The Morphologic and Immunohistochemical Profiling of Thyroid Nodules Submitted to Thermal Ablation  Avoids a Misdiagnosis of Carcinoma

The Morphologic and Immunohistochemical Profiling of Thyroid Nodules Submitted to Thermal Ablation Avoids a Misdiagnosis of Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Morphologic and Immunohistochemical Profiling of Thyroid Nodules Submitted to Thermal Ablation Avoids a Misdiagnosis of Carcinoma Pietro Tralongo, Fernanda Russotto, Valeria Zuccalà, Vincenzo Fiorentino, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8712675/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 Thermal ablation (TA) is a minimally invasive treatment for benign thyroid nodules, but it induces histological changes that can mimic malignancy. This study expands our previous series to characterize the histological and immunohistochemical changes occurring in these nodules after TA and to refine diagnostic criteria to prevent misdiagnosis of benign lesions as carcinoma. Twelve surgically excised thyroid nodules previously treated with laser thermal ablation were retrospectively analyzed. Histopathological examination focused on architecture, nuclear features, capsule characteristics, and degenerative changes. Immunohistochemistry was performed for galectin-3 (Gal-3), HBME-1, BRAF V600E, p53, and Ki-67. All nodules showed a fibrous capsule. Architecture was predominantly follicular (75%) or solid (25%). Sclerosis (83.3%) and subcapsular hemorrhage (91.7%) were common; necrosis and ischemia were rare. No true capsular or vascular invasion was observed. All cases were negative for Gal-3, and BRAF V600E. Eleven nodules were HBME-1-negative; one showed focal weak positivity; p53 was hyperexpressed in 3 cases. Ki-67 was consistently below 3%. TA induces characteristic degenerative and reactive changes that may simulate malignancy, including fibrosis, hemorrhage, and nuclear atypia. The consistent negativity of malignancy-associated immunohistochemical markers, together with low proliferative activity, confirms the benign nature of these alterations and supports the use of an integrated morphologic–immunohistochemical approach for accurate diagnosis. This expanded analysis reinforces that laser ablation induces reproducible histologic alterations in benign thyroid nodules that can simulate malignancy. Awareness of these changes, combined with a restricted immunohistochemical panel, allows confident recognition of treatment-related artifacts and prevents overdiagnosis of carcinoma. thermal ablation thyroid nodules laser ablation immunohistochemistry differential diagnosis Figures Figure 1 Figure 2 1. Introduction Nodular thyroid disease is a common benign condition with higher prevalence in women, iodine-deficient areas, and following radiation exposure [ 1 , 2 ]. The estimated prevalence widely ranges from 2–6% by palpation, 19–35% by ultrasonography, and up to 8–65% in autopsy series [ 1 ]. Most benign thyroid nodules (BTNs) are asymptomatic, but large or compressive lesions may provoke dysphagia, dyspnea, or cosmetic issues, thus requiring intervention [ 1 – 3 ]. The diagnostic confirmation is based on ultrasound-guided FNAC, according to the Bethesda system categorization. Treatment options include pharmaceutical suppression, usually not effective; surgical thyroidectomy; and TA methods, namely LA, radiofrequency ablation (RFA), and microwave ablation (MWA) [ 1 – 4 ]. TA causes controlled coagulative necrosis through hyperthermia, followed by fibrosis and partial reabsorption, and has better cosmetic results, quicker recovery, and less morbidity compared with surgery [ 2 , 5 – 7 ]. Long-term studies confirm volume reductions of 50–80% at 3–5 years, with regrowth rates of 4–20% often due to incomplete marginal ablation [ 8 , 9 ]. Indications include symptomatic solid BTNs (> 20 mL), autonomous "hot" nodules not suitable for radioiodine, or palliative approach to low-risk recurrent PTMC [ 1 , 10 , 11 ]. Recent guidelines from the American Thyroid Association, Korean Society of Thyroid Radiology, and European Thyroid Association support the use of TA for selected benign and low-risk malignant nodules [ 3 , 12 , 13 ]. Surgical excision may be indicated in cases of non-responders or persistent symptoms, where changes attributed to TA, such as fibrosis, subcapsular hemorrhage, nuclear clearing, architectural distortion, and pseudocapsular invasion, can simulate follicular neoplasm or carcinoma, leading to difficult diagnosis [ 1 , 14 – 17 ]. Artifacts resulting from thermal coagulation include the subsequent coagulative necrosis, sclerosis, and metaplastic oncocytic changes [ 18 , 19 ]. Ancillary IHC for Gal-3, HBME-1, BRAF V600E, and p53, along with Ki-67 proliferation indexing, is helpful in differentiating these artifacts from neoplastic progression [ 1 , 20 – 22 ]. Our group has previously reported histological findings in seven TA-treated nodules, drawing attention to diagnostic pitfalls and IHC utility [ 1 ]. The current study extends that series to a total of twelve cases, affording a much stronger characterization of morphologic and immunohistochemical features following laser ablation, with comparisons to emerging literature on post-TA histology [ 14 , 15 , 23 , 24 ]. 