Comparison of Immunohistochemistry and Direct Sanger Sequencing for Detection of the<i>BRAF</i><sup>V600E</sup>Mutation in Thyroid Neoplasm

Oh, Hye Seon; Kwon, Hyemi; Park, Suyeon; Kim, Mijin; Jeon, Min Ji; Kim, Tae Yong; Shong, Young Kee; Kim, Won; Choi, Jeongyong; Kim, Won Gu; Song, Dong Eun

doi:10.3803/enm.2018.33.1.62

Cited by 24 publications

(30 citation statements)

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“…Many techniques can be used for the detection of the BRAF V600E mutation, and the gold standard method for the molecular diagnosis of the BRAF V600E mutation is direct Sanger sequencing . However, Sanger sequencing identified only 7%‐20% of mutated alleles in a background of wild‐type alleles …”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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 technology that may facilitate the detection of rare point mutations in a background of wild-type alleles. The ddPCR technique is based on the partitioning of the sample into thousands of microreactions of a defined volume.…”

Section: Introductionmentioning

confidence: 99%

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

Wang

Sun²,

Jing

et al. 2019

Clinical Laboratory Analysis

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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.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Wang

Sun²,

Jing

et al. 2019

Clinical Laboratory Analysis

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“…Although Sanger sequencing has been widely acknowledged as the standard technique for detection of point mutation, from the literature review, we found out that the discordant rates are much higher when using Sanger sequencing (7-23%) to validate the performance of VE1 IHC [20,23,25,31,35,[38][39][40], than using a more sensitive molecular method, such as real-time PCR (0.1-8%) [19,22,23,[41][42][43][44][45]. Several groups achieved a discordance rate of < 2% when VE1 immunostaining was compared to real-time PCR [22,43].…”

Section: Discussionmentioning

confidence: 90%

“…There are different DNA-based techniques available for the detection of BRAF V600E mutation in PTC [17]. Sanger sequencing has been widely utilized as the gold standard method; however, it has a reported detection sensitivity of only 10-20% of mutant allele frequency, and therefore, this method alone might not be sufficient to detect the mutation in cases with low tumor cellularity [18][19][20]. Pyrosequencing and real-time polymerase chain reaction (PCR) are known to be more sensitive than Sanger sequencing, with a reported detection sensitivity of 5% and 1% mutant alleles, respectively [21][22][23].…”

Section: Introductionmentioning

confidence: 99%

VE1 Immunohistochemistry Improves the Limit of Genotyping for Detecting BRAFV600E Mutation in Papillary Thyroid Cancer

Choden

Keelawat

Jung

et al. 2020

Cancers

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Detection of BRAFV600E is useful for making diagnosis and risk stratification of papillary thyroid carcinoma (PTC). Molecular testing, however, is not always available for routine clinical use. To assess the clinical utility and reliability of VE1 immunohistochemistry (IHC) for detecting BRAFV600E mutation in PTC, VE1 IHC was performed on the tissue microarrays of 514 patients with PTC and was compared with Sanger sequencing results. Of 514 PTC cases, 433 (84.2%) were positive for VE1 expression. Among 6 discordant cases between VE1 IHC and Sanger sequencing, 3 initial VE1-false negative cases turned out to be true false negative on repeat testing, and 3 VE1-false positive cases showed BRAFV600E mutation using digital PCR analysis. PTCs with low variant allele fraction were positive for VE1 IHC but were not detected using sequencing. VE1 IHC showed 99.3% sensitivity, 100% specificity, 100% positive predictive value, and 96.4% negative predictive value. The BRAFV600E mutation was significantly associated with older age, multifocality, extrathyroidal extension, lymph node metastasis, and advanced tumor stage. In conclusion, VE1 IHC is a reliable method for detecting BRAFV600E mutation in PTC specimens.

