IMPORTANCE Thyroid cancer, predominantly papillary thyroid carcinoma (PTC), is common, but an estimated 30% of ultrasonography-guided fine-needle aspiration (FNA) biopsies of thyroid nodules are indeterminate. BRAF variation, associated with poor clinicopathological characteristics, is a useful molecular marker for diagnostics. OBJECTIVETo develop a sensitive molecular assay for BRAF V600E detection in remaining tissue of thyroid FNA biopsies to identify patients with cancer carrying a BRAF variation. DESIGN, SETTING, AND PARTICIPANTSThis diagnostic study used tumor tissue from surgical formalin-fixed, paraffin-embedded (FFPE) specimens and residual tissue from thyroid FNA biopsies for genomic DNA extraction. FFPE specimens served as the validation set, and residual tissue from FNA biopsies served as the test set. A molecular assay was developed for accurate detection of BRAF V600E variation using locked nucleic acid (LNA) probe-based droplet digital polymerase chain reaction (dPCR), and the assay was validated by BRAF V600E immunohistochemical staining (IHC). The study was conducted between February 2019 and May 2021. RESULTS A total of 271 specimens, including 77 FFPE specimens (with a follow-up of 48 matched surgical specimens) and 146 residual FNA samples, were collected from 223 patients (mean [SD] age, 53.8 [15.3] years; 174 [78.0%] women; 49 [22.0%] men). The molecular assay by dPCR was first established to specifically and accurately detect and quantify wild-type BRAF and variant BRAF in DNA from human follicular thyroid carcinoma-derived FTC-133 and papillary thyroid carcinomaderived BCPAP cells. The linearity of quantification of BRAF V600E was calculated (y = 0.7339x;R 2 = 0.9996) with sensitivity at 0.02 copies/μL and reproducibility in detecting variant DNA at various dilutions(coefficient of variance in 0.3% DNA, 9.63%; coefficient of variance in 1.0% DNA, 7.41%). In validation testing, the dPCR assay and IHC staining exhibited 100% specificity in concordantly identifying BRAF V600E in PTCs (κ = 0.873; P < .001) and sensitivity of 32.0% (95% CI, 19.1% to 44.9%) in dPCR and 26.0% (95% CI, 13.1% to 38.9%) in IHC staining, with an improvement by 23.08% in dPCR compared with the IHC staining. The dPCR assay further detected BRAF V600E in 39 of 146 residual FNA specimens (26.7%). At short-term follow-up, 48 patients, including 14 of 39 patients with BRAF variation and 34 of 107 patients without BRAF variation on residual FNA specimens, underwent resection. The dPCR assay of BRAF status in the matched surgical specimens showed BRAF V600E variations in 12 patients and wild-type BRAF in 36 patients, with a high agreement to that in residual tissue of FNA specimens (κ = 0.789; P < .001). Among 14 patients with BRAF variations on residual FNA, 13 were diagnosed with PTC and 1 was diagnosed with anaplastic thyroid cancer at the thyroidectomy.

Recognition of noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) that distinguishes them from invasive malignant encapsulated follicular variant of papillary thyroid carcinoma (EFVPTC) can prevent overtreatment of NIFTP patients. We and others have previously reported that programmed death-ligand 1 (PD-L1) is a useful biomarker in thyroid tumors; however, all reports to date have relied on manual scoring that is time consuming as well as subject to individual bias. Consequently, we developed a digital image analysis (DIA) protocol for cytoplasmic and membranous stain quantitation (ThyApp) and evaluated three tumor sampling methods [Systemic Uniform Random Sampling, hotspot nucleus, and hotspot nucleus/3,3′-Diaminobenzidine (DAB)]. A patient cohort of 153 cases consisting of 48 NIFTP, 44 EFVPTC, 26 benign nodules and 35 encapsulated follicular lesions/neoplasms with lymphocytic thyroiditis (LT) was studied. ThyApp quantitation of PD-L1 expression revealed a significant difference between invasive EFVPTC and NIFTP; but none between NIFTP and benign nodules. ThyApp integrated with hotspot nucleus tumor sampling method demonstrated to be most clinically relevant, consumed least processing time, and eliminated interobserver variance. In conclusion, the fully automatic DIA algorithm developed using a histomorphological approach objectively quantitated PD-L1 expression in encapsulated thyroid neoplasms and outperformed manual scoring in reproducibility and higher efficiency.

Background: non-invasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP), which is considered as low-risk cancer, should be distinguished from the malignant invasive encapsulated follicular variant of papillary thyroid carcinoma (EFVPTC). Improved discrimination of NIFTPs from invasive EFVPTCs using a molecular biomarker test could provide useful insights into pre- and post-surgical management of the indeterminate thyroid nodule. Galectin-3 (Gal-3), a β-galactosyl-binding molecule in the lectin group, is involved in different biological functions in well differentiated thyroid carcinomas. The aim of this study was to determine whether Gal-3 expression as a diagnostic marker could distinguish indolent NIFTP from invasive EFVPTC on tissue specimens from surgical thyroid nodules. Methods: immunohistochemical (IHC) analysis of cytoplasmic and nuclear Gal-3 expression was performed in formalin-fixed paraffin-embedded (FFPE) surgical tissues in four specific diagnostic subgroups- benign nodules, NIFTPs, EFVPTCs and lymphocytic/Hashimoto’s thyroiditis (LTs). Results: cytoplasmic Gal-3 expression (mean ± SD) was significantly increased in invasive EFVPTCs (4.80 ± 1.60) compared to NIFTPs (2.75 ± 1.58, p < 0.001) and benign neoplasms (2.09 ± 1.19, p < 0.001) with no significant difference between NIFTPs and benign lesions (p = 0.064). The presence of LT enhanced cytoplasmic Gal-3 expression (3.80 ± 1.32) compared to NIFTPs (p = 0.016) and benign nodules (p < 0.001). Nuclear Gal-3 expression in invasive EFVPTCs (1.84 ± 1.30) was significantly higher than in NIFTPs (1.00 ± 0.72, p = 0.001), but similar to benign nodules (1.44 ± 1.77, p = 0.215), thereby obviating its potential clinical application. Conclusions: our observations have indicated that increased cytoplasmic Gal-3 expression shows diagnostic potential in distinguishing NIFTP among encapsulated follicular variant nodules thereby serving as a possible ancillary test to H&E histopathological diagnostic criteria when LT interference is absent, to assist in the detection of the invasive EFVPTC among such nodules.