Genetic landscape of nodular goiter, thyroid nodules with intermediate features between nodular goiter and follicular adenoma

Genetic landscape of nodular goiter, thyroid nodules with intermediate features between nodular goiter and follicular adenoma | 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 Genetic landscape of nodular goiter, thyroid nodules with intermediate features between nodular goiter and follicular adenoma Ting Duan, Dingyuan Ren, Yanling Jin, Xiaming Lu, Zhenying Guo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4162113/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Nodular goiters (NG) and follicular adenoma (FA) are common thyroid benign nodules and thyroid nodules with intermediate features between nodular goiter and follicular (ING) is used to describe the disease with borderline features. The genetic landscape of these three diseases is poorly investigated comparatively. Methods Clinical information of NG, ING and FA was retrieved and reviewed. Cytology and histology of the pathologic archives were reviewed to confirm the diagnosis. DNA and RNA were extracted to be submitted to qPCR assay to detect BRAF, TERT, RET, RAS, PAX8 and NTRK genetic aberration. Results The demographic, clinical, image and cytologic features in NG and ING are similar. Most disease presents a benign clinical and cytologic behavior, causing no diagnostic difficulty. Low frequency (< 10%) RAS gene was found in NG and ING. Patients with FA are older than whom with NG or ING. FA lesion size is smaller than ones in NG or ING. Although sonography demonstrated most FA nodules as benign category, the cytologic evaluation demonstrated a considerable percentage (45%) of atypia disease. Additionally, much higher frequency of RAS gene abnormality has been found in FA. And PAX8-PPARG gene is found in 17.9% FA and in no NG/ING cases. Conclusion NG/ING and FA is in the same spectrum and represents two ends of the spectrum of thyroid benign nodular condition. Differentiation of ING from NG has little clinical significance and would not be recommended. FA is devoid from NG/ING clinically and genetically. Interpretation of genetic abnormality should be cautious. Thyroid nodules nodular goiters follicular adenomas genetic profile Figures Figure 1 Introduction Thyroid nodules is a common condition affecting approximately 19%-49% population in ultrasound examination 1 – 2 , in which most is benign condition and 11% is malignant condition 3 . Nodular goiters accounts for the most common types of thyroid nodules 4 , Most nodular goiters feature a mass in thyroid and can be easily identified by ultrasound examination, and will not cause a problem in the diagnosis 5 . Further examination, such as cytology based on fine needle aspiration (FNA) and molecular testing, would be needed if some suspicious features seen such as calcification 6 . Follicular adenoma represents the most common thyroid benign neoplasm, and can be easily manageable surgically. The diagnosis of follicular adenoma should be based on the intensive sampling and careful examination to exclude the follicular carcinoma featuring capsule invasion and vascular invasion 4 . For most circumstances, nodular goiter can be easily distinguished from follicular adenoma, in which well-defined borders, well established capsules and lack of disease in the adherent thyroid tissue would be seen. Nodular goiter is lacking well-defined borders, well established capsules and imparts a diffuse disease affecting the adherent thyroid tissue. However, borderline condition demonstrating the intermediate features between nodular goiter and follicular adenoma will cause a diagnosis dilemma 7 . Due to lack of uniformed terminology, various of diagnosis term would be used currently, such as adenomatoid nodular goiter 8 . This diagnosis dilemma may mainly be attributed to the unknown pathogenesis and biology of nodular goiter and follicular adenoma, and that the differential diagnosis mainly relies on the histology observation. In recent years, molecular testing of genetic abnormalities is being widely used as an ancillary study together with FNA cytology examination to identify the malignancies 6 . The molecular testing detects the genetic abnormalities, such as BFAF V600E, TERT promoter mutation, and RET gene rearrangement, which are mainly found in malignancies such as papillary carcinoma. And the genetic landscape of malignancies are well identified in recent studies 9 . However, the genetic landscape of benign thyroid nodules especially nodular goiter remains unclear, and nodular goiter are supposed not to carry any genetic abnormalities. In current studies, we report the genetic landscape of nodular goiter and thyroid nodules with intermediate features between nodular goiter and follicular adenoma using qPCR molecular test detecting BRAF, TERT, RET, RAS, PAX8 and NTRK, based on both FNA cytology samples and formalin-fixed paraffin-embedded (FFPE) samples. The genetic landscape may elaborate the genetic or biologic relationship nodular goiter and thyroid nodules with intermediate features between nodular goiter and follicular adenoma, facilitating the clinical diagnosis and management, as well as of terminology harmonization. Methods and Materials Patient and samples Clinical and pathologic information of nodular goiter and thyroid nodules with intermediate features between nodular goiter and follicular adenoma were retrieved from 2020 to 2023. Pathologic diagnosis was confirmed by expert pathologist. Cases would be eligible if both fresh cytology samples based on pre-operative FNA and post-operative FFPE samples are available. Demographic and clinicopathologic information were retrieved from the medical records. Ultrasound sonography records were collected based on the Thyroid imaging reporting and data system (TI-RAIDs). This study was approved by the institution ethic review board in the Zhejiang Province People’s Hospital. Cytology and histology Smear cytology samples based on the FNA were stained with hematoxylin plus eosin (H&E). The review and diagnosis of cytology samples was based on the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) 10 by expert pathologist. Tissues of nodular goiter and thyroid nodules with intermediate features between nodular goiter and follicular adenoma were intensively sampled with 5mm intervals. Slides with 4-um thickness were cut from the FFPE tissue blocks. Histology review was performed by expert pathologist. qPCR molecular testing The DNA and RNA of fresh cytology samples were extracted by Fresh DNA and RNA extraction kit (TiangenCo. Ltd, Shanghai, China) and the DNA and RNA of FFPE samples were extracted by FFPE DNA and RNA extraction kit (TiangenCo. Ltd, Shanghai, China). The thyroid cancer gene mutation detection kit is used for qualitatively detecting DNA point mutations and RNA fusion mutations for 12 thyroid cancer-related genes. The target genes include 8 DNA point mutations, which are BRAF V600E mutation, TERT promoter mutation C228T/C250T, KRAS G12C/ G12V/Q61R mutation, NRAS Q61R mutation, and HRAS Q61R mutation and 4 RNA fusion mutations: CCDC6-RET fusion, NCOA4-RET fusion, PAX8-PPARG fusion, ETV6-NTRK3 fusion. In brief, qPCR reactions were performed in a total volume of 20 µL containing 10 µL of 2 × qPCR Mix 3 µL of template DNA or RNA, 0.6 µL each of primers (each 10 µM), and 0.4 µL of probe (10 µM). The reaction was performed on applied biosystems 7500 real-time PCR system (ABI Life Technologies Corporation, USA). Molecular grade water was used as a negative control. The PCR reaction program was used as follows: 50°C for 15 min; 95°C for 15 min; followed by 15 cycles of 95°C for 15 s and 66°C for 40 s; and followed by 35 cycles of 95°C for 15 s and 60°C for 40 s. The data were analyzed using the 7500 software v 2.4 and the results were considered negative if cycle threshold (Ct) values were lower than cut-off. Statistics Baseline descriptive statistics and genetic mutation were summarized using medians (IQRs) and frequencies (percentages). Results Patients and demographics One hundred and four patients with nodular goiter (NG), 62 patients with thyroid nodules with intermediate features between nodular goiter (ING) and 28 patients with follicular adenoma (FA) were included. Most patients were young or middle-aged. The median age in the NG, ING and FA was 29 (range: 16–78), 28 ( range: 17–78) and 39 (range: 22–79), respectively, indicating FA may occur in the older population. The male and female percentage in the NG, ING and FA was 64% vs. 36%, 54% vs. 46% and 39% vs. 61, respectively. The demographics was summarized in Table 1 . Table 1 Demographic, clinical, image and cytologic features of NG, ING and FA NG ING FA Median age (range) 29 (16–78) 28 (17–78) 39 (22–79) Gender (%) Male 64 54 39 Female 36 46 61 Median lesion size (range, cm) 2.8 (1.2–6.4) 2.2 (1.1–7.4) 1.8 (1.2–5.1) Median time from disease onset to surgery (range, month ) 14 (3–44) 12 (1–36) 8 (1–20) TI-RAIDs category (%) 2 41 48 23 3 32 33 44 4a 27 16 25 4b and above 4 3 8 TBSRTC category 2 21 18 12 3 67 75 34 4 11 17 45 5 1 0 9 NG: Nodular goiter; ING: thyroid nodules with intermediate features between nodular goiter; FA: follicular adenoma; TI-RAIDs: Thyroid imaging reporting and data system; TBSRTC: Bethesda System for Reporting Thyroid Cytopathology. Clinical, image and cytologic features Median lesion size of NG, ING and FA was 2.8cm (range: 1.2cm-6.4cm), 2.2cm (range: 1.1cm-7.4cm) and 1.8cm (range: 1.2cm-5.1cm), and there is no significant difference in the lesion size among these disease. The Median time from disease onset to surgery was 14 months (range: 3 months–44 months), 12 months (range: 1 month-36 months ) and 8 (range:1 month-20 months) indicating the clinical evaluation of FA may trigger the surgery intervention comparing with the management of NG and ING. The clinical features were summarized in Table 1 . The ultrasonography finding based on the TI-RAIDs was summarized in Table 1 . Briefly, most lesions demonstrated a benign features and categories in the ultrasonography evaluation. The 4b and above suspected malignant lesion was 4%, 3% and 8% in the NG, ING and FA, respectively. Genetic profile The profile of BRAF V600E mutation, TERT promoter mutation C228T/C250T, KRAS G12C/ G12V/Q61R mutation, NRAS Q61R mutation, HRAS Q61R mutation, CCDC6-RET fusion, NCOA4-RET fusion, PAX8-PPARG fusion and ETV6-NTRK3 fusion in NG, ING and FA was summarized in Table 2 . RAS gene mutation was found in NG: KRAS G12C (2/104, 1.9%), KRAS G12V (2/104, 1.9%), KRAS Q61R (3/104, 2.9%), NRAS Q61R (2/104, 1.9%) and HRAS Q61R (2/104, 1.9%). BRAF V600E, which is considered to be the driving mutation of thyroid papillary carcinoma, was found with mutation in 2 out of 104 NGs (1.9%). We reviewed the clinical, radiologic and pathologic records and archives, and we found few indications suspicious of thyroid papillary carcinoma. The two patients have been being followed comprehensively and no recurrence has been found. Other gene are intact in NG group. Table 2 Genetic landscape of NG, ING and FA NG ING FA n = 104 n = 62 n = 28 NO. Frequency NO. Frequency NO. Frequency BRAF V600E 2 1.9% 1 1.6% 0 0.0% TERT C228T 0 0.0% 0 0.0% 0 0.0% TERT C250T 0 0.0% 0 0.0% 0 0.0% KRAS G12C 2 1.9% 2 3.2% 1 3.6% KRAS G12V 2 1.9% 3 4.8% 2 7.1% KRAS Q61R 3 2.9% 2 3.2% 3 10.7% NRAS Q61R 1 1.0% 6 9.7% 8 28.6% HRAS Q61R 1 1.0% 6 9.7% 9 32.1% CCDC6-RET 0 0.0% 0 0.0% 0 0.0% NCOA4-RET 0 0.0% 0 0.0% 1 3.6% PAX8-PPARG 0 0.0% 0 0.0% 5 17.9% ETV6-NTRK3 0 0.0% 0 0.0% 1 3.6% NG: Nodular goiter; ING: thyroid nodules with intermediate features between nodular goiter; FA: follicular adenoma; Similarly, RAS gene mutation was found in ING: KRAS G12C (1/62, 1.6%), KRAS G12V (3/62, 4.8%), KRAS Q61R (2/62, 3.2%), NRAS Q61R (6/62, 9.7%) and HRAS Q61R (6/62, 9.7%). One BRAF gene (1/62, 1.6%) was also found in ING. We reviewed the clinical, radiologic and pathologic records and archives, and we found few indications suspicious of thyroid papillary carcinoma. This patient has been being followed comprehensively and no recurrence has been found. Other gene are intact in ING group. The genetic landscape of FA is remarkable different from the one in the NG and ING. FA harbored the mutation of KRAS G12C (1/28, 3.6%), KRAS G12V (2/28, 7.1%), KRAS Q61R (3/28, 10.7%), NRAS Q61R (8/28,28.6%), and HRAS Q61R (9/28, 32.1%). Besides the SNV above, PAX8-PPARG fusion was identified in 5 out of 28 (17.9%) FA patients along with NCOA4-RET fusion (1/28, 3.6%) and ETV6-NTRK3 fusion (1/28, 3.6%). All the FA tissue were intensively sampled to rule out the possibility of malignancies. No recurrence or metastasis has been found. The grid figure of NG, ING and FA was provide in Fig. 1 A and the representative picture of qPCR assay to identify the mutation or fusion were provided in Fig. 1 B and Fig. 1 C. Discussion Nodular goiters (NG) and follicular adenoma (FA) represent the most common thyroid nodules, and most disease would not cause a diagnosis and treatment dilemma. Some nodular goiters may develop into the condition with features of follicular adenoma, such as the poorly developed capsule and more homogeneous follicles. A lot of