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.

The utilisation of digital droplet PCR to enhance the diagnosis of bladder and pancreaticobiliary tumours in cytology specimens

“…ddPCR requires much less input DNA than other molecular assays 4 . Studies have shown ddPCR is extremely sensitive and can detect low levels of mutant DNA, which were not detected by other less sensitive methods, that is standard PCR and Sanger sequencing 2,4,7,24 …”

“…4 Studies have shown ddPCR is extremely sensitive and can detect low levels of mutant DNA, which were not detected by other less sensitive methods, that is standard PCR and Sanger sequencing. 2,4,7,24 The detection of molecular alterations in cytology samples is now common practice. The presence of HER2 CNV in breast cancer, mutations in genes such as EGFR in lung cancers, KRAS in colorectal cancers and BRAF in melanoma and thyroid cancers are routinely tested at the time of diagnosis.…”

BRAF V600E Mutation Test in Fine Needle Aspiration (FNA) Specimens of Thyroid Nodules by Competitive Allele-Specific TaqMan PCR (castPCR)

“…It is estimated to be greater than 99%, which means that a positive BRAF V600E test result is very strong evidence that a thyroid nodule patient has PTC. Due to the high frequency of BRAF V600E mutation in PTC, the genetic test for diagnostic purposes has shown high sensitivity and specificity for the presence of tumor cells [2,26].…”

Diagnostic Value and Challenges of BRAF V600E Molecular Testing and Thyroid Fine-Needle Aspiration Cytology: A Retrospective Study from a Tertiary Institution in Southern Hunan Province, China

“…Its most common molecular event is an oncogenic alteration in the B-Raf proto-oncogene, serine/threonine kinase (BRAF), with valine-to-glutamate substitution at codon 600 (V600E) constituting the vast majority of the mutation [7]. Since approximately 78% of thyroid nodule FNA samples collected from Chinese patients contain the BRAF V600E mutation, several Chinese tertiary institutions utilize BRAF V600E molecular detection as ancillary testing to enhance the diagnostic efficiency of FNA cytology [8, 9].…”