2. Materials and Methods Between 2018 and 2024, we retrieved from the pathology archives of the University of Messina and “San Vincenzo” Hospital in Taormina (Italy) a series of twelve thyroid nodules that had undergone percutaneous laser ablation (LA) before surgical removal. These cases were selected according to strict criteria: all nodules had shown a definitively benign cytological diagnosis (Bethesda category II) on fine-needle aspiration cytology performed before ablation; complete clinical, ultrasonographic, and follow-up imaging data had to be available; and the final surgical histology had to confirm the benign nature of the lesion. All cases with a history of thyroid or extrathyroidal malignancies, incomplete documentation, or nodules treated with other thermal ablation techniques (e.g., radiofrequency or microwave ablation) were excluded. All hematoxylin–eosin-stained slides available from the resection specimens were independently reviewed by three experienced pathologists blind to the clinical and radiological data. Review was specifically directed at a number of histological parameters potentially modified by the thermal injury, namely: predominant architectural pattern (follicular versus solid/ trabecular), nuclear appearance (clear versus dark chromatin), nodule capsule integrity, presence and extension of degenerative phenomena (coagulative necrosis, fibrosis/sclerosis, hemorrhage, ischemic changes), signs of capsular or vascular invasion, and oncocytic metaplasia. Immunohistochemical studies were performed on 4-µm-thick sections of formalin-fixed, paraffin-embedded tissue to further characterize the ablated nodules and search for possible subtle signs of malignancy or proliferative activity masked by thermal effects. On a Ventana Benchmark automated platform, staining was performed with the following panel of antibodies: Galectin-3 (Gal-3, rabbit polyclonal, 1:200, overnight incubation), HBME-1 (mouse monoclonal, 1:100), BRAF V600E mutation-specific antibody (mouse monoclonal, VE1 clone, 1:100), p53 (mouse monoclonal, DO-7 clone, 1:50), and Ki-67 (mouse monoclonal, MIB-1 clone, 1:200). Heat-induced epitope retrieval was done in EDTA buffer at pH 8.0 for 30 minutes. Immunoreactivity was semi-quantitatively categorized as negative ( 20%). The proliferative fraction was also estimated more precisely by calculating the Ki-67 labeling index in ten randomly selected high-power fields with the aid of digital image analysis software (Aperio ScanScope). Interobserver reproducibility for the main morphological features was determined using Cohen's kappa coefficient. Calculated values > 0.80 were considered indicative of excellent agreement. The study was conducted in accordance with the Declaration of Helsinki and approved by the institutional ethics committee, under Protocol No. 2024/05. A waiver of informed consent was provided in light of the retrospective design and use of fully anonymized archival material. 3. Results The patient cohort consisted of 12 individuals with a mean age of 54 years, reflecting a relatively mature demographic typical for thyroid nodule presentations. There was a clear female predominance, with women comprising 66.7% of the group (n = 8/12) and men accounting for the remaining 33.3% (n = 4/12). The nodules themselves were solitary in the majority of cases at 66.7% (n = 8), while multinodular configurations were observed in 33.3% (n = 4); notably, a mean pre-TA volume of 25.4 mL (range 18–42 mL) and a post-TA surgical interval of 6–18 months (mean 10.2 months) were observed. Morphologically (as detailed in Table 1 and shown in Fig. 1 ), a fibrous capsule was present in nearly all cases at 91.7% (n = 11/12; mean thickness 0.5–1.2 mm), with only one instance lacking this feature (8.3%). Architectural patterns leaned heavily toward follicular structures in 83.3% of cases (n = 10/12), contrasted by solid arrangements in the remaining 16.7% (n = 2/12). Nuclear characteristics showed dark (basophilic, uniform) nuclei predominating in 66.7% (n = 8/12), while clear (pale, overlapping) nuclei appeared in 33.3% (n = 4/12). Degenerative alterations were common, including subcapsular hemorrhage in 91.7% (n = 11/12), often with evidence of organization and hemosiderin deposition; sclerosis or fibrosis in 75% (n = 9/12), typically zonal and proximate to the ablation site; focal ischemia marked by hyalinized vessels in 16.7% (n = 2/12); and coagulative necrosis, which was focal and limited to just 8.3% (n = 1/12). Importantly, there were no instances of true capsular invasion (0%) or vascular invasion (0%), underscoring the benign nature of these post-TA changes. Oncocytic metaplasia, characterized by eosinophilic and granular cytoplasm affecting 10–25% of cells, was evenly split at 50% prevalence (n = 6/12), often concentrated peripherally. Immunohistochemical (IHC) profiling was performed as shown in Table 2 (see also Fig. 2 ). Consistent negativity among all cases was noted for the following: Galectin-3 (100%, n = 12/12) and BRAF V600E mutation, consistent with non-neoplastic profiles. HBME-1 negativity was seen in 91.7% (n = 11/12), with only one case showing focal and weak membranous positivity (< 10% of cells) in microfollicles with atypia close to the ablation zone (8.3%). p53 positivity was identified in a quarter of cases (25%, n = 3/12), while the majority were negative (75%, n = 9/12). Proliferative activity, as determined by MIB-1 (Ki-67), was low (< 3%) in each case (100%, n = 12/12). Interobserver agreement was excellent with kappa values of 0.85 for morphology and 0.92 for IHC interpretation. These findings represent an extension of our previously reported series of seven cases [ 1 ], showing slightly increased rates of hemorrhage (91.7% vs. 85.7%) and a balanced prevalence of oncocytic changes (50% vs. 57.1%), perhaps due to an increase in sample size and possibly diverse post-treatment duration after TA, emphasizing the dynamic degeneration spectrum within ablated thyroid nodules. Table 1 Patients’ characteristics and nodules’ morphological features (N = 12) Patients' features Age, mean Gender n (%) Male Female Architecture n (%) Follicular Solid Nuclei n (%) Dark Clear Number of nodules n (%) Single nodule Plurinodular Capsule n (%) Yes No Necrosis n (%) Yes No Fibrosis n (%) Yes No Hemorrhage n (%) Yes No Ischemia n (%) Yes No Capsular invasion n (%) Yes No Vascular