Clinical value of multi-gene testing in distinguishing benign and malignant thyroid nodules

Background: The newly released 2022 WHO Classification of Neuroendocrine Neoplasms (version 5) and a recent update on thyroid tumor classifications have emphasized genetic testing to an unprecedented level. Fine needle aspiration (FNA) has been widely applied for the preoperative diagnosis of thyroid nodules. However, it is limited mainly to testing for a single gene-BRAFV600E, whereas multi-gene testing data are scarce, especially in the Asian population. This study aimed to explore the clinical value of multi-gene testing in the differential diagnosis of benign and malignant thyroid nodules based on the 2023 Bethesda System for Reporting Thyroid Cytopathology (BSRTC).A total of 615 thyroid nodules underwent ultrasound-guided fine-needle aspiration cytology (FNAC) were collected from Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The next-generation sequencing platform was applied for multi-gene testing. A panel of well-recognized commonly mutated genes in thyroid cancer were analyzed, including BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET.Gene mutations were identified in 324 nodules (52.7%), with BRAFV600E being the most prevalent driver gene alteration observed in this cohort (233/324; 79.1%), followed by RAS (77/324, 23.8%). The overall malignancy rate of gene mutations was 89.7% in our cohort, of which the lymph node metastasis rate was 45.3%. The combination of multi-gene testing and cytology resulted in 89.3% sensitivity, 95.2% specificity, 98.9% positive predictive value, 64.5% negative predictive value and 90.3% accuracy, which were significantly higher than those from mere cytology (sensitivity 68.6%, specificity 87.5%, positive predictive value 95.9%, negative predictive value 39.8%, accuracy 72.2%).Multi-gene testing could substantially enhance the detection rate of malignant thyroid nodules and protect patients with benign nodules from unnecessary surgeries. Multi-gene testing provides a valuable reference for individualized preoperative decision-making, which may serve as a crucial method for postoperative treatment and prognosis assessment.Thyroid neoplasms are common in the endocrine system, representing the most commonly diagnosed head and neck tumors. In the past few decades, the total incidence rate of thyroid neoplasm has increased about 2-fold and comprised 2% of the cancers.[1] Thyroid nodules are clinically prevalent and can be detected in approximately 20% to 76% of normal people by ultrasound techniques.[2] As the development of detecting and surgical techniques, an increasing number of thyroid nodules has been found as malignancy. In this context, early screening and differentiation of benign and malignant thyroid nodules are decisive for clinical decision-making. Currently, ultrasound-guided fine-needle aspiration cytology (FNAC) is widely applied in the preoperative diagnosis of benign and malignant thyroid nodules. It is interpreted based on The Bethesda System for Reporting Thyroid Cytopathology (BSRTC). However, a definitive diagnosis still cannot be achieved via the FNA for 15-30% of nodules.[3,4]With the recent advancement of molecular techniques to diagnose thyroid neoplasm, amounting evidence has proved that BRAFV600E -based FNA distinctly increases the detection rate of BRAF-mutated associated thyroid neoplasm.[5,6] However, studies on multi-gene testing have been mainly limited to the western population[7-10] and are less common in Asians. It is worth noting that different races may vary in molecular alterations in this type of tumor. Previous research showed that the common mutated gene: BRAFV600E, ranged from 29% to 83% (mean: 45%) in papillary thyroid cancer (PTC),[11,12] with higher alternation frequency in Asian people (70-80%)[13,14] and lower mutations in the Americans (40-50%),[15] although the rate has increased in the recent decade.[16]Given the growing need for individualized diagnosis and therapy, neither mere cytology nor single-gene testing is satisfactory for clinical practice. Herein, we established a 9-gene mutation panel (BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET) to sketch the gene mutation profile of thyroid neoplasms and to explore the diagnostic value of multi-gene testing in Chinese population based on BSRTC.Background: The newly released 2022 WHO Classification of Neuroendocrine Neoplasms (version 5) and a recent update on thyroid tumor classifications have emphasized genetic testing to an unprecedented level. Fine needle aspiration (FNA) has been widely applied for the preoperative diagnosis of thyroid nodules. However, it is limited mainly to testing for a single gene-BRAFV600E, whereas multi-gene testing data are scarce, especially in the Asian population. This study aimed to explore the clinical value of multi-gene testing in the differential diagnosis of benign and malignant thyroid nodules based on the 2023 Bethesda System for