Papillary thyroid carcinoma is the most common thyroid cancer of the endocrine system, with a relatively slow progression and a high survival rate (1, 2). Papillary thyroid cancer has been on the rise for three decades (3, 4). Fine-needle aspiration (FNA) cytology is the most commonly used method for diagnosing and categorizing thyroid carcinoma (1). Tumor cells in FNA biopsy samples vary in quantity, quality, and purity, making identification and diagnosis difficult (1). BRAF^V600E mutation has been established as an important molecular marker for papillary thyroid cancer diagnosis over the last decade, with a frequency of 65–80% (5, 6). As a result, a sensitive and accurate detection method for the BRAF^V600E mutation will aid in the early diagnosis of papillary thyroid carcinoma (7). For the detection of BRAF^V600E mutations, amplification-refractory mutation system (ARMS) and next-generation sequencing (NGS) are currently used, particularly NGS, which is a sensitive method in FNA samples with few mutant cells. However, NGS is expensive and inappropriate for the initial screening of all clinical patients. As a result, a more sensitive, low-cost, and accurate detection method is required.

The DNA mass spectroscopy (MS) method is a multiplexed medium-throughput ultra-sensitive mutation detection system. This method has been used successfully to detect mutations in patients with solid tumors, with a reported limit-of-detection frequency of 0.5% (8). It is far more sensitive and specific than other clinical methods currently in use. However, the detection of BRAF ^V600E mutations using MS has not been investigated in papillary thyroid carcinoma FNA samples. In this study, we used MS to detect the BRAF^V600E mutation in thyroid nodule FNA samples and compared its performance to that of NGS. Our study demonstrates the clinical significance of MS in the early detection of thyroid carcinoma.

We demonstrated in this study that both NGS and MS are effective methods for detecting BRAF ^V600E mutations in FNA biopsy samples of patients with thyroid nodules, with a strong inter-rater agreement, high specificity, and high PPV. Furthermore, the MS method demonstrated significant potential as a screening method for gene mutation detection and as an adjunct to postoperative pathological findings.

Several next-generation sequencing studies, including whole-genome sequencing (14), whole-exome sequencing (15, 16), and targeted sequencing (17, 18) have recently been conducted to investigate the genetic changes in papillary thyroid carcinoma. B-Raf proto-oncogene, serine/threonine kinase (BRAF^V600E), and telomerase reverse transcriptase (TERT) promoter mutations were the most frequently identified in papillary thyroid carcinoma. According to Cancer Genome Atlas (TCGA) cohort and several studies, the most common alterations are BRAF^V600E mutation in 62% (19, 20), TERT mutation in 22% (21), and RAS (including HRAS, NRAS, and KRAS) mutation in 13% (19). A high prevalence of BRAF^V600E mutations was also found in papillary thyroid carcinoma patients in a Chinese cohort study (22). In patients with papillary thyroid carcinoma, Khan and colleagues discovered that 94 percent of BRAF mutations were BRAF ^V600E mutations (21). Both the National Comprehensive Cancer Network (NCCN) and the Chinese Society of Clinical Oncology (CSCO) recommend testing for BRAF^V600E mutations as a supplement to clinical diagnosis. As a result, preliminary BRAF screening is required for all patients with thyroid nodules to aid clinical diagnosis. The BRAF^V600E mutation was found in 76.3% of the patients in this study, which was consistent with a Chinese cohort study (23).

TERT in 1.1%, and RAS (including HRAS, NRAS, and KRAS) mutation in 8.6% of the patients in this study.

Because of its high throughput, multi-gene coverage, and high precision, next-generation sequencing (NGS) is a widely known method for detecting solid tumor mutations. However, due to the high throughput and high cost of the equipment, NGS is not suitable for all thyroid tumors or nodules patients for initial screening, particularly for the detection of a BRAF gene (24). The MS method is a multiplexed ultrasensitive mutation detection system with a medium throughput. This method had previously been used successfully to detect mutations in patients with solid tumors, with a reported limit-of-detection frequency of 0.1% (8). In this study, we compared the efficacy of MS and NGS methods for detecting BRAF mutations. Our findings show that the MS method is a reliable and sensitive method for detecting BRAF^V600E mutations in thyroid tumors or nodules in patients’ FNA biopsy samples. Compared with NGS detection, the MS demonstrated greater sensitivity and specificity. However, the MS method did not detect three patients with BRAF^V600E. False-negative results were complicated because the study used retrospective samples. According to the NGS results, the BRAF mutation frequency in these three patients was relatively low. We hypothesized that the false-negative detection was a lack of sufficient tumor DNA in the remaining samples. The absence of false-positive cases demonstrates the MS method’s potential in clinical applications.