diagnostic terminologies have been proposed to describe this kind of conditions, however, this would not represent a distinct entity with a low intra-observer and inter-observer consistency. Moreover, the widely used diagnostic terminologies, such as the adenoid nodules cannot reflect the biology of the disease and cause unnecessary treatment and emotional suffering. We temporarily used the terminology of “intermediate features between nodular goiter and follicular adenoma” (ING) to describe this kind of condition. In current study, we found the clinic and demographic features of ING is similar to NG. The median time from disease onset to surgery of both disease is above 1 year, representing that only the disease with surgery indication would be managed surgically. With the respect of image, both NG and ING presented a benign feature without suspicious of malignancy: 96% and 97% cases presented with a TI-RAIDs of 2, 3 or 4a by sonography in NG and ING respectively, indicating that more intensive workup unnecessary for these disease. The cytologic evaluation based on FNA samples was consistent with the sonography:1% and 0% in NG and ING respectively was suspicious of malignancy (TBSRTC category 5). Taken together, we believe NG and ING is the same entity clinically. The genetic profile regarding the major driver mutations in thyroid caner also supports this: NG and ING harbors a low frequency mutation involving RAS gene, and this is consistent with the previous findings 11 . However, BRAF V600E is considered to be the hallmark genetic abnormality of papillary thyroid cancer and was rarely found in benign nodules such as NG or so-called “adenoid nodules” 12 . We double checked the diagnosis of these cases in NG and ING with BRAF V600E mutation and no special features indicating malignancy were found 13 . Therefore, we believe this is the BRAF V600E mutation without clinical significance and requires further investigations. These comprehensive evidence from the clinical, image and genetic side, NG and ING are the same disease with variable morphologic features, and differentiation of these two diseases would provide few clinical and biologic significance. Thus, we recommend abandoning the poorly defined terminology of ING. The follicular adenoma (FA) cohort in current study presented different features. The median age of disease onset was higher than NG and ING with approximately a decade. The median lesion diameter is smaller and had a shorter time from disease onset to surgery. We believe these would be attributed to the suspicious features to proceed to the surgical intervention. However, sonography finding and diagnosis category of FA is similar to the NG/ING group and most cases were diagnosed as TI-RAIDs of 2, 3 or 4a. Only a few of FA cases (8%) would be suspicious of malignancy, which is consistent with other real world evidence 14 . On the contrary, cytologic finding of FA is more worrying than NG/ING, as 45% was diagnosed as TBSRTC category 4 and 9% was diagnosed as TBSRTC category 5, driving considerable patients to be managed surgically. Genetically, FA cohort demonstrated the classic genetic profile of follicular benign tumor: mutation involving KRAS G12C, KRAS G12V, KRAS Q61R, NRAS Q61R, HRAS Q61R, arrangement involving PAX8-PPARG and intact BRAF V600E. Interestingly, NCOA4-RET and ETV6-NTRK3 were identified in one case. As NCOA4-RET and ETV6-NTRK3 were considered to be the hallmark of thyroid malignancies, we double checked the clinical, image and pathologic archives of these two cases and did not find any features of malignancies. Patients are ongoing followed intensively. As the widely used molecular testing in thyroid nodules diagnosis, the understanding of genetic aberration is re-shaping. RET gene fusion can be found in benign nodules instead of exclusively in malignancies 15 . We believe that the clinical interpretation should be made comprehensively and determining benign or malignant disease independent of clinical information and only based on genetic testing may be harmful. Based on the evidence from the comparison of FA with NG/ING, FA and NG/ING is the two ends of the same disease spectrum, and FA may be the well-developed disease which derived from its early phase condition of NG and ING. Considering of this, it is not necessary to exclusively diagnose these disease using one of specific terminology and it is vital to accept the gray zone disease. Comprehensive evaluation and follow up is critical to manage these disease instead of choosing a diagnostic terminology in dilemma. To conclude, the current study investigated and described the clinical, image and genetic features of three groups of benign thyroid diseases: NG, ING and FA. NG/ING and FA is in the same disease spectrum and represents two ends of the spectrum of thyroid benign nodular condition. Differentiation of ING from NG has little clinical significance and would not be recommended. FA is devoid from NG/ING clinically and genetically. Interpretation of genetic abnormality should be cautious and comprehensive. Declarations Funding: This study was supported by grants from Zhejiang Provincial Health Commission, 2023 Zhejiang Provincial Medical and Health Science and Technology Plan(2023KY463). Declaration of Conflicting Interests All authors disclosed no conflicts of interest. Author contribution D.T, J.YL. and L.XM. collected the clinical data; R.DY. analyzed the clinical data; D.T. conducted the histology review, molecular assay; J.YL. analyzed the assay data; D.T, and G.ZY. developed the manuscript; All the authors reviewed and confirmed the manuscript. References Zou B, Sun L, Wang X, Chen Z. The Prevalence of Single and Multiple Thyroid Nodules and Its Association with Metabolic Diseases in Chinese: A Cross-Sectional Study. Int J Endocrinol. 2020;2020:5381012. Jiang H, Tian Y, Yan W, Kong Y, Wang H, Wang A, et al. The Prevalence of Thyroid Nodules and an Analysis of Related Lifestyle Factors in Beijing Communities. Int J Environ Res Public Health. 2016;13(4):442. Cawood TJ, Mackay GR, Hunt PJ, O'Shea D, Skehan S, Ma Y. TIRADS Management Guidelines in the Investigation of Thyroid Nodules; Illustrating the Concerns, Costs, and Performance. J Endocr Soc. 2020;4(4):bvaa031. Juhlin CC, Mete O, Baloch ZW. The 2022 WHO Classification of thyroid tumors: novel concepts in nomenclature and grading. Endocr Relat Cancer. 2022:ERC-22-0293. Holzer K, Bartsch DK. Struma nodosa [Nodular goiter]. Chirurg. 2020;91(9):712–719. Marotta V, Bifulco M, Vitale M. Significance of RAS Mutations in Thyroid Benign Nodules and Non-Medullary Thyroid Cancer. Cancers (Basel). 