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

  • Endocrine Methods and Techniques
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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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Funding

The study was supported by “Sapienza” University of Rome (grant RP11916B8919C921 to M.S.).

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Author notes

  1. These authors contributed equally: Antonella Verrienti, Valeria Pecce

Authors and Affiliations

  1. Department of Translational and Precision Medicine, “Sapienza” University of Rome, Rome, Italy

    Antonella Verrienti, Valeria Pecce, Luana Abballe, Valeria Ramundo, Rosa Falcone, Farzaneh Inanloo Nigi Jak, Giorgio Grani & Marialuisa Sponziello

  2. Division of Anatomic Pathology and Histology, Catholic University of Sacred Heart, Foundation “Agostino Gemelli” University Hospital IRCCS, Rome, Italy

    Chiara Brunelli & Guido Fadda

  3. Department of Radiological, Oncological and Pathological Sciences, “Sapienza” University of Rome, Rome, Italy

    Daniela Bosco, Valeria Ascoli, Raffaella Carletti & Cira Di Gioia

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  1. Antonella Verrienti
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  2. Valeria Pecce
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  3. Luana Abballe
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  4. Valeria Ramundo
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  5. Rosa Falcone
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  7. Chiara Brunelli
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  8. Guido Fadda
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  10. Valeria Ascoli
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  13. Giorgio Grani
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  14. Marialuisa Sponziello
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Corresponding author

Correspondence to Luana Abballe.

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All procedures involving humans were in accordance with the ethical standards of the institutional and/or national research committee (Ethics Committee of Azienda Universitaria Policlinico Umberto I, Rome, Italy; Prot. 806/16, Rif. 4233) and with the 1964 Helsinki Declaration and its later amendments.

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Verrienti, A., Pecce, V., Abballe, L. et al. Analytical validation of a novel targeted next-generation sequencing assay for mutation detection in thyroid nodule aspirates and tissue. Endocrine 69, 451–455 (2020). https://doi.org/10.1007/s12020-020-02372-8

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