invasion n (%) Yes No Presence of oncocytic cells n (%) Yes No Table 2 Immunohistochemical profile (N = 12) Markers Results Total n.12 (%) Galectin-3 Positive 0 Negative 12 (100%) HBME-1 Positive 1 (8.3%) Negative 11 (91.7%) p53 Positive 3 (25%) Negative 9 (75%) BRAF-V600E Positive 0 Negative 12 (100%) MIB-1 (Ki-67) < 3% 12 (100%) 4. Discussion The current series of twelve cytologically benign thyroid nodules subjected to laser ablation confirms, refines, and significantly expands our initial seven-case experience [ 1 ] while demonstrating near-complete histological overlap with post-ablation changes described across the thermal energy modalities of radiofrequency, microwave, and HIFU [ 6 , 14 – 16 , 23 – 26 ]. The most consistent and diagnostically impactful changes remain the formation of a well-defined fibrous capsule (in 91.7% of cases, n = 11/12), prominent subcapsular hemorrhage (91.7%, n = 11/12, often with hemosiderin-laden macrophages indicating organization), and dense zonal fibrosis/sclerosis centered on the ablation site (75%, n = 9/12). These features represent the stereotypical repair response to laser-induced coagulative necrosis and are virtually indistinguishable from those observed following RFA or MWA [ 14 – 16 , 23 – 25 ]. Architectural rearrangement toward solid or microfollicular patterns (16.7%, n = 2/12 predominantly solid; the remainder retaining a follicular pattern) combined with nuclear clearing (33.3%, n = 4/12) can raise concern for an encapsulated follicular neoplasm or follicular variant of papillary thyroid carcinoma. However, several reproducible clues reliably indicate a reactive, post-ablative process rather than true neoplasia: (i) strict zonal distribution of atypical features, with maximum intensity near the ablation center and gradual fading toward the periphery; (ii) complete absence of genuine capsular or vascular invasion (0% in all cases), despite occasional pseudoinvasion of the fibrous capsule by reactive follicles or hemorrhage; (iii) presence of oncocytic metaplasia in half of the cases (50%, n = 6/12), typically peripheral and affecting 10–30% of cells—a well-recognized reactive phenomenon following thermal injury [ 1 , 15 , 18 ]; and (iv) negligible mitotic activity and uniformly low Ki-67 labeling index (< 3% in 100% of cases). Immunohistochemistry provides critical reassurance. Complete negativity for galectin-3 (100%), BRAF V600E (100%), and low proliferative activity (Ki-67 < 3% in all cases) strongly supports benignancy, since these markers maintain high specificity for malignancy even in thermally altered tissue [ 20 – 22 ]. The single case with focal, weak HBME-1 membranous positivity (8.3%, < 10% of cells in microfollicular areas adjacent to the ablation zone) mirrors sporadic reactivity described after RFA and MWA [ 15 , 21 , 24 ] and does not correlate with invasive behavior or malignant transformation. p53 positivity, observed in 25% of cases (n = 3/12), was weak and limited, possibly consistent with stress-related rather than mutational overexpression. [ 27 ] Such comparisons across thermal ablation techniques are remarkably consistent. Thus, identical degenerative patterns after microwave ablation were documented by both Spiezia et al. [ 14 ] and Romanelli et al. [ 24 ]; Bernardi et al. [ 15 ] underscored the finding of pseudoinvasion as a common RFA-related mimic of malignancy; and Pacella et al. [ 26 ], in the largest published surgical series after laser ablation to date, emphasized that diagnostic errors were completely eliminated by familiarity with these changes combined with a limited IHC panel. Our current cohort reinforces the notion that the histological response is largely modality-independent, reflecting a shared final common pathway of thermal coagulative necrosis followed by organized repair. The clinical implications are impactful: failure to recognize these pseudomalignant changes carries the risk of overdiagnosis of carcinoma with possible unnecessary completion thyroidectomy, central-neck dissection, and radioiodine therapy, with attendant lifelong morbidity. In turn, true malignant transformation after ablation of cytologically benign nodules remains exceedingly rare; indeed, the clean IHC profile further reduces this already low theoretical risk. Study limitations include the retrospective design, relatively modest sample size (although still among the largest surgical series published after laser ablation), and inclusion of laser ablation only, which precludes direct head-to-head histological comparison with RFA or MWA in the same patients. However, the striking uniformity of findings across different energy sources in the literature strongly supports the broad applicability of these observations to all thermal ablation techniques currently in clinical use. 5. Conclusions Laser thermal ablation of benign thyroid nodules causes distinctive and reproducible histological changes, including dense fibrosis, subcapsular hemorrhage, pseudocapsular invasion, and oncocytic metaplasia, which can mimic malignancy. The combination of judicious morphological assessment with a limited immunohistochemical panel (Gal-3, HBME-1, BRAF V600E, p53, Ki-67) is a dependable way to distinguish treatment-related changes from a real neoplastic transformation. When pathologists interpret resected specimens after thermal ablation, they need to recognize these features to avoid an overdiagnosis of carcinoma and ensure the most appropriate patient management. Declarations Declaration of interest All authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Human Ethics and Consent to Participate declarations Ethical review and approval were waived for this study due to its retrospective nature. All patient data were collected anonymously according to the Declaration of Helsinki, and the study adhered to Good Clinical Practice guidelines. Written informed consent, as part of the routine diagnosis and treatment procedures, was obtained from patients or their guardians according to the Declaration of Helsinki, and the study adhered to Good Clinical Practice guidelines. Funding This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Author Contribution Conceptualization: P. T. and G.F.; Methodology: V.Z., V.F., E.M., and S.R.; Formal analysis and investigation: ; Writing - original draft preparation: P.T.; Writing - review and editing: E.D.R., M.M., and G.F.; Resources: F.R., M.G.M., M. F., L.P., W.G., G.R., M.B., and E.G.; Supervision: M.M. and G.F. . References Russotto, F., Fiorentino, V., Pizzimenti, C., et al. 