2021;13(15):3785. Qureshi IA, Khabaz MN, Baig M, Begum B, Abdelrehaman AS, Hussain MB. Histopathological findings in goiter: A review of 624 thyroidectomies. Neuro Endocrinol Lett. 2015;36(1):48–52. Schreiner AM, Yang GC. Adenomatoid nodules are the main cause for discrepant histology in 234 thyroid fine-needle aspirates reported as follicular neoplasm. Diagn Cytopathol. 2012;40(5):375–9. Liang J, Cai W, Feng D, Teng H, Mao F, Jiang Y, Hu S, Li X, Zhang Y, Liu B, Sun ZS. Genetic landscape of papillary thyroid carcinoma in the Chinese population. J Pathol. 2018;244(2):215–226. Cibas ES, Ali SZ. The 2017 Bethesda System for Reporting Thyroid Cytopathology. Thyroid. 2017;27(11):1341–1346. Clinkscales W, Ong A, Nguyen S, Harruff EE, Gillespie MB. Diagnostic Value of RAS Mutations in Indeterminate Thyroid Nodules. Otolaryngol Head Neck Surg. 2017;156(3):472–479. Ye L, Zhou X, Huang F, Wang W, Qi Y, Xu H, Yang S, Shen L, Fei X, Xie J, Cao M, Zhou Y, Zhu W, Wang S, Ning G, Wang W. The genetic landscape of benign thyroid nodules revealed by whole exome and transcriptome sequencing. Nat Commun. 2017;8:15533. Yu L, Ma L, Tu Q, Zhang YI, Chen Y, Yu D, Yang S. Clinical significance of BRAF V600E mutation in 154 patients with thyroid nodules. Oncol Lett. 2015;9(6):2633–2638. Zhu Y, Song Y, Xu G, Fan Z, Ren W. Causes of misdiagnoses by thyroid fine-needle aspiration cytology (FNAC): our experience and a systematic review. Diagn Pathol. 2020;15(1):1. Sapio MR, Guerra A, Marotta V, Campanile E, Formisano R, Deandrea M, Motta M, Limone PP, Fenzi G, Rossi G, Vitale M. High growth rate of benign thyroid nodules bearing RET/PTC rearrangements. J Clin Endocrinol Metab. 2011;96(6):E916-9. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4162113","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":283868563,"identity":"fcf9df8b-c509-45a4-b41d-95b94d0d261f","order_by":0,"name":"Ting Duan","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital(Affiliated People's Hospital, Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Duan","suffix":""},{"id":283868564,"identity":"fb518e60-6593-489d-8f78-fc990b670b85","order_by":1,"name":"Dingyuan Ren","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital(Affiliated People's Hospital, Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Dingyuan","middleName":"","lastName":"Ren","suffix":""},{"id":283868566,"identity":"acb5d9e7-4c0e-4c9f-a595-07d2b35b765e","order_by":2,"name":"Yanling Jin","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital(Affiliated People's Hospital, Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yanling","middleName":"","lastName":"Jin","suffix":""},{"id":283868568,"identity":"8513b6ed-1348-4a45-86ea-2f8e0d6ce113","order_by":3,"name":"Xiaming Lu","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital(Affiliated People's Hospital, Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xiaming","middleName":"","lastName":"Lu","suffix":""},{"id":283868570,"identity":"1aff1d5a-b7ca-4a4d-ab98-787bd493d414","order_by":4,"name":"Zhenying Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYBACNhiDn5n54APStEi2syUbkGadwXkeMwGiVPJJ5Bi/5qmwyzM+zGDGwFBjE03YYRI5ZtY8Z5KLzQ4zpD1gOJaW20BQC88ZM2PetgOJ2w4zHDdgbDhMgpbNzYxtEsRpYe8xfgzSsoGZmY1YLW1ljHPOJCfOOMzGbJBAjF/km5k3f3hTYZfY33/+44MPNTaEtTAwcJhJwNkJhJWDAPvjD8QpHAWjYBSMghELAMT9OeOE6V90AAAAAElFTkSuQmCC","orcid":"","institution":"Zhejiang Provincial People's Hospital(Affiliated People's Hospital, Hangzhou Medical College","correspondingAuthor":true,"prefix":"","firstName":"Zhenying","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2024-03-25 09:20:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4162113/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4162113/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53750837,"identity":"f8632fca-477c-4ff4-9f17-c8332e26692f","added_by":"auto","created_at":"2024-03-29 18:41:51","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":409252,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic profile of NG, ING and FA. \u003cstrong\u003e(A)\u003c/strong\u003e: Grid figure of genetic abnormality in NG, ING and FA. \u003cstrong\u003e(B)\u003c/strong\u003e: Representative melt plot of RET-CDC6 fusion; \u003cstrong\u003e(C)\u003c/strong\u003e: Representative melt plot of BRAF V600E wild type.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4162113/v1/aa0bfcbcd614a0eb66b019dd.jpeg"},{"id":54215787,"identity":"bec9dae9-13d0-4776-8a39-e081df4af94b","added_by":"auto","created_at":"2024-04-06 14:44:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":388102,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4162113/v1/9831e66a-93dc-4683-a9b0-71ae75d6285e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eGenetic landscape of nodular goiter, thyroid nodules with intermediate features between nodular goiter and follicular adenoma\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThyroid nodules is a common condition affecting approximately 19%-49% population in ultrasound examination\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, in which most is benign condition and 11% is malignant condition\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Nodular goiters accounts for the most common types of thyroid nodules\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, Most nodular goiters feature a mass in thyroid and can be easily identified by ultrasound examination, and will not cause a problem in the diagnosis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Further examination, such as cytology based on fine needle aspiration (FNA) and molecular testing, would be needed if some suspicious features seen such as calcification\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Follicular adenoma represents the most common thyroid benign neoplasm, and can be easily manageable surgically. The diagnosis of follicular adenoma should be based on the intensive sampling and careful examination to exclude the follicular carcinoma featuring capsule invasion and vascular invasion \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor most circumstances, nodular goiter can be easily distinguished from follicular adenoma, in which well-defined borders, well established capsules and lack of disease in the adherent thyroid tissue would be seen. Nodular goiter is lacking well-defined borders, well established capsules and imparts a diffuse disease affecting the adherent thyroid tissue. However, borderline condition demonstrating the intermediate features between nodular goiter and follicular adenoma will cause a diagnosis dilemma\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Due to lack of uniformed terminology, various of diagnosis term would be used currently, such as adenomatoid nodular goiter\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This diagnosis dilemma may mainly be attributed to the unknown pathogenesis and biology of nodular goiter and follicular adenoma, and that the differential diagnosis mainly relies on the histology observation.