Reporting Thyroid Cytopathology (BSRTC).A total of 615 thyroid nodules underwent ultrasound-guided fine-needle aspiration cytology (FNAC) were collected from Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The next-generation sequencing platform was applied for multi-gene testing. A panel of well-recognized commonly mutated genes in thyroid cancer were analyzed, including BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET.Gene mutations were identified in 324 nodules (52.7%), with BRAFV600E being the most prevalent driver gene alteration observed in this cohort (233/324; 79.1%), followed by RAS (77/324, 23.8%). The overall malignancy rate of gene mutations was 89.7% in our cohort, of which the lymph node metastasis rate was 45.3%. The combination of multi-gene testing and cytology resulted in 89.3% sensitivity, 95.2% specificity, 98.9% positive predictive value, 64.5% negative predictive value and 90.3% accuracy, which were significantly higher than those from mere cytology (sensitivity 68.6%, specificity 87.5%, positive predictive value 95.9%, negative predictive value 39.8%, accuracy 72.2%).Multi-gene testing could substantially enhance the detection rate of malignant thyroid nodules and protect patients with benign nodules from unnecessary surgeries. Multi-gene testing provides a valuable reference for individualized preoperative decision-making, which may serve as a crucial method for postoperative treatment and prognosis assessment.Thyroid neoplasms are common in the endocrine system, representing the most commonly diagnosed head and neck tumors. In the past few decades, the total incidence rate of thyroid neoplasm has increased about 2-fold and comprised 2% of the cancers.[1] Thyroid nodules are clinically prevalent and can be detected in approximately 20% to 76% of normal people by ultrasound techniques.[2] As the development of detecting and surgical techniques, an increasing number of thyroid nodules has been found as malignancy. In this context, early screening and differentiation of benign and malignant thyroid nodules are decisive for clinical decision-making. Currently, ultrasound-guided fine-needle aspiration cytology (FNAC) is widely applied in the preoperative diagnosis of benign and malignant thyroid nodules. It is interpreted based on The Bethesda System for Reporting Thyroid Cytopathology (BSRTC). However, a definitive diagnosis still cannot be achieved via the FNA for 15-30% of nodules.[3,4]With the recent advancement of molecular techniques to diagnose thyroid neoplasm, amounting evidence has proved that BRAFV600E -based FNA distinctly increases the detection rate of BRAF-mutated associated thyroid neoplasm.[5,6] However, studies on multi-gene testing have been mainly limited to the western population[7-10] and are less common in Asians. It is worth noting that different races may vary in molecular alterations in this type of tumor. Previous research showed that the common mutated gene: BRAFV600E, ranged from 29% to 83% (mean: 45%) in papillary thyroid cancer (PTC),[11,12] with higher alternation frequency in Asian people (70-80%)[13,14] and lower mutations in the Americans (40-50%),[15] although the rate has increased in the recent decade.[16]Given the growing need for individualized diagnosis and therapy, neither mere cytology nor single-gene testing is satisfactory for clinical practice. Herein, we established a 9-gene mutation panel (BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET) to sketch the gene mutation profile of thyroid neoplasms and to explore the diagnostic value of multi-gene testing in Chinese population based on BSRTC.Background: The newly released 2022 WHO Classification of Neuroendocrine Neoplasms (version 5) and a recent update on thyroid tumor classifications have emphasized genetic testing to an unprecedented level. Fine needle aspiration (FNA) has been widely applied for the preoperative diagnosis of thyroid nodules. However, it is limited mainly to testing for a single gene-BRAFV600E, whereas multi-gene testing data are scarce, especially in the Asian population. This study aimed to explore the clinical value of multi-gene testing in the differential diagnosis of benign and malignant thyroid nodules based on the 2023 Bethesda System for Reporting Thyroid Cytopathology (BSRTC).A total of 615 thyroid nodules underwent ultrasound-guided fine-needle aspiration cytology (FNAC) were collected from Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The next-generation sequencing platform was applied for multi-gene testing. A panel of well-recognized commonly mutated genes in thyroid cancer were analyzed, including BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET.Gene mutations were identified in 324 nodules (52.7%), with BRAFV600E being the most prevalent driver gene alteration observed in this cohort (233/324; 79.1%), followed by RAS (77/324, 23.8%). The overall malignancy rate of gene mutations was 89.7% in our cohort, of which the lymph node