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Development of a Molecular Assay for Detection and Quantification of theBRAFVariation in Residual Tissue From Thyroid Nodule Fine-Needle Aspiration Biopsy Specimens

Chazen

MacMillan

et al. 2021

JAMA Netw Open

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IMPORTANCE Thyroid cancer, predominantly papillary thyroid carcinoma (PTC), is common, but an estimated 30% of ultrasonography-guided fine-needle aspiration (FNA) biopsies of thyroid nodules are indeterminate. BRAF variation, associated with poor clinicopathological characteristics, is a useful molecular marker for diagnostics. OBJECTIVETo develop a sensitive molecular assay for BRAF V600E detection in remaining tissue of thyroid FNA biopsies to identify patients with cancer carrying a BRAF variation. DESIGN, SETTING, AND PARTICIPANTSThis diagnostic study used tumor tissue from surgical formalin-fixed, paraffin-embedded (FFPE) specimens and residual tissue from thyroid FNA biopsies for genomic DNA extraction. FFPE specimens served as the validation set, and residual tissue from FNA biopsies served as the test set. A molecular assay was developed for accurate detection of BRAF V600E variation using locked nucleic acid (LNA) probe-based droplet digital polymerase chain reaction (dPCR), and the assay was validated by BRAF V600E immunohistochemical staining (IHC). The study was conducted between February 2019 and May 2021. RESULTS A total of 271 specimens, including 77 FFPE specimens (with a follow-up of 48 matched surgical specimens) and 146 residual FNA samples, were collected from 223 patients (mean [SD] age, 53.8 [15.3] years; 174 [78.0%] women; 49 [22.0%] men). The molecular assay by dPCR was first established to specifically and accurately detect and quantify wild-type BRAF and variant BRAF in DNA from human follicular thyroid carcinoma-derived FTC-133 and papillary thyroid carcinomaderived BCPAP cells. The linearity of quantification of BRAF V600E was calculated (y = 0.7339x;R 2 = 0.9996) with sensitivity at 0.02 copies/μL and reproducibility in detecting variant DNA at various dilutions(coefficient of variance in 0.3% DNA, 9.63%; coefficient of variance in 1.0% DNA, 7.41%). In validation testing, the dPCR assay and IHC staining exhibited 100% specificity in concordantly identifying BRAF V600E in PTCs (κ = 0.873; P < .001) and sensitivity of 32.0% (95% CI, 19.1% to 44.9%) in dPCR and 26.0% (95% CI, 13.1% to 38.9%) in IHC staining, with an improvement by 23.08% in dPCR compared with the IHC staining. The dPCR assay further detected BRAF V600E in 39 of 146 residual FNA specimens (26.7%). At short-term follow-up, 48 patients, including 14 of 39 patients with BRAF variation and 34 of 107 patients without BRAF variation on residual FNA specimens, underwent resection. The dPCR assay of BRAF status in the matched surgical specimens showed BRAF V600E variations in 12 patients and wild-type BRAF in 36 patients, with a high agreement to that in residual tissue of FNA specimens (κ = 0.789; P < .001). Among 14 patients with BRAF variations on residual FNA, 13 were diagnosed with PTC and 1 was diagnosed with anaplastic thyroid cancer at the thyroidectomy.

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Comparison of Immunohistochemistry and Direct Sanger Sequencing for Detection of theBRAF^V600EMutation in Thyroid Neoplasm

Cited by 24 publications

References 35 publications

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

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

VE1 Immunohistochemistry Improves the Limit of Genotyping for Detecting BRAFV600E Mutation in Papillary Thyroid Cancer

Development of a Molecular Assay for Detection and Quantification of theBRAFVariation in Residual Tissue From Thyroid Nodule Fine-Needle Aspiration Biopsy Specimens

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Comparison of Immunohistochemistry and Direct Sanger Sequencing for Detection of theBRAFV600EMutation in Thyroid Neoplasm

Cited by 24 publications

References 35 publications

Comparison of droplet digital PCR and direct Sanger sequencing for the detection of the BRAFV600E mutation in papillary thyroid carcinoma

Comparison of droplet digital PCR and direct Sanger sequencing for the detection of the BRAFV600E mutation in papillary thyroid carcinoma

VE1 Immunohistochemistry Improves the Limit of Genotyping for Detecting BRAFV600E Mutation in Papillary Thyroid Cancer

Development of a Molecular Assay for Detection and Quantification of theBRAFVariation in Residual Tissue From Thyroid Nodule Fine-Needle Aspiration Biopsy Specimens

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Product

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Comparison of Immunohistochemistry and Direct Sanger Sequencing for Detection of theBRAF^V600EMutation in Thyroid Neoplasm

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

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