**4. Discussion**

Indeterminate thyroid nodules are highly prevalent in daily clinical practice and represent up to 30% of all clinically assessed thyroid nodules [41], whereas only less than 20% of this group are malignant. Invasive EFVPTC, noninvasive EFVPTC (not meeting criteria for NIFTP diagnosis) and infiltrative FVPTC were designated as malignant lesions, making the diagnosis of thyroid cancer very difficult in cases where histological hallmarks of invasion are not evident [15–17,37]. A subset of encapsulated follicular tumors, formerly considered to be noninvasive encapsulated/well demarcated follicular variant of PTC, has been reclassified under a new histological nomenclature, NIFTP [11]. The incidence of NIFTP was as high as 13.3% of all PTC cases in North American and European populations [42] and 16.8% of all well-differentiated thyroid cancers in Ontario, Canada [24]. Histopathological examination of the entire capsulated tumor after its resection according to the rigid diagnostic criteria remains the gold standard for NIFTP diagnosis. Malignant behavior (lymph node and/or distant metastasis) has been reported in NIFTP patients [21,43,44]. NIFTP is not entirely considered as a benign thyroid neoplasm, but correct classification of noninvasive EFVPTC that would qualify NITFP is required to ensure an extremely low rate of adverse oncologic outcomes. Molecular marker testing has risen as an auxiliary tool to distinguish malignant invasive EFVPTCs from more indolent NIFTPs and benign nodules [11,25], potentially assisting pathologists in the management of indeterminate thyroid nodules [27]. Scores on the base of the levels of protein marker expression in thyroid nodules may objectively distinguish malignant lesions from indeterminate thyroid nodules, thereby aiding the correct diagnosis and consequent avoidance of over-treatment of NIFTP lesions when the score is low. Our previous study has shown that the degree of cytoplasmic PD-L1 expression could serve as a useful adjunct to traditional H&E histopathology assessment of such nodules, among which with a low expression of cytoplasmic PD-L1 can be considered as benign nodules or NIFTP [25].

Gal-3 expression has been recognized as a promising diagnostic molecular marker of thyroid malignancy due to its differential expression between thyroid carcinomas and normal or benign thyroid tissues [29,45–47]. However, the reclassification of EFVPTC without capsular or vascular invasion to NIFTP not only affects how pathologists evaluate and report this subset of thyroid neoplasms but also raises the need for rebuilding the clinical, histologic, cytologic and molecular parameters for this new entity and accordingly establishing new molecular tests [22]. Therefore, the use of ancillary testing with protein markers previously developed, such as Gal-3 and HBME1, requires to be re-evaluated in the era of NIFTP. In the present study, our data have shown that cytoplasmic Gal-3 expression is significantly increased in invasive EFVPTCs as compared to NIFTPs or benign thyroid nodules in thyroid resection specimens. Concurrently, though there was no significant difference between NIFTPs and benign nodules, cytoplasmic Gal-3 expression in the former was higher than that in the latter, supporting NIFTP cannot be simply considered as a benign lesion. Chronic inflammation can be associated with 30–58% of PTC [48,49]. We also noted that the presence of LT enhances cytoplasmic Gal-3 expression and henceforward the LT increased expression needs to be interpreted with caution. This observation is consistent with the result from another report which showed the increased expression of Gal-3 in an inflammatory environment [50]. Patients with LT were usually under prolonged stimuli from chronic inflammation. The mechanism underlying modulation of Gal-3 expression in thyroid with chronic inflammatory process remains to be determined. Localization of Gal-3 in papillary carcinomas has been reported in both the cytoplasm and nucleus [40,51,52], however, our findings and other's [29] showed predominant expression of Gal-3 in the cytoplasm in PTC rather than the nucleus. Nuclear expression has been observed in some benign thyroid conditions in our study and reports of others [51,52]. The increased cytoplasmic Gal-3 in invasive EFVPTC might contribute to thyroid cancer development through the induction of the capsular, vascular and/or extrathyroidal invasive activity. The detailed correlation between Gal-3 expression and the degree of invasion was not able to be analyzed since most EFVPTC cases were presented with minimal capsular invasion in the current study. Recently, genetic alterations were intensively studied, such as *BRAF*, *RAS* and *TERT* promoter mutations and *RET/PTC* and *PAX8/PPARγ* rearrangements [53,54]. NIFTPs are commonly detected with the frequent occurrence of RAS mutations and lack of BRAF*V600E* and TERT promoter mutations [23]. Whether the level of Gal-3 expression can be associated with such mutational status for better identifying NIFTP requires further investigation. Our observations have suggested that cytoplasmic Gal-3 expression can be considered as an ancillary aid to H&E diagnostic criteria in distinguishing invasive EFVPTC from NIFTP and benign nodules.

After four decades of steady increase, thyroid cancer incidence rate reached a plateau and possibly started to decline between 2013 and 2020 in the United States [55]. This reverse trend in the incidence of thyroid cancers has been correlating with the increasing understanding of over-diagnosis and the indolent nature of many thyroid nodules that were more likely classified as cancers previously. NIFTP has emerged as a low risk tumor with an indolent clinical course. The present study was focused on evaluating the diagnostic value of the Gal-3 cytoplasmic expression in the histological tissue samples between NIFTP and EFVPTC. To our knowledge, this is the first report showing the diagnostic value of increased cytoplasmic Gal-3 expression in ruling out the indolent NIFTP from invasive EFVPTC. The Gal-3 test proposed here does not replace conventional surgical histopathological examination but represents an auxiliary diagnostic method, especially for cases where morphologic features of invasion are equivocal, that may affect clinical decision-making with regard to completion thyroidectomy, central lymph node dissection, and adjunctive radioiodine therapy. In practice, Gal-3 staining alone add little to histology evaluation when the diagnosis of NIFTP could be achieved via complete resection of the nodules for histological examination of the entire capsule to rule out invasion. However, in pre-surgical fine needle aspiration (FNA) biopsies, NIFTP can belong to any of four categories of the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC), including benign, atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS), follicular neoplasm or suspicious for a follicular neoplasm (FN/SFN) and suspicious for malignancy (SFM) [56]. The definitive diagnosis of NIFTP cannot be made based on

the observation of the preoperative cytology specimens, while molecular tests would be highly useful to improve the accuracy in the diagnostics of NIFTP in FNA biopsies. Gal-3 test could have clinically significant utility in assisting in preoperative diagnosis if it were successfully applied to cytology specimens [57]. We are aware of the limitation of our study which is based upon a single patient cohort from a single tertiary care center. Future studies in a larger patient cohort from multiple centers are needed to validate our observations and conclusions. Furthermore, the NIFTP cases were re-classified based on a thorough review of pathology reports and assessment of H&E slides in this study. NIFTP diagnosis is challenging for pathologist and a potential misclassification error might exist particularly when specimens were managed in a way the entire tumor capsule could not be fully assessed for invasion based on pathology review of slides. We are also cognizant that the clinical outcome analysis for each subtype was limited due to incomplete followup information, hence the possible association of cytoplasmic Gal-3 expression with the long-term prognoses of NIFTP verses invasive EFVPTC remains further investigation.