\u003c/p\u003e \u003cp\u003eIn recent years, molecular testing of genetic abnormalities is being widely used as an ancillary study together with FNA cytology examination to identify the malignancies\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The molecular testing detects the genetic abnormalities, such as BFAF V600E, TERT promoter mutation, and RET gene rearrangement, which are mainly found in malignancies such as papillary carcinoma. And the genetic landscape of malignancies are well identified in recent studies\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, the genetic landscape of benign thyroid nodules especially nodular goiter remains unclear, and nodular goiter are supposed not to carry any genetic abnormalities.\u003c/p\u003e \u003cp\u003eIn current studies, we report the genetic landscape of nodular goiter and thyroid nodules with intermediate features between nodular goiter and follicular adenoma using qPCR molecular test detecting BRAF, TERT, RET, RAS, PAX8 and NTRK, based on both FNA cytology samples and formalin-fixed paraffin-embedded (FFPE) samples. The genetic landscape may elaborate the genetic or biologic relationship nodular goiter and thyroid nodules with intermediate features between nodular goiter and follicular adenoma, facilitating the clinical diagnosis and management, as well as of terminology harmonization.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient and samples\u003c/h2\u003e \u003cp\u003eClinical and pathologic information of nodular goiter and thyroid nodules with intermediate features between nodular goiter and follicular adenoma were retrieved from 2020 to 2023. Pathologic diagnosis was confirmed by expert pathologist. Cases would be eligible if both fresh cytology samples based on pre-operative FNA and post-operative FFPE samples are available. Demographic and clinicopathologic information were retrieved from the medical records. Ultrasound sonography records were collected based on the Thyroid imaging reporting and data system (TI-RAIDs). This study was approved by the institution ethic review board in the Zhejiang Province People\u0026rsquo;s Hospital.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCytology and histology\u003c/h2\u003e \u003cp\u003eSmear cytology samples based on the FNA were stained with hematoxylin plus eosin (H\u0026amp;E). The review and diagnosis of cytology samples was based on the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e by expert pathologist. Tissues of nodular goiter and thyroid nodules with intermediate features between nodular goiter and follicular adenoma were intensively sampled with 5mm intervals. Slides with 4-um thickness were cut from the FFPE tissue blocks. Histology review was performed by expert pathologist.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eqPCR molecular testing\u003c/h2\u003e \u003cp\u003eThe DNA and RNA of fresh cytology samples were extracted by Fresh DNA and RNA extraction kit (TiangenCo. Ltd, Shanghai, China) and the DNA and RNA of FFPE samples were extracted by FFPE DNA and RNA extraction kit (TiangenCo. Ltd, Shanghai, China).\u003c/p\u003e \u003cp\u003eThe thyroid cancer gene mutation detection kit is used for qualitatively detecting DNA point mutations and RNA fusion mutations for 12 thyroid cancer-related genes. The target genes include 8 DNA point mutations, which are BRAF V600E mutation, TERT promoter mutation C228T/C250T, KRAS G12C/ G12V/Q61R mutation, NRAS Q61R mutation, and HRAS Q61R mutation and 4 RNA fusion mutations: CCDC6-RET fusion, NCOA4-RET fusion, PAX8-PPARG fusion, ETV6-NTRK3 fusion. In brief, qPCR reactions were performed in a total volume of 20 \u0026micro;L containing 10 \u0026micro;L of 2 \u0026times; qPCR Mix 3 \u0026micro;L of template DNA or RNA, 0.6 \u0026micro;L each of primers (each 10 \u0026micro;M), and 0.4 \u0026micro;L of probe (10 \u0026micro;M). The reaction was performed on applied biosystems 7500 real-time PCR system (ABI Life Technologies Corporation, USA). Molecular grade water was used as a negative control. The PCR reaction program was used as follows: 50\u0026deg;C for 15 min; 95\u0026deg;C for 15 min; followed by 15 cycles of 95\u0026deg;C for 15 s and 66\u0026deg;C for 40 s; and followed by 35 cycles of 95\u0026deg;C for 15 s and 60\u0026deg;C for 40 s. The data were analyzed using the 7500 software v 2.4 and the results were considered negative if cycle threshold (Ct) values were lower than cut-off.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eBaseline descriptive statistics and genetic mutation were summarized using medians (IQRs) and frequencies (percentages).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePatients and demographics\u003c/h2\u003e \u003cp\u003eOne hundred and four patients with nodular goiter (NG), 62 patients with thyroid nodules with intermediate features between nodular goiter (ING) and 28 patients with follicular adenoma (FA) were included. Most patients were young or middle-aged. The median age in the NG, ING and FA was 29 (range: 16\u0026ndash;78), 28 ( range: 17\u0026ndash;78) and 39 (range: 22\u0026ndash;79), respectively, indicating FA may occur in the older population. The male and female percentage in the NG, ING and FA was 64% vs. 36%, 54% vs. 46% and 39% vs. 61, respectively. The demographics was 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\u003eDemographic, clinical, image and cytologic features of NG, ING and FA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eING\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMedian age (range)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29 (16\u0026ndash;78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28 (17\u0026ndash;78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39 (22\u0026ndash;79)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMale\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFemale\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMedian lesion size (range, cm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.8 (1.2\u0026ndash;6.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.2 (1.1\u0026ndash;7.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.8 (1.2\u0026ndash;5.