Dataset Information

Detection of BRAF V600E in Fine-Needle Aspiration Samples of Thyroid Nodules by Droplet Digital PCR.

ABSTRACT:

Background

BRAF exon 15 p.V600E (BRAF V600E) mutation has been established as an important molecular marker for papillary thyroid carcinoma diagnosis by ultrasound-guided fine-needle aspiration biopsy (FNAB). Sanger sequencing is the gold standard for detecting BRAF V600E mutations but fails to identify low-frequency mutations. However, droplet digital PCR (ddPCR) is a popular new method for detecting low-frequency mutations. Here, we compare the efficiency of droplet digital PCR (ddPCR) and Sanger sequencing for detection of the BRAF V600E mutation in thyroid fine-needle aspiration (FNA) samples.

Methods

Thyroid fine-needle aspiration samples from 278 patients with 310 thyroid nodules were collected. Sanger sequencing and ddPCR were conducted to detect the BRAF V600E mutation.

Results

The BRAF V600E mutation was found in 94 nodules (30.32%) by ddPCR and 40 nodules (12.90%) by Sanger sequencing in 310 FNA samples. A total of 119 nodules were confirmed PTC by postsurgical pathology. Among which the BRAF mutation was found in 80 (67.23%) nodules by ddPCR and 31 (26.05%) by Sanger sequencing. All nodules carrying the mutation detected by Sanger sequencing (SS+) were verified by ddPCR (ddPCR+). Also, all nodules with no mutation detected by ddPCR were interpreted as wild-type by Sanger sequencing (SS-). In addition. Almost all SS+/ddPCR + nodules (95.00%; 38/40) and SS-/ddPCR + nodules (100.00%; 54/54) displayed a BRAF mutation rate of >5% and <15%, respectively, indicating easy misdetection by Sanger sequencing when the mutation rate is between 5 and 15%.

Conclusion

ddPCR has higher sensitivity than Sanger sequencing and we propose ddPCR as a supplement to Sanger sequencing in molecular testing of BRAF using FNAB samples.

SUBMITTER: Lu SY

PROVIDER: S-EPMC8983273 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

Detection of <i>BRAF</i> V600E in Fine-Needle Aspiration Samples of Thyroid Nodules by Droplet Digital PCR.

Lu Sang-Yu SY Chen Ying-Chao YC Feng Jia-Lin JL Zhou Qin-Yi QY Chen Jing J Zhu Chen-Fang CF Guo Miao-Miao MM Zhang Man-Man MM Zhang Qian-Yue QY Lu Meng M Yang Liu L Wu Jing J Zhao Shuang-Xia SX Song Huai-Dong HD Ye Xiao-Ping XP

International journal of endocrinology 20220329

<h4>Background</h4><i>BRAF</i> exon 15 p.V600E (<i>BRAF</i> V600E) mutation has been established as an important molecular marker for papillary thyroid carcinoma diagnosis by ultrasound-guided fine-needle aspiration biopsy (FNAB). Sanger sequencing is the gold standard for detecting <i>BRAF</i> V600E mutations but fails to identify low-frequency mutations. However, droplet digital PCR (ddPCR) is a popular new method for detecting low-frequency mutations. Here, we compare the efficiency of drople ...[more]

PMID: 35392249

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Project description:BackgroundThe BRAFV600E mutation is valuable for the diagnosis, prognosis, and therapy of papillary thyroid cancer (PTC). However, studies related to this mutation have involved only a small number of patients. Therefore, we performed a large-scale analysis from a single institute to evaluate the accuracy of combined fine-needle aspiration (FNA) and BRAFV600E mutation tests for PTC diagnosis.MethodsA total of 4600 patients with thyroid nodules who underwent both FNA cytology and BRAFV600E mutation analysis on FNA specimens were enrolled. The association between the BRAFV600E mutation and clinicopathological features was analyzed. A separate analysis was performed for the 311 patients who underwent repeated FNA for comparison of cytological evaluation and BRAFV600E mutation results. The diagnostic efficacy of the BRAFV600E mutation test and cytologic diagnoses was evaluated for 516 patients who underwent preoperative FNA tests in comparison with conclusive postoperative histopathologic results.ResultsThe cytology results of all 4600 FNA samples were categorized according to The Bethesda System for Reporting Thyroid Cytology (TBSRTC) stages I-VI, which accounted for 11.76%, 60.02%, 6.46%, 3.61%, 6.71%, and 11.43% of the samples, respectively. The BRAFV600E mutation was detected in 762 (16.57%) FNA samples, with rates of 1.48%, 0.87%, 20.20%, 3.01%, 66.02%, and 87.81% for TBSRTC I-VI lesions, respectively. Among the 311 repeat FNA cases, 81.0% of the BRAFV600E -positive and 4.3% of the BRAFV600E -negative specimens with an initial indication of cytological non-malignancy were ultimately diagnosed as malignant by repeat FNA (p < 0.001). Among the 516 patients who underwent thyroidectomy, the sensitivity and specificity of the BRAFV600E mutation test alone for PTC diagnosis were 76.71% and 100.0%, respectively, which increased to 96.62% and 88.03%, respectively, when combining the BRAFV600E mutation test with cytology. BRAFV600E mutation was significantly associated with lymph node metastasis (p < 0.001), but not with age, gender, or tumor size.ConclusionsThe BRAFV600E mutation test in FNA samples has potential to reduce false negatives in PTC diagnosis, and therefore plays an important role in the diagnosis of thyroid nodules, especially those with an indeterminate or nondiagnostic cytology, which should be considered for repeat FNA.