Discriminating Interpatient Variabilities of RAS Gene Variants for Precision Detection of Thyroid Cancer.

Importance: Interpatient variabilities in genomic variants may reflect differences in tumor statuses among individuals. Objectives: To delineate interpatient variabilities in RAS variants in thyroid tumors based on the fifth World Health Organization classification of thyroid neoplasms and assess their diagnostic significance in cancer detection among patients with thyroid nodules. Design, Setting, and Participants: This prospective diagnostic study analyzed surgically resected thyroid tumors obtained from February 2016 to April 2022 and residual thyroid fine-needle aspiration (FNA) biopsies obtained from January 2020 to March 2021, at Mount Sinai Hospital, Toronto, Ontario, Canada. Data were analyzed from June 20, 2022, to October 15, 2023. Exposures: Quantitative detection of interpatient disparities of RAS variants (ie, NRAS, HRAS, and KRAS) was performed along with assessment of BRAF V600E and TERT promoter variants (C228T and C250T) by detecting their variant allele fractions (VAFs) using digital polymerase chain reaction assays. Main Outcomes and Measures: Interpatient differences in RAS, BRAF V600E, and TERT promoter variants were analyzed and compared with surgical histopathologic diagnoses. Malignancy rates, sensitivity, specificity, positive predictive values, and negative predictive values were calculated. Results: A total of 438 surgically resected thyroid tumor tissues and 249 thyroid nodule FNA biopsies were obtained from 620 patients (470 [75.8%] female; mean [SD] age, 50.7 [15.9] years). Median (IQR) follow-up for patients who underwent FNA biopsy analysis and subsequent resection was 88 (50-156) days. Of 438 tumors, 89 (20.3%) were identified with the presence of RAS variants, including 51 (11.6%) with NRAS, 29 (6.6%) with HRAS, and 9 (2.1%) with KRAS. The interpatient differences in these variants were discriminated at VAF levels ranging from 0.15% to 51.53%. The mean (SD) VAF of RAS variants exhibited no significant differences among benign nodules (39.2% [11.2%]), noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTPs) (25.4% [14.3%]), and malignant neoplasms (33.4% [13.8%]) (P = .28), although their distribution was found in 41.7% of NIFTPs and 50.7% of invasive encapsulated follicular variant papillary thyroid carcinomas (P < .001). RAS variants alone, regardless of a low or high VAF, were significantly associated with neoplasms at low risk of tumor recurrence (60.7% of RAS variants vs 26.9% of samples negative for RAS variants; P < .001). Compared with the sensitivity of 54.2% (95% CI, 48.8%-59.4%) and specificity of 100% (95% CI, 94.8%-100%) for BRAF V600E and TERT promoter variant assays, the inclusion of RAS variants into BRAF and TERT promoter variant assays improved sensitivity to 70.5% (95% CI, 65.4%-75.2%), albeit with a reduction in specificity to 88.8% (95% CI, 79.8%-94.1%) in distinguishing malignant neoplasms from benign and NIFTP tumors. Furthermore, interpatient differences in 5 gene variants (NRAS, HRAS, KRAS, BRAF, and TERT) were discriminated in 54 of 126 indeterminate FNAs (42.9%) and 18 of 76 nondiagnostic FNAs (23.7%), and all tumors with follow-up surgical pathology confirmed malignancy. Conclusions and Relevance: This diagnostic study delineated interpatient differences in RAS variants present in thyroid tumors with a variety of histopathological diagnoses. Discrimination of interpatient variabilities in RAS in combination with BRAF V600E and TERT promoter variants could facilitate cytology examinations in preoperative precision malignancy diagnosis among patients with thyroid nodules.

Facilitation of Definitive Cancer Diagnosis With Quantitative Molecular Assays of BRAF V600E and TERT Promoter Variants in Patients With Thyroid Nodules.