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Clinical & developmental immunology, 2013, 846584. https://doi.org/10.1155/2013/846584 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-8712675","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":585290503,"identity":"23ad6e76-2b19-4742-bb4d-d355a51a8a6f","order_by":0,"name":"Pietro 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Messina","correspondingAuthor":false,"prefix":"","firstName":"Emanuela","middleName":"","lastName":"Germanà","suffix":""},{"id":585290519,"identity":"04d890ca-e85f-4566-98f9-385f50a03be3","order_by":11,"name":"Emilia Magliolo","email":"","orcid":"","institution":"“San Vincenzo” Hospital","correspondingAuthor":false,"prefix":"","firstName":"Emilia","middleName":"","lastName":"Magliolo","suffix":""},{"id":585290520,"identity":"c9faa339-9352-48e9-8291-d8baf361582a","order_by":12,"name":"Serenella Ristagno","email":"","orcid":"","institution":"“San Vincenzo” Hospital","correspondingAuthor":false,"prefix":"","firstName":"Serenella","middleName":"","lastName":"Ristagno","suffix":""},{"id":585290523,"identity":"ae5174bb-51bd-464a-add5-f0d1c5ad37b7","order_by":13,"name":"Esther Diana Rossi","email":"","orcid":"","institution":"Catholic University of the Sacred Heart","correspondingAuthor":false,"prefix":"","firstName":"Esther","middleName":"Diana","lastName":"Rossi","suffix":""},{"id":585290525,"identity":"6f21d673-efea-44cd-84cc-f2e8f99ed286","order_by":14,"name":"Maurizio Martini","email":"","orcid":"","institution":"University of Messina","correspondingAuthor":false,"prefix":"","firstName":"Maurizio","middleName":"","lastName":"Martini","suffix":""},{"id":585290528,"identity":"f1fd8b06-11ed-4fa6-9c2e-9e29c81f64e0","order_by":15,"name":"Guido Fadda","email":"","orcid":"","institution":"University of Messina","correspondingAuthor":false,"prefix":"","firstName":"Guido","middleName":"","lastName":"Fadda","suffix":""}],"badges":[],"createdAt":"2026-01-27 16:24:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8712675/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8712675/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101874471,"identity":"65c32da7-a1cb-433f-8758-c906d487cafd","added_by":"auto","created_at":"2026-02-04 13:58:40","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3156188,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological findings of TA-treated thyroid nodules showing the presence of clear nuclei ((A), hematoxylin and eosin staining, 100× magnification), a fibrous capsule with follicular architecture ((B), hematoxylin and eosin staining, 50× magnification), oncocytic cells ((C), hematoxylin and eosin staining, 200× magnification), sclerosis with occasional presence of multinucelated giant cells ((D), hematoxylin and eosin staining, 100× magnification). TA: thermal ablation\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8712675/v1/75832dc861fc08f103ef8ec8.jpeg"},{"id":101874470,"identity":"7833cd22-3482-49e4-940e-de18989fd23a","added_by":"auto","created_at":"2026-02-04 13:58:40","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":872025,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of absent expression of Galectin-3 ((A), 200× magnification; scale bar, 84 µm). Hyperexpression of p53 was observed in a subset of cases ((B), 200× magnification; scale bar, 42 µm). TA: thermal ablation.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8712675/v1/d7b24ff939c7e78baf3b0dde.jpeg"},{"id":104834888,"identity":"d23c2289-960c-499a-821b-7f2122eb9d38","added_by":"auto","created_at":"2026-03-17 17:34:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4598887,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8712675/v1/2390d7a1-08a9-4b08-82a8-9bc92240aa24.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Morphologic and Immunohistochemical Profiling of Thyroid Nodules Submitted to Thermal Ablation Avoids a Misdiagnosis of Carcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNodular thyroid disease is a common benign condition with higher prevalence in women, iodine-deficient areas, and following radiation exposure [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The estimated prevalence widely ranges from 2\u0026ndash;6% by palpation, 19\u0026ndash;35% by ultrasonography, and up to 8\u0026ndash;65% in autopsy series [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Most benign thyroid nodules (BTNs) are asymptomatic, but large or compressive lesions may provoke dysphagia, dyspnea, or cosmetic issues, thus requiring intervention [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The diagnostic confirmation is based on ultrasound-guided FNAC, according to the Bethesda system categorization. Treatment options include pharmaceutical suppression, usually not effective; surgical thyroidectomy; and TA methods, namely LA, radiofrequency ablation (RFA), and microwave ablation (MWA) [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTA causes controlled coagulative necrosis through hyperthermia, followed by fibrosis and partial reabsorption, and has better cosmetic results, quicker