Furthermore, we compared the efficiency of MS and NGS methods in detecting multiple-gene mutations. The MS method maintained high specificity and sensitivity, as expected. However, 11.8% of patients were reported negative because they had mutations that were not found in the MS panel. Intriguingly, MS results from NGS were consistent in a patient with BRAF^V600E and TERT mutations. Previous research has shown that patients with both BRAF^V600E and TERT mutations have a poor prognosis, so simultaneous multigene screening is necessary (13).

The MS and NGS results were also evaluated using the postoperative pathological findings as the standard. The MS method had higher sensitivity and specificity with pathological findings, and the majority of false-negative patients had out-of-panel mutations. Although the 11-gene panel of NGS demonstrated higher sensitivity due to the greater number of non-BRAF mutations covered, NGS demonstrated lower specificity due to non-BRAF mutations detected in benign lesions (12, 25). Three of the five patients with benign lesions had RAS mutations, and one had an NTRK3 fusion. Previous research has shown that these mutations can be found in both malignant and benign lesions (11, 12, 25). In addition, all malignant in our study were PTC. Previous studies have confirmed that non-BRAF genes were more common in other subtype thyroid carcinoma patients, which may have higher diagnostic value. For example, RAS mutation in follicular thyroid carcinoma was more common than BRAF (12). Moreover, 7 patients had the results of clinical diagnosis in 11 patients with non-BRAF mutations. Of the 7 patients, 4 (57.14%) patients were benign lesions, and 3 of 4 patients harbored RAS mutations. Although several studies have demonstrated that multigene testing can improve the specificity of clinically assisted diagnosis (26–28). Previous research and CSCO guidelines have shown that RAS mutations have an unsatisfactory clinical impact on the management of thyroid nodules (29). As a result, the NCCN and CSCO guidelines recommend BRAF as the primary screening gene for adjunctive diagnosis, and these non-BRAF genes are not required for initial screening. These findings imply that the MS method can be used as a primary screening method for molecular detection in patients.

There were a few limitations to this study as well. Due to the small number of patients in our cohort who had TERT and TP53 mutations, the feasibility of the MS method in this population needs to be investigated further. Second, the importance of multiple-gene detection should be discussed further. Because our study was retrospective, residual DNA from NGS detection was used as samples in MS detection, limiting the sample size of this study. The possibility of full-process detection of clinical FNA samples should be investigated further.

Finally, our findings showed that the MS method was a precise and dependable alternative for detecting BRAF mutations in patients with thyroid nodules. Furthermore, the MS method as primary screening for molecular detection was promising.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Ethics Committee of Jiangsu Hospital of Integrated Traditional Chinese and Western Medicine and Jiangsu University Affiliated People’s Hospital. All procedures were in accordance with the 1964 Declaration of Helsinki and its later amendments. The patients/participants provided their written informed consent to participate in this study.

X-QQ and EA contributed to the conception and design of the study. Y-GW and F-JX contributed to the acquisition of data. L-LZ, R-LY, and X-YL assisted in sample management and experiment operation. F-JX, NY, MX, and D-JG organized the database. NY, F-JX, YY and MX contributed to the confirmation of the authenticity of the data. Y-GW, X-QQ, EA, WX-P and D-JG performed data analysis and interpretation. EA performed review and editing. All authors contributed to the manuscript and read and approved the final manuscript. All authors contributed to the article and approved the submitted version.

This study was financially supported by National Natural Science Foundation of China (Project No. 81971629) and Zhenjiang Commission of Science and Technology (Project No. SH2020046).

NY was employed by Dian Diagnostics Group Co., Ltd and MX was employed by Zhejiang Digena Diagnosis Technology Co. Ltd.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.