metastasis rate was 45.3%. The combination of multi-gene testing and cytology resulted in 89.3% sensitivity, 95.2% specificity, 98.9% positive predictive value, 64.5% negative predictive value and 90.3% accuracy, which were significantly higher than those from mere cytology (sensitivity 68.6%, specificity 87.5%, positive predictive value 95.9%, negative predictive value 39.8%, accuracy 72.2%).Multi-gene testing could substantially enhance the detection rate of malignant thyroid nodules and protect patients with benign nodules from unnecessary surgeries. Multi-gene testing provides a valuable reference for individualized preoperative decision-making, which may serve as a crucial method for postoperative treatment and prognosis assessment.Thyroid neoplasms are common in the endocrine system, representing the most commonly diagnosed head and neck tumors. In the past few decades, the total incidence rate of thyroid neoplasm has increased about 2-fold and comprised 2% of the cancers.[1] Thyroid nodules are clinically prevalent and can be detected in approximately 20% to 76% of normal people by ultrasound techniques.[2] As the development of detecting and surgical techniques, an increasing number of thyroid nodules has been found as malignancy. In this context, early screening and differentiation of benign and malignant thyroid nodules are decisive for clinical decision-making. Currently, ultrasound-guided fine-needle aspiration cytology (FNAC) is widely applied in the preoperative diagnosis of benign and malignant thyroid nodules. It is interpreted based on The Bethesda System for Reporting Thyroid Cytopathology (BSRTC). However, a definitive diagnosis still cannot be achieved via the FNA for 15-30% of nodules.[3,4]With the recent advancement of molecular techniques to diagnose thyroid neoplasm, amounting evidence has proved that BRAFV600E -based FNA distinctly increases the detection rate of BRAF-mutated associated thyroid neoplasm.[5,6] However, studies on multi-gene testing have been mainly limited to the western population[7-10] and are less common in Asians. It is worth noting that different races may vary in molecular alterations in this type of tumor. Previous research showed that the common mutated gene: BRAFV600E, ranged from 29% to 83% (mean: 45%) in papillary thyroid cancer (PTC),[11,12] with higher alternation frequency in Asian people (70-80%)[13,14] and lower mutations in the Americans (40-50%),[15] although the rate has increased in the recent decade.[16]Given the growing need for individualized diagnosis and therapy, neither mere cytology nor single-gene testing is satisfactory for clinical practice. Herein, we established a 9-gene mutation panel (BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET) to sketch the gene mutation profile of thyroid neoplasms and to explore the diagnostic value of multi-gene testing in Chinese population based on BSRTC.Background: The newly released 2022 WHO Classification of Neuroendocrine Neoplasms (version 5) and a recent update on thyroid tumor classifications have emphasized genetic testing to an unprecedented level. Fine needle aspiration (FNA) has been widely applied for the preoperative diagnosis of thyroid nodules. However, it is limited mainly to testing for a single gene-BRAFV600E, whereas multi-gene testing data are scarce, especially in the Asian population. This study aimed to explore the clinical value of multi-gene testing in the differential diagnosis of benign and malignant thyroid nodules based on the 2023 Bethesda System for Reporting Thyroid Cytopathology (BSRTC).A total of 615 thyroid nodules underwent ultrasound-guided fine-needle aspiration cytology (FNAC) were collected from Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The next-generation sequencing platform was applied for multi-gene testing. A panel of well-recognized commonly mutated genes in thyroid cancer were analyzed, including BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET.Gene mutations were identified in 324 nodules (52. 7%), with BRAFV600E being the most prevalent driver gene alteration observed in this cohort (233/324; 79.1%), followed by RAS (77/324, 23.8%). The overall malignancy rate of gene mutations was 89.7% in our cohort, of which the lymph node metastasis rate was 45.3%. The combination of multi-gene testing and cytology resulted in 89.3% sensitivity, 95.2% specificity, 98.9% positive predictive value, 64.5% negative predictive value and 90.3% accuracy, which were significantly higher than those from mere cytology (sensitivity 68.6%, specificity 87.5%, positive predictive value 95.9%, negative predictive value 39.8%, accuracy 72.2%).Multi-gene testing could substantially enhance the detection rate of malignant thyroid nodules and protect patients with benign nodules from unnecessary surgeries. Multi-gene testing provides a valuable reference for individualized preoperative decision-making, which may serve as a crucial method for postoperative treatment and prognosis assessment.Thyroid neoplasms are common in the endocrine system, representing the most commonly diagnosed head and neck tumors. In the past few decades, the total incidence rate of thyroid neoplasm has increased about 2-fold and comprised 2% of the cancers.