Dataset Information

Utility of droplet digital polymerase chain reaction for TERT and BRAF mutational profiling of thyroid nodules.

ABSTRACT:

Background

Mutations involving BRAF and TERT are important predictors of disease severity in thyroid cancer, but molecular testing is limited by cost and lack of adequate tissue sample. This study aimed to assess the utility of BRAFV600E and TERT testing using droplet digital PCR (ddPCR) as a diagnostic and prognostic tool for thyroid fine needle aspirate biopsy (FNAB).

Methods

Patients with thyroid nodules were prospectively enrolled from March 2015 to September 2018. Pre-operative FNAB was collected for standard cytology and molecular testing. BRAFV600E and TERT levels were analyzed by ddPCR. Cytology (Bethesda system) and ddPCR results were correlated to surgical pathology.

Results

A total of 222 patients were enrolled, of which 124 received thyroid surgery. Pre-operative cytology alone with Bethesda ≥5 was 100% specific and 70% sensitive for malignancy on final surgical pathology. BRAFV600E positivity or TERT overexpression was 100% specific and 60.0% sensitive. Combining cytology (Bethesda ≥5) with BRAFV600E and TERT testing increased the sensitivity of a malignant diagnosis to 80.0%. High TERT levels and/or BRAFV600E was associated with aggressive or advanced stage pathology.

Conclusions

Combining cytology with ddPCR analysis of BRAFV600E and TERT can improve the diagnostic accuracy of thyroid FNAB, and help predict aggressive pathology.

SUBMITTER: Rosvall BR

PROVIDER: S-EPMC8547072 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Publications

Utility of droplet digital polymerase chain reaction for TERT and BRAF mutational profiling of thyroid nodules.

Rosvall Brandon R BR Kostiuk Morris M Williams Jordana J Matkin Ashlee A Harris Jeffrey J Seikaly Hadi H O'Connell Daniel A DA Biron Vincent L VL

BMC cancer 20211026 1

<h4>Background</h4>Mutations involving BRAF and TERT are important predictors of disease severity in thyroid cancer, but molecular testing is limited by cost and lack of adequate tissue sample. This study aimed to assess the utility of BRAFV600E and TERT testing using droplet digital PCR (ddPCR) as a diagnostic and prognostic tool for thyroid fine needle aspirate biopsy (FNAB).<h4>Methods</h4>Patients with thyroid nodules were prospectively enrolled from March 2015 to September 2018. Pre-operati ...[more]

PMID: 34702207

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Project description:Digital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. This holds true in particular when independent transgene and plant-specific reference gene copy numbers have to be combined to determine the content of GM material in a sample. Droplets which show fluorescent units ranging between those of explicit positive and negative droplets are called 'rain'. Signals of such droplets can hinder analysis and the correct setting of a threshold. In this manuscript, a computer-based algorithm has been carefully designed to evaluate assay performance and facilitate objective criteria for assay optimization. Optimized assays in return minimize the impact of rain on ddPCR analysis. We developed an Excel based 'experience matrix' that reflects the assay parameters of GMO ddPCR tests performed in our laboratory. Parameters considered include singleplex/duplex ddPCR, assay volume, thermal cycler, probe manufacturer, oligonucleotide concentration, annealing/elongation temperature, and a droplet separation evaluation. We additionally propose an objective droplet separation value which is based on both absolute fluorescence signal distance of positive and negative droplet populations and the variation within these droplet populations. The proposed performance classification in the experience matrix can be used for a rating of different assays for the same GMO target, thus enabling employment of the best suited assay parameters. Main optimization parameters include annealing/extension temperature and oligonucleotide concentrations. The droplet separation value allows for easy and reproducible assay performance evaluation. The combination of separation value with the experience matrix simplifies the choice of adequate assay parameters for a given GMO event.

Dataset Information

Utility of droplet digital polymerase chain reaction for TERT and BRAF mutational profiling of thyroid nodules.

Background

Methods

Results

Conclusions

Publications

Utility of droplet digital polymerase chain reaction for TERT and BRAF mutational profiling of thyroid nodules.

Similar Datasets

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