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMedian time from disease onset to surgery (range, month\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (3\u0026ndash;44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (1\u0026ndash;36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (1\u0026ndash;20)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTI-RAIDs category (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e4a\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e4b and above\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTBSRTC category\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\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\u003e45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNG: Nodular goiter; ING: thyroid nodules with intermediate features between nodular goiter; FA: follicular adenoma; TI-RAIDs: Thyroid imaging reporting and data system; TBSRTC: Bethesda System for Reporting Thyroid Cytopathology.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eClinical, image and cytologic features\u003c/h2\u003e \u003cp\u003eMedian lesion size of NG, ING and FA was 2.8cm (range: 1.2cm-6.4cm), 2.2cm (range: 1.1cm-7.4cm) and 1.8cm (range: 1.2cm-5.1cm), and there is no significant difference in the lesion size among these disease. The Median time from disease onset to surgery was 14 months (range: 3 months\u0026ndash;44 months), 12 months (range: 1 month-36 months ) and 8 (range:1 month-20 months) indicating the clinical evaluation of FA may trigger the surgery intervention comparing with the management of NG and ING. The clinical features were summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe ultrasonography finding based on the TI-RAIDs was summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Briefly, most lesions demonstrated a benign features and categories in the ultrasonography evaluation. The 4b and above suspected malignant lesion was 4%, 3% and 8% in the NG, ING and FA, respectively.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eGenetic profile\u003c/h2\u003e \u003cp\u003eThe profile of BRAF V600E mutation, TERT promoter mutation C228T/C250T, KRAS G12C/ G12V/Q61R mutation, NRAS Q61R mutation, HRAS Q61R mutation, CCDC6-RET fusion, NCOA4-RET fusion, PAX8-PPARG fusion and ETV6-NTRK3 fusion in NG, ING and FA was summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. RAS gene mutation was found in NG: KRAS G12C (2/104, 1.9%), KRAS G12V (2/104, 1.9%), KRAS Q61R (3/104, 2.9%), NRAS Q61R (2/104, 1.9%) and HRAS Q61R (2/104, 1.9%). BRAF V600E, which is considered to be the driving mutation of thyroid papillary carcinoma, was found with mutation in 2 out of 104 NGs (1.9%). We reviewed the clinical, radiologic and pathologic records and archives, and we found few indications suspicious of thyroid papillary carcinoma. The two patients have been being followed comprehensively and no recurrence has been found. Other gene are intact in NG group.\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\u003eGenetic landscape of NG, ING and FA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eNG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eING\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eFA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNO.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNO.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNO.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBRAF V600E\u003c/b\u003e\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\u003e1.9%\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\u003e1.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTERT C228T\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0%\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\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTERT C250T\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0%\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\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKRAS G12C\u003c/b\u003e\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\u003e1.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.6%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKRAS G12V\u003c/b\u003e\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\u003e1.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.1%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKRAS Q61R\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.7%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNRAS Q61R\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28.6%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHRAS Q61R\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32.1%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCCDC6-RET\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0%\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\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNCOA4-RET\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0%\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\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.6%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePAX8-PPARG\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0%\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\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.9%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eETV6-NTRK3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0%\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\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.6%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNG: Nodular goiter; ING: thyroid nodules with intermediate features between nodular goiter; FA: follicular adenoma;\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSimilarly, RAS gene mutation was found in ING: KRAS G12C (1/62, 1.6%), KRAS G12V (3/62, 4.8%), KRAS Q61R (2/62, 3.2%), NRAS Q61R (6/62, 9.7%) and HRAS Q61R (6/62, 9.7%). One BRAF gene (1/62, 1.6%) was also found in ING. We reviewed the clinical, radiologic and pathologic records and archives, and we found few indications suspicious of thyroid papillary carcinoma. This patient has been being followed comprehensively and no recurrence has been found. Other gene are intact in ING group.\u003c/p\u003e \u003cp\u003eThe genetic landscape of FA is remarkable different from the one in the NG and ING. FA harbored the mutation of KRAS G12C (1/28, 3.6%), KRAS G12V (2/28, 7.1%), KRAS Q61R (3/28, 10.7%), NRAS Q61R (8/28,28.6%), and HRAS Q61R (9/28, 32.1%). Besides the SNV above, PAX8-PPARG fusion was identified in 5 out of 28 (17.9%) FA patients along with NCOA4-RET fusion (1/28, 3.6%) and ETV6-NTRK3 fusion (1/28, 3.6%). All the FA tissue were intensively sampled to rule out the possibility of malignancies. No recurrence or metastasis has been found.