[1] Thyroid nodules are clinically prevalent and can be detected in approximately 20% to 76% of normal people by ultrasound techniques.[2] As the development of detecting and surgical techniques, an increasing number of thyroid nodules has been found as malignancy. In this context, early screening and differentiation of benign and malignant thyroid nodules are decisive for clinical decision-making. Currently, ultrasound-guided fine-needle aspiration cytology (FNAC) is widely applied in the preoperative diagnosis of benign and malignant thyroid nodules. It is interpreted based on The Bethesda System for Reporting Thyroid Cytopathology (BSRTC). However, a definitive diagnosis still cannot be achieved via the FNA for 15-30% of nodules.[3,4]With the recent advancement of molecular techniques to diagnose thyroid neoplasm, amounting evidence has proved that BRAFV600E -based FNA distinctly increases the detection rate of BRAF-mutated associated thyroid neoplasm.[5,6] However, studies on multi-gene testing have been mainly limited to the western population[7-10] and are less common in Asians. It is worth noting that different races may vary in molecular alterations in this type of tumor. Previous research showed that the common mutated gene: BRAFV600E, ranged from 29% to 83% (mean: 45%) in papillary thyroid cancer (PTC),[11,12] with higher alternation frequency in Asian people (70-80%)[13,14] and lower mutations in the Americans (40-50%),[15] although the rate has increased in the recent decade.[16]Given the growing need for individualized diagnosis and therapy, neither mere cytology nor single-gene testing is satisfactory for clinical practice. Herein, we established a 9-gene mutation panel (BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET) to sketch the gene mutation profile of thyroid neoplasms and to explore the diagnostic value of multi-gene testing in Chinese population based on BSRTC.Background: The newly released 2022 WHO Classification of Neuroendocrine Neoplasms (version 5) and a recent update on thyroid tumor classifications have emphasized genetic testing to an unprecedented level. Fine needle aspiration (FNA) has been widely applied for the preoperative diagnosis of thyroid nodules. However, it is limited mainly to testing for a single gene-BRAFV600E, whereas multi-gene testing data are scarce, especially in the Asian population. This study aimed to explore the clinical value of multi-gene testing in the differential diagnosis of benign and malignant thyroid nodules based on the 2023 Bethesda System for Reporting Thyroid Cytopathology (BSRTC).A total of 615 thyroid nodules underwent ultrasound-guided fine-needle aspiration cytology (FNAC) were collected from Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The next-generation sequencing platform was applied for multi-gene testing. A panel of well-recognized commonly mutated genes in thyroid cancer were analyzed, including BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET.Gene mutations were identified in 324 nodules (52.7%), with BRAFV600E being the most prevalent driver gene alteration observed in this cohort (233/324; 79.1%), followed by RAS (77/324, 23.8%). The overall malignancy rate of gene mutations was 89.7% in our cohort, of which the lymph node metastasis rate was 45.3%. The combination of multi-gene testing and cytology resulted in 89.3% sensitivity, 95.2% specificity, 98.9% positive predictive value, 64.5% negative predictive value and 90.3% accuracy, which were significantly higher than those from mere cytology (sensitivity 68.6%, specificity 87.5%, positive predictive value 95.9%, negative predictive value 39.8%, accuracy 72.2%).Multi-gene testing could substantially enhance the detection rate of malignant thyroid nodules and protect patients with benign nodules from unnecessary surgeries. Multi-gene testing provides a valuable reference for individualized preoperative decision-making, which may serve as a crucial method for postoperative treatment and prognosis assessment.Thyroid neoplasms are common in the endocrine system, representing the most commonly diagnosed head and neck tumors. In the past few decades, the total incidence rate of thyroid neoplasm has increased about 2-fold and comprised 2% of the cancers.[1] Thyroid nodules are clinically prevalent and can be detected in approximately 20% to 76% of normal people by ultrasound techniques.[2] As the development of detecting and surgical techniques, an increasing number of thyroid nodules has been found as malignancy. In this context, early screening and differentiation of benign and malignant thyroid nodules are decisive for clinical decision-making. Currently, ultrasound-guided fine-needle aspiration cytology (FNAC) is widely applied in the preoperative diagnosis of benign and malignant thyroid nodules. It is interpreted based on The Bethesda System for Reporting Thyroid Cytopathology (BSRTC). However, a definitive diagnosis still cannot be achieved via the FNA for 15-30% of nodules.