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06.06.2020 | Endocrine Methods and Techniques

Analytical validation of a novel targeted next-generation sequencing assay for mutation detection in thyroid nodule aspirates and tissue

verfasst von: Antonella Verrienti, Valeria Pecce, Luana Abballe, Valeria Ramundo, Rosa Falcone, Farzaneh Inanloo Nigi Jak, Chiara Brunelli, Guido Fadda, Daniela Bosco, Valeria Ascoli, Raffaella Carletti, Cira Di Gioia, Giorgio Grani, Marialuisa Sponziello

Erschienen in: Endocrine | Ausgabe 2/2020

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Abstract

Purpose

The identification of somatic mutations in cancer specimens enables detection of molecular markers for personalized treatment. We recently developed a novel molecular assay and evaluated its clinical performance as an ancillary molecular method for indeterminate thyroid nodule cytology. Herein we describe the analytical validation of the novel targeted next-generation sequencing (NGS) assay in thyroid samples from different sources.

Methods

We present validation data of a novel NGS-based panel on 463 thyroid samples, including 310 fine-needle aspiration (FNA) specimens from different sources (FNA collected in preservative solution, liquid-based, and stained smears), 10 fresh frozen, and 143 formalin-fixed paraffin-embedded (FFPE) thyroid tissue specimens. Sequencing performance in the different samples was evaluated along with reproducibility, repeatability, minimum nucleic acid input to detect variants, and analytical sensitivity of the assay.

Results

All thyroid samples achieved high sequencing performance, with a mean base coverage depth ranging from 2228 × (in liquid-based FNA) to 3661 × (in FNA stained smears), and coverage uniformity ranging from 86% (in FFPE) to 95% (in FNA collected in preservative solution), with all target regions covered above the minimum depth required to call a variant (500×). The minimum nucleic acid input was 1 ng. Analytic sensitivity for mutation detection was 2–5% mutant allele frequency.

Conclusions

This validation study of a novel NGS-based assay for thyroid nodules demonstrated that the assay can be reliably used on multiple thyroid sample types, including FNA from different sources and FF and FFPE thyroid samples, thus providing a robust and reliable assay to genotype thyroid nodules, which may improve thyroid cancer diagnosis and care.

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Titel: Analytical validation of a novel targeted next-generation sequencing assay for mutation detection in thyroid nodule aspirates and tissue
verfasst von: Antonella Verrienti
Valeria Pecce
Luana Abballe
Valeria Ramundo
Rosa Falcone
Farzaneh Inanloo Nigi Jak
Chiara Brunelli
Guido Fadda
Daniela Bosco
Valeria Ascoli
Raffaella Carletti
Cira Di Gioia
Giorgio Grani
Marialuisa Sponziello
Publikationsdatum: 06.06.2020
Verlag: Springer US
Erschienen in: Endocrine / Ausgabe 2/2020
Print ISSN: 1355-008X
Elektronische ISSN: 1559-0100
DOI: https://doi.org/10.1007/s12020-020-02372-8

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Analytical validation of a novel targeted next-generation sequencing assay for mutation detection in thyroid nodule aspirates and tissue

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