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

Abstract

BackgroundThe 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.MethodsSamples 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.ResultsThe 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.ConclusionsddPCR was a reliable, highly sensitive alternative method for the detection of the BRAF V600E mutation in PTC patients.

Prognostic value ofBRAF/MIR‐17 signature and B‐Raf protein expression in patients with colorectal cancer: A pilot study

“…Also, it will be interesting to explore the association of BRAF/MIR17 signature with the most common BRAF V600E mutation by the newly emerged PCR techniques to add to its clinical utility. 49,50 Based on these limitations, future large-scale studies supported by functional in vitro and gene mutation exploring studies are warranted to confirm and validate the results.…”

Superiority of Droplet Digital PCR Over Real-Time Quantitative PCR for JAK2 V617F Allele Mutational Burden Assessment in Myeloproliferative Neoplasms: A Retrospective Study

“…By using highly sensitive methods, like ddPCR, we did not find a JAK2 V617F mutation in the control population, probably due to the low number of control samples used in our study. An increasing number of techniques have been developed for JAK2 V617F mutation identification by using molecular approaches including qPCR, such as high-resolution melt (HRM) [19], allele-specific oligonucleotide (ASO-PCR) [20], DNA direct sequencing [21], and recently ddPCR [22]. These techniques have different levels of LoD and specificity.…”

“…These techniques have different levels of LoD and specificity. Direct sequencing has a reported LoD of approximately 15% mutant alleles [21], ASO-RT-PCR has a LoD of around 1% [22], while ASO-ddPCR and locked nucleic acid (LNA) RT-PCR have greater sensitivity for detecting rare mutations, with a limit of detection of about 0.1% [23][24][25]. Several studies have suggested that the JAK2 V617F mutational load correlates with disease course and could therefore be a predictive marker.…”

VE1 immunohistochemistry is an adjunct tool for detection of BRAFV600E mutation: Validation in thyroid cancer patients

“…In routine molecular pathology laboratories, different molecular methodologies such as Sanger sequencing, allele‐specific PCR (AS‐PCR), droplet digital PCR (ddPCR) and polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) are employed for the detection of BRAF V600E mutation 7,15‐18 . Although different techniques have different performance rates and sensitivities, the technique performs to detect the mutation may have significant correlation with prevalance of BRAF V600E .…”