[3,4]With the recent advancement of molecular techniques to diagnose thyroid neoplasm, amounting evidence has proved that BRAFV600E -based FNA distinctly increases the detection rate of BRAF-mutated associated thyroid neoplasm.[5,6] However, studies on multi-gene testing have been mainly limited to the western population[7-10] and are less common in Asians. It is worth noting that different races may vary in molecular alterations in this type of tumor. Previous research showed that the common mutated gene: BRAFV600E, ranged from 29% to 83% (mean: 45%) in papillary thyroid cancer (PTC),[11,12] with higher alternation frequency in Asian people (70-80%)[13,14] and lower mutations in the Americans (40-50%),[15] although the rate has increased in the recent decade. [16]Given the growing need for individualized diagnosis and therapy, neither mere cytology nor single-gene testing is satisfactory for clinical practice. Herein, we established a 9-gene mutation panel (BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET) to sketch the gene mutation profile of thyroid neoplasms and to explore the diagnostic value of multi-gene testing in Chinese population based on BSRTC.Background: The newly released 2022 WHO Classification of Neuroendocrine Neoplasms (version 5) and a recent update on thyroid tumor classifications have emphasized genetic testing to an unprecedented level. Fine needle aspiration (FNA) has been widely applied for the preoperative diagnosis of thyroid nodules. However, it is limited mainly to testing for a single gene-BRAFV600E, whereas multi-gene testing data are scarce, especially in the Asian population. This study aimed to explore the clinical value of multi-gene testing in the differential diagnosis of benign and malignant thyroid nodules based on the 2023 Bethesda System for Reporting Thyroid Cytopathology (BSRTC).A total of 615 thyroid nodules underwent ultrasound-guided fine-needle aspiration cytology (FNAC) were collected from Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The next-generation sequencing platform was applied for multi-gene testing. A panel of well-recognized commonly mutated genes in thyroid cancer were analyzed, including BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET.Gene mutations were identified in 324 nodules (52.7%), with BRAFV600E being the most prevalent driver gene alteration observed in this cohort (233/324; 79.1%), followed by RAS (77/324, 23.8%). The overall malignancy rate of gene mutations was 89.7% in our cohort, of which the lymph node metastasis rate was 45.3%. The combination of multi-gene testing and cytology resulted in 89.3% sensitivity, 95.2% specificity, 98.9% positive predictive value, 64.5% negative predictive value and 90.3% accuracy, which were significantly higher than those from mere cytology (sensitivity 68.6%, specificity 87.5%, positive predictive value 95.9%, negative predictive value 39.8%, accuracy 72.2%).Multi-gene testing could substantially enhance the detection rate of malignant thyroid nodules and protect patients with benign nodules from unnecessary surgeries. Multi-gene testing provides a valuable reference for individualized preoperative decision-making, which may serve as a crucial method for postoperative treatment and prognosis assessment.Thyroid neoplasms are common in the endocrine system, representing the most commonly diagnosed head and neck tumors. In the past few decades, the total incidence rate of thyroid neoplasm has increased about 2-fold and comprised 2% of the cancers.[1] Thyroid nodules are clinically prevalent and can be detected in approximately 20% to 76% of normal people by ultrasound techniques.[2] As the development of detecting and surgical techniques, an increasing number of thyroid nodules has been found as malignancy. In this context, early screening and differentiation of benign and malignant thyroid nodules are decisive for clinical decision-making. Currently, ultrasound-guided fine-needle aspiration cytology (FNAC) is widely applied in the preoperative diagnosis of benign and malignant thyroid nodules. It is interpreted based on The Bethesda System for Reporting Thyroid Cytopathology (BSRTC). However, a definitive diagnosis still cannot be achieved via the FNA for 15-30% of nodules. [3,4]With the recent advancement of molecular techniques to diagnose thyroid neoplasm, amounting evidence has proved that BRAFV600E -based FNA distinctly increases the detection rate of BRAF-mutated associated thyroid neoplasm.