recovery, and less morbidity compared with surgery [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Long-term studies confirm volume reductions of 50\u0026ndash;80% at 3\u0026ndash;5 years, with regrowth rates of 4\u0026ndash;20% often due to incomplete marginal ablation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Indications include symptomatic solid BTNs (\u0026gt;\u0026thinsp;20 mL), autonomous \"hot\" nodules not suitable for radioiodine, or palliative approach to low-risk recurrent PTMC [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Recent guidelines from the American Thyroid Association, Korean Society of Thyroid Radiology, and European Thyroid Association support the use of TA for selected benign and low-risk malignant nodules [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSurgical excision may be indicated in cases of non-responders or persistent symptoms, where changes attributed to TA, such as fibrosis, subcapsular hemorrhage, nuclear clearing, architectural distortion, and pseudocapsular invasion, can simulate follicular neoplasm or carcinoma, leading to difficult diagnosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Artifacts resulting from thermal coagulation include the subsequent coagulative necrosis, sclerosis, and metaplastic oncocytic changes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Ancillary IHC for Gal-3, HBME-1, BRAF V600E, and p53, along with Ki-67 proliferation indexing, is helpful in differentiating these artifacts from neoplastic progression [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur group has previously reported histological findings in seven TA-treated nodules, drawing attention to diagnostic pitfalls and IHC utility [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The current study extends that series to a total of twelve cases, affording a much stronger characterization of morphologic and immunohistochemical features following laser ablation, with comparisons to emerging literature on post-TA histology [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eBetween 2018 and 2024, we retrieved from the pathology archives of the University of Messina and \u0026ldquo;San Vincenzo\u0026rdquo; Hospital in Taormina (Italy) a series of twelve thyroid nodules that had undergone percutaneous laser ablation (LA) before surgical removal. These cases were selected according to strict criteria: all nodules had shown a definitively benign cytological diagnosis (Bethesda category II) on fine-needle aspiration cytology performed before ablation; complete clinical, ultrasonographic, and follow-up imaging data had to be available; and the final surgical histology had to confirm the benign nature of the lesion. All cases with a history of thyroid or extrathyroidal malignancies, incomplete documentation, or nodules treated with other thermal ablation techniques (e.g., radiofrequency or microwave ablation) were excluded.\u003c/p\u003e \u003cp\u003eAll hematoxylin\u0026ndash;eosin-stained slides available from the resection specimens were independently reviewed by three experienced pathologists blind to the clinical and radiological data. Review was specifically directed at a number of histological parameters potentially modified by the thermal injury, namely: predominant architectural pattern (follicular versus solid/ trabecular), nuclear appearance (clear versus dark chromatin), nodule capsule integrity, presence and extension of degenerative phenomena (coagulative necrosis, fibrosis/sclerosis, hemorrhage, ischemic changes), signs of capsular or vascular invasion, and oncocytic metaplasia.\u003c/p\u003e \u003cp\u003eImmunohistochemical studies were performed on 4-\u0026micro;m-thick sections of formalin-fixed, paraffin-embedded tissue to further characterize the ablated nodules and search for possible subtle signs of malignancy or proliferative activity masked by thermal effects. On a Ventana Benchmark automated platform, staining was performed with the following panel of antibodies: Galectin-3 (Gal-3, rabbit polyclonal, 1:200, overnight incubation), HBME-1 (mouse monoclonal, 1:100), BRAF V600E mutation-specific antibody (mouse monoclonal, VE1 clone, 1:100), p53 (mouse monoclonal, DO-7 clone, 1:50), and Ki-67 (mouse monoclonal, MIB-1 clone, 1:200). Heat-induced epitope retrieval was done in EDTA buffer at pH 8.0 for 30 minutes. Immunoreactivity was semi-quantitatively categorized as negative (\u0026lt;\u0026thinsp;5% of cells), focal/weak (5\u0026ndash;20%), or diffuse/strong (\u0026gt;\u0026thinsp;20%). The proliferative fraction was also estimated more precisely by calculating the Ki-67 labeling index in ten randomly selected high-power fields with the aid of digital image analysis software (Aperio ScanScope).\u003c/p\u003e \u003cp\u003eInterobserver reproducibility for the main morphological features was determined using Cohen's kappa coefficient. Calculated values\u0026thinsp;\u0026gt;\u0026thinsp;0.80 were considered indicative of excellent agreement.\u003c/p\u003e \u003cp\u003e The study was conducted in accordance with the Declaration of Helsinki and approved by the institutional ethics committee, under Protocol No. 2024/05. A waiver of informed consent was provided in light of the retrospective design and use of fully anonymized archival material.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe patient cohort consisted of 12 individuals with a mean age of 54 years, reflecting a relatively mature demographic typical for thyroid nodule presentations. There was a clear female predominance, with women comprising 66.7% of the group (n\u0026thinsp;=\u0026thinsp;8/12) and men accounting for the remaining 33.3% (n\u0026thinsp;=\u0026thinsp;4/12). The nodules themselves were solitary in the majority of cases at 66.7% (n\u0026thinsp;=\u0026thinsp;8), while multinodular configurations were observed in 33.3% (n\u0026thinsp;=\u0026thinsp;4); notably, a mean pre-TA volume of 25.4 mL (range 18\u0026ndash;42 mL) and a post-TA surgical interval of 6\u0026ndash;18 months (mean 10.2 months) were observed.