Comparison of droplet digital PCR and direct Sanger sequencing for the detection of the BRAF V600E mutation in papillary thyroid carcinoma

Abstract Background The BRAF V600E mutation status is a useful diagnostic and prognostic marker for papillary thyroid carcinoma (PTC). Although it is a commonly used method, Sanger sequencing has several limitations in detecting the BRAF V600E mutation. The aim of this study was to evaluate the efficiency of droplet digital PCR (ddPCR) as an alternative method for the detection of the BRAF V600E mutation in PTC patients. Methods Samples from a total of 120 patients with PTC and 30 patients with benign nodular thyroid disease who underwent thyroid surgery were collected. The BRAF V600E mutation status of the PTC patients was tested by Sanger sequencing and ddPCR. Results The BRAF V600E mutation was detected in 67 samples (44.67%) by Sanger sequencing and 92 samples (61.33%) by ddPCR. The detection of the mutation by the two methods was inconsistent in twenty‐five samples (16.67%). The sensitivity and specificity of the ddPCR method were 100% and 69.88%, respectively, and the positive predictive and negative predictive values were 72.83% and 100%, respectively. The concordance rate between the two methods in detecting the BRAF V600E mutation was 83.33%. Neither Sanger sequencing nor ddPCR detected BRAF V600E in 30 patients with benign nodular thyroid disease. The 92 samples with the BRAF V600E mutation were detected by ddPCR at a fractional abundance from 0.28% to 45.40% as follows: ≥10% (59 samples, 64.13%), 5%‐10% (8 samples, 8.70%), and ≤5% (25 samples, 27.17%). The BRAF V600E mutation was detected in all 59 samples at a fractional abundance ≥10% and in four samples at a fractional abundance from 5% to 10%, and no BRAF V600E mutation was detected at a fractional abundance ≤5% by Sanger sequencing. Conclusions ddPCR was a reliable, highly sensitive alternative method for the detection of the BRAF V600E mutation in PTC patients.

specificity of the ddPCR method were 100% and 69.88%, respectively, and the positive predictive and negative predictive values were 72.83% and 100%, respectively.
Conclusions: ddPCR was a reliable, highly sensitive alternative method for the detection of the BRAF V600E mutation in PTC patients.

K E Y W O R D S
BRAF V600E mutation, ddPCR, papillary thyroid carcinoma, Sanger sequencing

| INTRODUC TI ON
Thyroid cancer (TC) is the most common type of endocrine malignancy, and papillary thyroid carcinoma (PTC) accounts for the vast majority (90%) of thyroid malignancies. 1,2 BRAF V600E has a T1799A point mutation in exon 15, resulting in the substitution of valine for glutamic acid at amino acid 600 (V600E), and it is the most common driver mutation of PTC. In addition, the BRAF V600E mutation is associated with aggressive clinical features and a higher risk of recurrence of PTC 3 and may helpful in detecting malignancy in some thyroid nodules. 4,5 Many techniques can be used for the detection of the BRAF V600E mutation, [6][7][8] and the gold standard method for the molecular diagnosis of the BRAF V600E mutation is direct Sanger sequencing. 2 However, Sanger sequencing identified only 7%-20% of mutated alleles in a background of wild-type alleles. 8,9 Droplet digital PCR (ddPCR) is a recently introduced technol- The limit of detection of ddPCR is 0.0005%. 11 Therefore, ddPCR analysis sensitively detects and quantifies low-abundance gene mutations like the BRAF V600E mutation.
In the present study, the BRAF V600E mutation was detected by ddPCR and Sanger sequencing. ddPCR was a better method than Sanger sequencing in detecting the BRAF V600E mutation in PTC samples.

| Patients
A total of 150 patients who underwent thyroid surgery between November 2016 and February 2019 in the Jiangsu Cancer Hospital were included in this study. All specimens were obtained after thyroid surgery, fixed in formalin, and embedded in paraffin. These samples were from 120 patients with PTC and 30 patients with benign nodular thyroid disease.
Tumor staging was performed according to the seventh edition of the tumor-node-metastasis (TNM) classification by the American Joint Committee on Cancer (8th edition). All patients who participated in the study gave their informed consent.

| DNA extraction
Hematoxylin-eosin slides prepared from specimens were examined by two experienced pathologists to estimate the areas enriched in tumor cell populations. Tissue was scraped from this preselected area and transferred to an Eppendorf tube for DNA extraction using the QIAamp FFPE Tissue Kit (Qiagen). Premix Taq™ Hot Start Version (Takara). The purified PCR products were sequenced using the forward primer above and a BigDye

Patients with benign nodular thyroid disease
Terminator v 3.1 kit (Thermo Fisher). Capillary separation and data collection were performed on an ABI 3500 Genetic Analyzer.

| ddPCR analysis to detect the BRAF V600E mutation
The

| Clinical characteristics
The clinical characteristics of the patients are shown in Table 1.
Of the included patients, 62 (41.33%) were male and 88 (58.67%) were female, and the average age was 57 years (ranged from 17 to 72).

| D ISCUSS I ON
The BRAF V600E mutation is the most common driver mutation in PTC and acts as a useful diagnostic and prognostic marker for PTC. [12][13][14] Sanger sequencing is the gold standard for detecting the BRAF V600E mutation and is a commonly used method in laboratories.
Because of its relatively low sensitivity, the detection of mutations requires a large amount of tumor DNA in the samples. ddPCR is a relatively new method with multiple advantages, such as improved sensitivity and absolute quantification. 15 Table 5.
The BRAF V600E mutation was found in 55.83% of PTC patients by Sanger sequencing and 76.67% by ddPCR in the present study.
A higher mutation rate was detected by ddPCR. These results were consistent with previous studies that showed a BRAF V600E mutation rate of 29%-90%. 2,14,20,21 In the present study, the BRAF V600E mutation was only detected in PTC patients and not in patients with benign nodular thyroid disease, which was also consistent with previous studies. 22,23 It is important to note that mutations at a fractional abundance