Importance: Molecular testing of the presence of pathogenic genomic variants in a tumor without quantifying the variant allele fraction (VAF) does not differentiate the variation extent among tumors, often resulting in an inconclusive diagnosis because of interpatient variability. Objective: To examine the association between the quantification of VAFs of BRAF V600E and TERT promoter variants and a definitive cancer diagnosis of thyroid tumors. Design, Setting, and Participants: This diagnostic study analyzed a cohort of 378 surgically resected thyroid tumors with a maximum dimension of 1 cm or larger between March 15, 2016, and March 16, 2020, and a separate cohort of 217 residual thyroid fine-needle aspiration (FNA) biopsy specimens obtained from January 22, 2020, to March 2, 2021, at Mount Sinai Hospital, Toronto, Ontario, Canada. Data analysis was conducted between February 1, 2021, and February 1, 2023. Exposures: Quantitative VAF assays of BRAF V600E and TERT promoter variants (C228T and C250T) were performed by digital polymerase chain reaction molecular assays. Main Outcomes and Measures: The VAFs of BRAF V600E and TERT promoter variants were correlated with tumor histologic diagnoses and histopathologic features to delineate the association of VAF assays with tumor malignancy. The receiver operating characteristic curve analysis, sensitivity, specificity, positive predictive value, negative predictive value, and logistic regression analysis based on follow-up histopathologic types were used to determine the diagnostic utility of the quantitative molecular assays. Results: A total of 595 specimens, including 378 surgically resected thyroid tumors and 217 thyroid nodule FNA biopsy specimens, were collected from 580 patients (436 [75.2%] female with a mean [SD] age of 50 [16] years and 144 [24.8%] male with a mean [SD] age of 55 [14] years). Sensitive VAF assays of 378 thyroid tumors revealed the presence of the BRAF V600E variant in 162 tumors (42.9%), with 26 (16.0%) at a low VAF of 1% or less and 136 (84.0%) at a high VAF of greater than 1%, and the presence of TERT promoter variants in 49 tumors (13.0%), including 45 C228T variants (91.8%), 15 (33.3%) of which were quantified as having a low VAF (≤1%) and 30 (66.7%) as having a high VAF (>1%), and 4 C250T variants (8.2%) with VAFs between 40.0% and 47.0%. All tumors detected with BRAF V600E and/or TERT promoter variants, whether at low or high VAFs, received a definitive cancer diagnosis. Further analysis delineated a significant association between high VAFs of either variant individually or different VAF levels for both variants in coexistence and aggressive histopathologic features of tumors. Excluding low VAFs assisted in identifying patients at an intermediate-to-high risk of recurrence (odds ratio, 5.3; 95% CI, 1.9-14.6; P = .001). The VAF assays on the residual FNA biopsy specimens showed a high agreement to those on surgical tissues (κ = 0.793, P < .001) and stratified malignancy in 40 of 183 indeterminate FNA cases (21.9%), with a sensitivity of 93.8% (95% CI, 67.7%-99.7%), specificity of 90.0% (95% CI, 75.4%-96.7%), positive predictive value of 78.9% (95% CI, 53.9%-93.0%), and negative predictive value of 97.3% (95% CI, 84.2%-99.9%). Conclusions and Relevance: This diagnostic study suggests that sensitive quantitative VAF assays of BRAF V600E and TERT promoter variants can elucidate the interpatient variability in tumors and facilitate a definitive cancer diagnosis of thyroid nodules by differentiating the variation extent of genomic variants, even at low VAFs.

Development of a Molecular Assay for Detection and Quantification of the BRAF Variation in Residual Tissue From Thyroid Nodule Fine-Needle Aspiration Biopsy Specimens.

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. Objective: To 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 Participants: This 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 carcinoma-derived BCPAP cells. The linearity of quantification of BRAF V600E was calculated (y = 0.7339x; R2 = 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. Conclusions and Relevance: This diagnostic study developed a sensitive molecular assay for detection and quantification of BRAF V600E variation in residual tissue from thyroid FNA biopsies to identify patients with cancer harboring BRAF V600E in a cost-effective manner, highlighting the clinical value of molecular assay of the remaining FNA tissue in the management of thyroid nodules.

Diagnostic Value of Galectin-3 in Distinguishing Invasive Encapsulated Carcinoma from Noninvasive Follicular Thyroid Neoplasms with Papillary-Like Nuclear Features (NIFTP).

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 ß-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.

Programmed death-ligand 1 expression by digital image analysis advances thyroid cancer diagnosis among encapsulated follicular lesions.

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.