\u003c/p\u003e \u003cp\u003eMorphologically (as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), a fibrous capsule was present in nearly all cases at 91.7% (n\u0026thinsp;=\u0026thinsp;11/12; mean thickness 0.5\u0026ndash;1.2 mm), with only one instance lacking this feature (8.3%). Architectural patterns leaned heavily toward follicular structures in 83.3% of cases (n\u0026thinsp;=\u0026thinsp;10/12), contrasted by solid arrangements in the remaining 16.7% (n\u0026thinsp;=\u0026thinsp;2/12). Nuclear characteristics showed dark (basophilic, uniform) nuclei predominating in 66.7% (n\u0026thinsp;=\u0026thinsp;8/12), while clear (pale, overlapping) nuclei appeared in 33.3% (n\u0026thinsp;=\u0026thinsp;4/12). Degenerative alterations were common, including subcapsular hemorrhage in 91.7% (n\u0026thinsp;=\u0026thinsp;11/12), often with evidence of organization and hemosiderin deposition; sclerosis or fibrosis in 75% (n\u0026thinsp;=\u0026thinsp;9/12), typically zonal and proximate to the ablation site; focal ischemia marked by hyalinized vessels in 16.7% (n\u0026thinsp;=\u0026thinsp;2/12); and coagulative necrosis, which was focal and limited to just 8.3% (n\u0026thinsp;=\u0026thinsp;1/12). Importantly, there were no instances of true capsular invasion (0%) or vascular invasion (0%), underscoring the benign nature of these post-TA changes. Oncocytic metaplasia, characterized by eosinophilic and granular cytoplasm affecting 10\u0026ndash;25% of cells, was evenly split at 50% prevalence (n\u0026thinsp;=\u0026thinsp;6/12), often concentrated peripherally.\u003c/p\u003e \u003cp\u003eImmunohistochemical (IHC) profiling was performed as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (see also Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Consistent negativity among all cases was noted for the following: Galectin-3 (100%, n\u0026thinsp;=\u0026thinsp;12/12) and BRAF V600E mutation, consistent with non-neoplastic profiles. HBME-1 negativity was seen in 91.7% (n\u0026thinsp;=\u0026thinsp;11/12), with only one case showing focal and weak membranous positivity (\u0026lt;\u0026thinsp;10% of cells) in microfollicles with atypia close to the ablation zone (8.3%). p53 positivity was identified in a quarter of cases (25%, n\u0026thinsp;=\u0026thinsp;3/12), while the majority were negative (75%, n\u0026thinsp;=\u0026thinsp;9/12). Proliferative activity, as determined by MIB-1 (Ki-67), was low (\u0026lt;\u0026thinsp;3%) in each case (100%, n\u0026thinsp;=\u0026thinsp;12/12). Interobserver agreement was excellent with kappa values of 0.85 for morphology and 0.92 for IHC interpretation.\u003c/p\u003e \u003cp\u003eThese findings represent an extension of our previously reported series of seven cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], showing slightly increased rates of hemorrhage (91.7% vs. 85.7%) and a balanced prevalence of oncocytic changes (50% vs. 57.1%), perhaps due to an increase in sample size and possibly diverse post-treatment duration after TA, emphasizing the dynamic degeneration spectrum within ablated thyroid nodules.\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\u003ePatients\u0026rsquo; characteristics and nodules\u0026rsquo; morphological features (N\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatients' features\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge, mean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\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\u0026nbsp;\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\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArchitecture \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFollicular\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSolid\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNuclei \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDark\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eClear\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of nodules \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSingle nodule\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePlurinodular\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCapsule\u0026nbsp;\u003cem\u003en\u003c/em\u003e\u0026nbsp;(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNo\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNecrosis \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNo\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFibrosis \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNo\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemorrhage \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNo\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIschemia \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNo\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCapsular invasion \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNo\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVascular invasion \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNo\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePresence of oncocytic cells \u003cem\u003en\u003c/em\u003e (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNo\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \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\u003eImmunohistochemical profile (N\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMarkers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResults\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal n.12 (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGalectin-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\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\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (100%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBME-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (8.3%)\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\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (91.