[5,6] However, studies on multi-gene testing have been mainly limited to the western population[7-10] and are less common in Asians. It is worth noting that different races may vary in molecular alterations in this type of tumor. Previous research showed that the common mutated gene: BRAFV600E, ranged from 29% to 83% (mean: 45%) in papillary thyroid cancer (PTC),[11,12] with higher alternation frequency in Asian people (70-80%)[13,14] and lower mutations in the Americans (40-50%),[15] although the rate has increased in the recent decade.[16]Given the growing need for individualized diagnosis and therapy, neither mere cytology nor single-gene testing is satisfactory for clinical practice. Herein, we established a 9-gene mutation panel (BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET) to sketch the gene mutation profile of thyroid neoplasms and to explore the diagnostic value of multi-gene testing in Chinese population based on BSRTC.Background: The newly released 2022 WHO Classification of Neuroendocrine Neoplasms (version 5) and a recent update on thyroid tumor classifications have emphasized genetic testing to an unprecedented level. Fine needle aspiration (FNA) has been widely applied for the preoperative diagnosis of thyroid nodules. However, it is limited mainly to testing for a single gene-BRAFV600E, whereas multi-gene testing data are scarce, especially in the Asian population. This study aimed to explore the clinical value of multi-gene testing in the differential diagnosis of benign and malignant thyroid nodules based on the 2023 Bethesda System for Reporting Thyroid Cytopathology (BSRTC).A total of 615 thyroid nodules underwent ultrasound-guided fine-needle aspiration cytology (FNAC) were collected from Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The next-generation sequencing platform was applied for multi-gene testing. A panel of well-recognized commonly mutated genes in thyroid cancer were analyzed, including BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET.Gene mutations were identified in 324 nodules (52.7%), with BRAFV600E being the most prevalent driver gene alteration observed in this cohort (233/324; 79.1%), followed by RAS (77/324, 23.8%). The overall malignancy rate of gene mutations was 89.7% in our cohort, of which the lymph node metastasis rate was 45.3%. The combination of multi-gene testing and cytology resulted in 89.3% sensitivity, 95.2% specificity, 98.9% positive predictive value, 64.5% negative predictive value and 90.3% accuracy, which were significantly higher than those from mere cytology (sensitivity 68.6%, specificity 87.5%, positive predictive value 95.9%, negative predictive value 39.8%, accuracy 72.2%).Multi-gene testing could substantially enhance the detection rate of malignant thyroid nodules and protect patients with benign nodules from unnecessary surgeries. Multi-gene testing provides a valuable reference for individualized preoperative decision-making, which may serve as a crucial method for postoperative treatment and prognosis assessment.Thyroid neoplasms are common in the endocrine system, representing the most commonly diagnosed head and neck tumors. In the past few decades, the total incidence rate of thyroid neoplasm has increased about 2-fold and comprised 2% of the cancers. [1] Thyroid nodules are clinically prevalent and can be detected in approximately 20% to 76% of normal people by ultrasound techniques.[2] As the development of detecting and surgical techniques, an increasing number of thyroid nodules has been found as malignancy. In this context, early screening and differentiation of benign and malignant thyroid nodules are decisive for clinical decision-making. Currently, ultrasound-guided fine-needle aspiration cytology (FNAC) is widely applied in the preoperative diagnosis of benign and malignant thyroid nodules. It is interpreted based on The Bethesda System for Reporting Thyroid Cytopathology (BSRTC). However, a definitive diagnosis still cannot be achieved via the FNA for 15-30% of nodules.[3,4]With the recent advancement of molecular techniques to diagnose thyroid neoplasm, amounting evidence has proved that BRAFV600E -based FNA distinctly increases the detection rate of BRAF-mutated associated thyroid neoplasm.[5,6] However, studies on multi-gene testing have been mainly limited to the western population[7-10] and are less common in Asians. It is worth noting that different races may vary in molecular alterations in this type of tumor. Previous research showed that the common mutated gene: BRAFV600E, ranged from 29% to 83% (mean: 45%) in papillary thyroid cancer (PTC),[11,12] with higher alternation frequency in Asian people (70-80%)[13,14] and lower mutations in the Americans (40-50%),[15] although the rate has increased in the recent decade.[16]Given the growing need for individualized diagnosis and therapy, neither mere cytology nor single-gene testing is satisfactory for clinical practice. Herein, we established a 9-gene mutation panel (BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET) to sketch the gene mutation profile of thyroid neoplasms and to explore the diagnostic value of multi-gene testing in Chinese population based on BSRTC.