Reliable preoperative diagnosis of thyroid nodules remained challenging because of the inconclusiveness of fine-needle aspiration (FNA) cytology. In the present study, 583 formalin-fixed paraffin embedded (FFPE) thyroid nodule tissues and 161 FNA specimens were enrolled retrospectively. Then BRAF V600E, EZH1 Q571R, SPOP P94R, and ZNF148 mutations among these samples were identified using Sanger sequencing. Based on this four-gene genomic classifier, we proposed a two-step modality to diagnose thyroid nodules to differentiate benign and malignant thyroid nodules. In the FFPE group, thyroid cancers were effectively diagnosed in 37.7% (220/583) of neoplasms by the primary BRAF V600E testing, and 15.7% (57/363) of thyroid nodules could be further determined as benign by subsequent EZH1 Q571R, SPOP P94R, and ZNF148 (we called them “ESZ”) mutation testing. In the FNA group, 161 BRAF wild-type specimens were classified according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC). A total of 7 mutated samples fell within Bethesda categories III–IV, and the mutation rate of “ESZ” in Bethesda III–IV categories was 8.4%. The two-step genomic classifier could further improve thyroid nodule diagnosis, which may inform more optimal patient management.

由于细针抽吸（FNA）细胞学检查的不确定性，甲状腺结节的可靠术前诊断仍然具有挑战性。在本研究中，回顾性纳入了 583 例福尔马林固定石蜡包埋 (FFPE) 甲状腺结节组织和 161 例 FNA 标本。然后使用桑格测序鉴定这些样本中的BRAF V600E、EZH1 Q571R、SPOP P94R 和ZNF148突变。基于这种四基因基因组分类器，我们提出了一种诊断甲状腺结节的两步模式，以区分良性和恶性甲状腺结节。在 FFPE 组中，通过初次BRAF V600E 检测，37.7%（220/583）的肿瘤被有效诊断为甲状腺癌，并且通过随后的EZH1 Q571R、SPOP可以进一步确定 15.7%（57/363）的甲状腺结节为良性。 P94R 和ZNF148（我们称之为“ESZ”）突变测试。在 FNA 组中，161 个BRAF野生型标本根据贝塞斯达甲状腺细胞病理学报告系统 (TBSRTC) 进行分类。共有7个突变样本属于Bethesda III-IV类别，其中“ESZ”在Bethesda III-IV类别中的突变率为8.4%。两步基因组分类器可以进一步改善甲状腺结节的诊断，这可以为更优化的患者管理提供信息。