\u003c/em\u003e expression, only one was scored as negative with both antibodies (1/10; 10%), while the remaining 9 cases (9/10; 90%) exhibited various degrees of positivity for at least one antibody. Of the 19 \u003cem\u003eTERT\u003c/em\u003e wild-type tumors, no case (0/19; 0%) was scored as negative using the RM236 clone, and two cases (2/19; 11%) using the 4D9 clone. The differences between \u003cem\u003eTERT\u003c/em\u003e promoter mutated and wildtype groups were non-significant (Fisher\u0026rsquo;s Exact test P\u0026thinsp;=\u0026thinsp;0.35 and 0.59 respectively). The sensitivity and specificity for 5hmC IHC to detect mutated cases were 10% and 100% for RM236 and 20% and 89% for 4D9 respectively.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003e5hmC IHC is not a highly sensitive marker for the detection of \u003cem\u003eTERT\u003c/em\u003e promoter mutations in follicular thyroid tumors. Further analyses in larger cohorts are warranted.\u003c/p\u003e","manuscriptTitle":"5hmC immunohistochemistry: a predictor of TERT promoter mutational status in follicular thyroid carcinoma?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-21 14:20:08","doi":"10.21203/rs.3.rs-2642987/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-03-15T12:35:00+00:00","index":0,"fulltext":""},{"type":"editorAssigned","content":"","date":"2023-03-03T14:51:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Endocrine","date":"2023-03-01T07:17:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"endocrine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"endo","sideBox":"Learn more about [Endocrine](https://www.springer.com/journal/12020)","snPcode":"12020","submissionUrl":"https://submission.nature.com/new-submission/12020/3","title":"Endocrine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ba0a936a-a9b2-48d8-bf35-2b55d2f8d22c","owner":[],"postedDate":"March 21st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2023-03-21T14:20:08+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-21 14:20:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2642987","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2642987","identity":"rs-2642987","version":["v1"]},"buildId":"re_ckhLnmML6MCF96OHNJ","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}