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (25%)\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\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (75%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBRAF-V600E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\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\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (100%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMIB-1 (Ki-67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (100%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe current series of twelve cytologically benign thyroid nodules subjected to laser ablation confirms, refines, and significantly expands our initial seven-case experience [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] while demonstrating near-complete histological overlap with post-ablation changes described across the thermal energy modalities of radiofrequency, microwave, and HIFU [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe most consistent and diagnostically impactful changes remain the formation of a well-defined fibrous capsule (in 91.7% of cases, n\u0026thinsp;=\u0026thinsp;11/12), prominent subcapsular hemorrhage (91.7%, n\u0026thinsp;=\u0026thinsp;11/12, often with hemosiderin-laden macrophages indicating organization), and dense zonal fibrosis/sclerosis centered on the ablation site (75%, n\u0026thinsp;=\u0026thinsp;9/12). These features represent the stereotypical repair response to laser-induced coagulative necrosis and are virtually indistinguishable from those observed following RFA or MWA [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eArchitectural rearrangement toward solid or microfollicular patterns (16.7%, n\u0026thinsp;=\u0026thinsp;2/12 predominantly solid; the remainder retaining a follicular pattern) combined with nuclear clearing (33.3%, n\u0026thinsp;=\u0026thinsp;4/12) can raise concern for an encapsulated follicular neoplasm or follicular variant of papillary thyroid carcinoma. However, several reproducible clues reliably indicate a reactive, post-ablative process rather than true neoplasia: (i) strict zonal distribution of atypical features, with maximum intensity near the ablation center and gradual fading toward the periphery; (ii) complete absence of genuine capsular or vascular invasion (0% in all cases), despite occasional pseudoinvasion of the fibrous capsule by reactive follicles or hemorrhage; (iii) presence of oncocytic metaplasia in half of the cases (50%, n\u0026thinsp;=\u0026thinsp;6/12), typically peripheral and affecting 10\u0026ndash;30% of cells\u0026mdash;a well-recognized reactive phenomenon following thermal injury [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]; and (iv) negligible mitotic activity and uniformly low Ki-67 labeling index (\u0026lt;\u0026thinsp;3% in 100% of cases).\u003c/p\u003e \u003cp\u003eImmunohistochemistry provides critical reassurance. Complete negativity for galectin-3 (100%), BRAF V600E (100%), and low proliferative activity (Ki-67\u0026thinsp;\u0026lt;\u0026thinsp;3% in all cases) strongly supports benignancy, since these markers maintain high specificity for malignancy even in thermally altered tissue [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The single case with focal, weak HBME-1 membranous positivity (8.3%, \u0026lt;\u0026thinsp;10% of cells in microfollicular areas adjacent to the ablation zone) mirrors sporadic reactivity described after RFA and MWA [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and does not correlate with invasive behavior or malignant transformation. p53 positivity, observed in 25% of cases (n\u0026thinsp;=\u0026thinsp;3/12), was weak and limited, possibly consistent with stress-related rather than mutational overexpression. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eSuch comparisons across thermal ablation techniques are remarkably consistent. Thus, identical degenerative patterns after microwave ablation were documented by both Spiezia et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and Romanelli et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]; Bernardi et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] underscored the finding of pseudoinvasion as a common RFA-related mimic of malignancy; and Pacella et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], in the largest published surgical series after laser ablation to date, emphasized that diagnostic errors were completely eliminated by familiarity with these changes combined with a limited IHC panel. Our current cohort reinforces the notion that the histological response is largely modality-independent, reflecting a shared final common pathway of thermal coagulative necrosis followed by organized repair.\u003c/p\u003e \u003cp\u003eThe clinical implications are impactful: failure to recognize these pseudomalignant changes carries the risk of overdiagnosis of carcinoma with possible unnecessary completion thyroidectomy, central-neck dissection, and radioiodine therapy, with attendant lifelong morbidity. In turn, true malignant transformation after ablation of cytologically benign nodules remains exceedingly rare; indeed, the clean IHC profile further reduces this already low theoretical risk.