\u003c/p\u003e \u003cp\u003eThe grid figure of NG, ING and FA was provide in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and the representative picture of qPCR assay to identify the mutation or fusion were provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNodular goiters (NG) and follicular adenoma (FA) represent the most common thyroid nodules, and most disease would not cause a diagnosis and treatment dilemma. Some nodular goiters may develop into the condition with features of follicular adenoma, such as the poorly developed capsule and more homogeneous follicles. A lot of diagnostic terminologies have been proposed to describe this kind of conditions, however, this would not represent a distinct entity with a low intra-observer and inter-observer consistency. Moreover, the widely used diagnostic terminologies, such as the adenoid nodules cannot reflect the biology of the disease and cause unnecessary treatment and emotional suffering. We temporarily used the terminology of \u0026ldquo;intermediate features between nodular goiter and follicular adenoma\u0026rdquo; (ING) to describe this kind of condition.\u003c/p\u003e \u003cp\u003eIn current study, we found the clinic and demographic features of ING is similar to NG. The median time from disease onset to surgery of both disease is above 1 year, representing that only the disease with surgery indication would be managed surgically. With the respect of image, both NG and ING presented a benign feature without suspicious of malignancy: 96% and 97% cases presented with a TI-RAIDs of 2, 3 or 4a by sonography in NG and ING respectively, indicating that more intensive workup unnecessary for these disease. The cytologic evaluation based on FNA samples was consistent with the sonography:1% and 0% in NG and ING respectively was suspicious of malignancy (TBSRTC category 5). Taken together, we believe NG and ING is the same entity clinically. The genetic profile regarding the major driver mutations in thyroid caner also supports this: NG and ING harbors a low frequency mutation involving RAS gene, and this is consistent with the previous findings\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, BRAF V600E is considered to be the hallmark genetic abnormality of papillary thyroid cancer and was rarely found in benign nodules such as NG or so-called \u0026ldquo;adenoid nodules\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. We double checked the diagnosis of these cases in NG and ING with BRAF V600E mutation and no special features indicating malignancy were found\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Therefore, we believe this is the BRAF V600E mutation without clinical significance and requires further investigations. These comprehensive evidence from the clinical, image and genetic side, NG and ING are the same disease with variable morphologic features, and differentiation of these two diseases would provide few clinical and biologic significance. Thus, we recommend abandoning the poorly defined terminology of ING.\u003c/p\u003e \u003cp\u003eThe follicular adenoma (FA) cohort in current study presented different features. The median age of disease onset was higher than NG and ING with approximately a decade. The median lesion diameter is smaller and had a shorter time from disease onset to surgery. We believe these would be attributed to the suspicious features to proceed to the surgical intervention. However, sonography finding and diagnosis category of FA is similar to the NG/ING group and most cases were diagnosed as TI-RAIDs of 2, 3 or 4a. Only a few of FA cases (8%) would be suspicious of malignancy, which is consistent with other real world evidence\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. On the contrary, cytologic finding of FA is more worrying than NG/ING, as 45% was diagnosed as TBSRTC category 4 and 9% was diagnosed as TBSRTC category 5, driving considerable patients to be managed surgically. Genetically, FA cohort demonstrated the classic genetic profile of follicular benign tumor: mutation involving KRAS G12C, KRAS G12V, KRAS Q61R, NRAS Q61R, HRAS Q61R, arrangement involving PAX8-PPARG and intact BRAF V600E. Interestingly, NCOA4-RET and ETV6-NTRK3 were identified in one case. As NCOA4-RET and ETV6-NTRK3 were considered to be the hallmark of thyroid malignancies, we double checked the clinical, image and pathologic archives of these two cases and did not find any features of malignancies. Patients are ongoing followed intensively. As the widely used molecular testing in thyroid nodules diagnosis, the understanding of genetic aberration is re-shaping. RET gene fusion can be found in benign nodules instead of exclusively in malignancies\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. We believe that the clinical interpretation should be made comprehensively and determining benign or malignant disease independent of clinical information and only based on genetic testing may be harmful. Based on the evidence from the comparison of FA with NG/ING, FA and NG/ING is the two ends of the same disease spectrum, and FA may be the well-developed disease which derived from its early phase condition of NG and ING. Considering of this, it is not necessary to exclusively diagnose these disease using one of specific terminology and it is vital to accept the gray zone disease. Comprehensive evaluation and follow up is critical to manage these disease instead of choosing a diagnostic terminology in dilemma.\u003c/p\u003e \u003cp\u003eTo conclude, the current study investigated and described the clinical, image and genetic features of three groups of benign thyroid diseases: NG, ING and FA. NG/ING and FA is in the same disease spectrum and represents two ends of the spectrum of thyroid benign nodular condition. Differentiation of ING from NG has little clinical significance and would not be recommended. FA is devoid from NG/ING clinically and genetically. Interpretation of genetic abnormality should be cautious and comprehensive.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was supported by grants from Zhejiang Provincial Health Commission, 2023 Zhejiang Provincial Medical and Health Science and Technology Plan(2023KY463).