Reclassification of Thyroid Neoplasms with Aid of Genetic Alterations and Their Surrogate Markers

Summary: Introduction: ThyroSeq testing is critical for stratifying the risk of thyroid malignancy and guiding clinical management. However, little is known about how ThyroSeq's genetic data and surrogate markers specifically influence reclassification. This study aims to investigate the role of ThyroSeq testing in conjunction with available surrogate immunostaining markers, such as BRAFV600E, NRAS Q61R, and IMP3, in aiding the reclassification of pathologic diagnoses for thyroid neoplasms. Materials and Methods: Between September 2017 and December 2021, 55 thyroid fine-needle aspirations with an indeterminate diagnosis were evaluated by ThyroSeq v3 testing. The ThyroSeq report estimates the risk of malignancy (ROM) and detects specific mutations. These ROM categories are classified as high ROM, intermediate to intermediate-high (Int-High), and intermediate-low (Int-Low) based on the ThyroSeq report. Subsequent pathological diagnoses were reviewed following resections. Available surrogate markers, BRAFV600E (Clone: VE1, Abcam, Cambridge, MA), NRAS Q61R (Clone: SP174, Abcam, Cambridge, MA), and IMP3 (Clone: 69.1, Dako Carpinteria, CA), were used. Immunostaining was performed on corresponding unstained slides from selected blocks. The stained slides were then assessed. Statistical methods from Fisher exact test were used to evaluate the performance of immunostaining. Results: The cohort comprised 42 females and 13 males, with lesion size averaging 3.2 cm and ages 21 to 84. 20 cases (36%) were in High ROM, with 70% harboring BRAFV600E mutation (including 5% with NTRK3 fusion), retaining a diagnosis of classic papillary thyroid carcinoma (cPTC); 15% remaining follicular-patterned neoplasms, and rare reclassifications occurred (3 cases, 15%). 34 (62%) Intermediate to High-Int ROM, 70% were reclassified. Among this ROM group, 29/34 (85%) harbored RAS-like alterations. 67 % (6/9) of cPTCs reclassified to follicular-patterned neoplasms. Within 74% (25/34) of follicular-patterned neoplasms, 32.3% (11/34) were reclassified, and 29.4% (10/34) remained unchanged. 1 case (2%) in the Int-Low ROM reclassified (Table 1). BRAF mutation (n=14) matched immunostaining. 1 NTRK fusion showed false-positive. NRAS mutation (n=21) matched immunostaining with 17% false-positive rate. THADA mutation (n=3) matched IMP3 with 1 DICER alteration false-positive (Table 1 and Fig. 1A-F). Conclusions: This study demonstrates the value of integrating molecular ThyroSeq testing with a reevaluation of morphological findings. This combined approach proves useful for reclassifying the pathological diagnosis of thyroid neoplasms. Our results show that immunostaining for BRAFV600E, NRAS Q61R, and IMP3 exhibited high sensitivity (100%, 93.8% and 100%, respectively) and specificity (95.1%, 94.9% and 91.7%, respectively), suggesting their potential utility as surrogate markers in reclassification, particularly for challenging cases. Further research is warranted to explore the clinical utility of these markers and to refine the classification and management of thyroid lesions.