\u003c/p\u003e \u003cp\u003eStudy limitations include the retrospective design, relatively modest sample size (although still among the largest surgical series published after laser ablation), and inclusion of laser ablation only, which precludes direct head-to-head histological comparison with RFA or MWA in the same patients. However, the striking uniformity of findings across different energy sources in the literature strongly supports the broad applicability of these observations to all thermal ablation techniques currently in clinical use.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eLaser thermal ablation of benign thyroid nodules causes distinctive and reproducible histological changes, including dense fibrosis, subcapsular hemorrhage, pseudocapsular invasion, and oncocytic metaplasia, which can mimic malignancy. The combination of judicious morphological assessment with a limited immunohistochemical panel (Gal-3, HBME-1, BRAF V600E, p53, Ki-67) is a dependable way to distinguish treatment-related changes from a real neoplastic transformation. When pathologists interpret resected specimens after thermal ablation, they need to recognize these features to avoid an overdiagnosis of carcinoma and ensure the most appropriate patient management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclaration of interest\u003c/h2\u003e \u003cp\u003eAll authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.\u003c/p\u003e \u003ch2\u003eHuman Ethics and Consent to Participate declarations\u003c/h2\u003e \u003cp\u003eEthical review and approval were waived for this study due to its retrospective nature. All patient data were collected anonymously according to the Declaration of Helsinki, and the study adhered to Good Clinical Practice guidelines.\u003c/p\u003e \u003cp\u003e Written informed consent, as part of the routine diagnosis and treatment procedures, was obtained from patients or their guardians according to the Declaration of Helsinki, and the study adhered to Good Clinical Practice guidelines.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: P. T. and G.F.; Methodology: V.Z., V.F., E.M., and S.R.; Formal analysis and investigation: ; Writing - original draft preparation: P.T.; Writing - review and editing: E.D.R., M.M., and G.F.; Resources: F.R., M.G.M., M. F., L.P., W.G., G.R., M.B., and E.G.; Supervision: M.M. and G.F. .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRussotto, F., Fiorentino, V., Pizzimenti, C., et al. (2024) \u0026lsquo;Histologic Evaluation of Thyroid Nodules Treated with Thermal Ablation: An Institutional Experience\u0026rsquo;, International Journal of Molecular Sciences, 25(18), 10182. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms251810182\u003c/span\u003e\u003cspan address=\"10.3390/ijms251810182\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValcavi, R., Riganti, F., Bertani, A., et al. 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Clinical \u0026amp; developmental immunology, 2013, 846584. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2013/846584\u003c/span\u003e\u003cspan address=\"10.1155/2013/846584\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":"thermal ablation, thyroid nodules, laser ablation, immunohistochemistry, differential diagnosis","lastPublishedDoi":"10.21203/rs.3.rs-8712675/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8712675/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThermal ablation (TA) is a minimally invasive treatment for benign thyroid nodules, but it induces histological changes that can mimic malignancy. This study expands our previous series to characterize the histological and immunohistochemical changes occurring in these nodules after TA and to refine diagnostic criteria to prevent misdiagnosis of benign lesions as carcinoma. Twelve surgically excised thyroid nodules previously treated with laser thermal ablation were retrospectively analyzed. Histopathological examination focused on architecture, nuclear features, capsule characteristics, and degenerative changes. Immunohistochemistry was performed for galectin-3 (Gal-3), HBME-1, BRAF V600E, p53, and Ki-67.\u003c/p\u003e \u003cp\u003eAll nodules showed a fibrous capsule. Architecture was predominantly follicular (75%) or solid (25%). Sclerosis (83.3%) and subcapsular hemorrhage (91.7%) were common; necrosis and ischemia were rare. No true capsular or vascular invasion was observed. All cases were negative for Gal-3, and BRAF V600E. Eleven nodules were HBME-1-negative; one showed focal weak positivity; p53 was hyperexpressed in 3 cases. Ki-67 was consistently below 3%. TA induces characteristic degenerative and reactive changes that may simulate malignancy, including fibrosis, hemorrhage, and nuclear atypia. The consistent negativity of malignancy-associated immunohistochemical markers, together with low proliferative activity, confirms the benign nature of these alterations and supports the use of an integrated morphologic\u0026ndash;immunohistochemical approach for accurate diagnosis.\u003c/p\u003e \u003cp\u003eThis expanded analysis reinforces that laser ablation induces reproducible histologic alterations in benign thyroid nodules that can simulate malignancy. Awareness of these changes, combined with a restricted immunohistochemical panel, allows confident recognition of treatment-related artifacts and prevents overdiagnosis of carcinoma.\u003c/p\u003e","manuscriptTitle":"The Morphologic and Immunohistochemical Profiling of Thyroid Nodules Submitted to Thermal Ablation Avoids a Misdiagnosis of Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 13:58:29","doi":"10.21203/rs.3.rs-8712675/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":"6d5ee3b9-c4f6-487e-b1f8-07a79f5987df","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T18:39:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 13:58:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8712675","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8712675","identity":"rs-8712675","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}