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Conflicting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors disclosed no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.T, J.YL. and L.XM. collected the clinical data;\u003c/p\u003e\n\u003cp\u003eR.DY. analyzed the clinical data;\u003c/p\u003e\n\u003cp\u003eD.T. conducted the histology review, molecular assay;\u003c/p\u003e\n\u003cp\u003eJ.YL. analyzed the assay data;\u003c/p\u003e\n\u003cp\u003eD.T, and G.ZY. developed the manuscript;\u003c/p\u003e\n\u003cp\u003eAll the authors reviewed and confirmed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZou B, Sun L, Wang X, Chen Z. The Prevalence of Single and Multiple Thyroid Nodules and Its Association with Metabolic Diseases in Chinese: A Cross-Sectional Study. Int J Endocrinol. 2020;2020:5381012.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang H, Tian Y, Yan W, Kong Y, Wang H, Wang A, et al. The Prevalence of Thyroid Nodules and an Analysis of Related Lifestyle Factors in Beijing Communities. Int J Environ Res Public Health. 2016;13(4):442.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCawood TJ, Mackay GR, Hunt PJ, O'Shea D, Skehan S, Ma Y. TIRADS Management Guidelines in the Investigation of Thyroid Nodules; Illustrating the Concerns, Costs, and Performance. J Endocr Soc. 2020;4(4):bvaa031.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJuhlin CC, Mete O, Baloch ZW. The 2022 WHO Classification of thyroid tumors: novel concepts in nomenclature and grading. Endocr Relat Cancer. 2022:ERC-22-0293.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolzer K, Bartsch DK. Struma nodosa [Nodular goiter]. Chirurg. 2020;91(9):712\u0026ndash;719.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarotta V, Bifulco M, Vitale M. Significance of RAS Mutations in Thyroid Benign Nodules and Non-Medullary Thyroid Cancer. Cancers (Basel). 2021;13(15):3785.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQureshi IA, Khabaz MN, Baig M, Begum B, Abdelrehaman AS, Hussain MB. Histopathological findings in goiter: A review of 624 thyroidectomies. Neuro Endocrinol Lett. 2015;36(1):48\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchreiner AM, Yang GC. Adenomatoid nodules are the main cause for discrepant histology in 234 thyroid fine-needle aspirates reported as follicular neoplasm. Diagn Cytopathol. 2012;40(5):375\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang J, Cai W, Feng D, Teng H, Mao F, Jiang Y, Hu S, Li X, Zhang Y, Liu B, Sun ZS. Genetic landscape of papillary thyroid carcinoma in the Chinese population. J Pathol. 2018;244(2):215\u0026ndash;226.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCibas ES, Ali SZ. The 2017 Bethesda System for Reporting Thyroid Cytopathology. Thyroid. 2017;27(11):1341\u0026ndash;1346.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClinkscales W, Ong A, Nguyen S, Harruff EE, Gillespie MB. Diagnostic Value of RAS Mutations in Indeterminate Thyroid Nodules. Otolaryngol Head Neck Surg. 2017;156(3):472\u0026ndash;479.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe L, Zhou X, Huang F, Wang W, Qi Y, Xu H, Yang S, Shen L, Fei X, Xie J, Cao M, Zhou Y, Zhu W, Wang S, Ning G, Wang W. The genetic landscape of benign thyroid nodules revealed by whole exome and transcriptome sequencing. Nat Commun. 2017;8:15533.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu L, Ma L, Tu Q, Zhang YI, Chen Y, Yu D, Yang S. Clinical significance of BRAF V600E mutation in 154 patients with thyroid nodules. Oncol Lett. 2015;9(6):2633\u0026ndash;2638.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Y, Song Y, Xu G, Fan Z, Ren W. Causes of misdiagnoses by thyroid fine-needle aspiration cytology (FNAC): our experience and a systematic review. Diagn Pathol. 2020;15(1):1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSapio MR, Guerra A, Marotta V, Campanile E, Formisano R, Deandrea M, Motta M, Limone PP, Fenzi G, Rossi G, Vitale M. High growth rate of benign thyroid nodules bearing RET/PTC rearrangements. J Clin Endocrinol Metab. 2011;96(6):E916-9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Thyroid nodules, nodular goiters, follicular adenomas, genetic profile","lastPublishedDoi":"10.21203/rs.3.rs-4162113/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4162113/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNodular goiters (NG) and follicular adenoma (FA) are common thyroid benign nodules and thyroid nodules with intermediate features between nodular goiter and follicular (ING) is used to describe the disease with borderline features. The genetic landscape of these three diseases is poorly investigated comparatively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical information of NG, ING and FA was retrieved and reviewed. Cytology and histology of the pathologic archives were reviewed to confirm the diagnosis. DNA and RNA were extracted to be submitted to qPCR assay to detect BRAF, TERT, RET, RAS, PAX8 and NTRK genetic aberration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe demographic, clinical, image and cytologic features in NG and ING are similar. Most disease presents a benign clinical and cytologic behavior, causing no diagnostic difficulty. Low frequency (\u0026lt; 10%) RAS gene was found in NG and ING. Patients with FA are older than whom with NG or ING. FA lesion size is smaller than ones in NG or ING. Although sonography demonstrated most FA nodules as benign category, the cytologic evaluation demonstrated a considerable percentage (45%) of atypia disease. Additionally, much higher frequency of RAS gene abnormality has been found in FA. And PAX8-PPARG gene is found in 17.9% FA and in no NG/ING cases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNG/ING and FA is in the same spectrum and represents two ends of the spectrum of thyroid benign nodular condition. Differentiation of ING from NG has little clinical significance and would not be recommended. FA is devoid from NG/ING clinically and genetically. Interpretation of genetic abnormality should be cautious.\u003c/p\u003e","manuscriptTitle":"Genetic landscape of nodular goiter, thyroid nodules with intermediate features between nodular goiter and follicular adenoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-29 18:41:46","doi":"10.21203/rs.3.rs-4162113/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c5aa0a30-3ed9-48fb-a6f5-6b7496f5195c","owner":[],"postedDate":"March 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-06T14:44:24+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-29 18:41:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4162113","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4162113","identity":"rs-4162113","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}