RESUMO
Molecular analysis is used to evaluate the risk of malignancy for thyroid fine-needle aspirates, identified as indeterminate by microscopic cytology, on the basis of the detection of various oncogenic DNA mutations and fusion transcripts or on the use of various mRNAs or miRNA-based classifier algorithms. Our approach has been to use a combination test using the detection of oncogenic mutations/fusion transcripts and an miRNA expression-based classifier algorithm. To improve the performance of the combination test, the next-generation sequencing (NGS)-based mutational panel was expanded from the detection of 5 oncogenes to 10 oncogenes and tumor suppressor genes and the detection of fusion transcripts was increased from 6 to 38. Herein, we describe the assay development of the expanded panel NGS test and optimization of various steps for the library preparation of multiplexed target genes to maintain quality parameters for sequencing and to improve the robustness of the test for use in clinical testing in a College of American Pathologists/Clinical Laboratory Improvements Amendments-certified laboratory. Technical hurdles in NGS library preparation for the sequencing of both normal and high guanine-cytosine-rich regions, and balanced amplification of various amplicons in highly multiplexed PCRs, were successfully overcome. Analytical validation as a laboratory-developed test (ThyGeNEXT) included the demonstration of assay reproducibility, lower limit of detection, as well as other fundamental quality parameters.
Assuntos
Biomarcadores , Análise Mutacional de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Nódulo da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/etiologia , Alelos , Substituição de Aminoácidos , Biomarcadores Tumorais , Biópsia por Agulha Fina , Análise Mutacional de DNA/métodos , Diagnóstico Diferencial , Frequência do Gene , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/genéticaRESUMO
BACKGROUND: Limited data are available on the analysis of somatic mutations in metastatic lymph nodes in adult and pediatric patients with papillary thyroid carcinomas. METHODS: A total of 92, microdissected, formalin-fixed, paraffin-embedded tissue specimens from 39 patients were analyzed for the presence of somatic mutations utilizing the ThyGenX next-generation sequencing test. RESULTS: Somatic mutations were detected in 67% of papillary thyroid carcinoma specimens. The majority of patients with synchronous and all 6 patients with radioactive iodine-resistant (metachronous) metastatic lymph nodes contained BRAF mutations. Four patients had mutations detected in their metastatic lymph nodes that were not detected in their primary tumors. For the most part, BRAF mutations were seen in adults, and RAS mutations were seen in children. CONCLUSION: Findings of different mutations in metastatic lymph nodes compared with the primary papillary thyroid carcinomas are probably the result of tumor heterogeneity. The presence of the BRAF mutation in metastatic lymph nodes might be responsible for the recurrence of papillary thyroid carcinomas and resistance to radioactive iodine therapy. The good prognosis observed in papillary thyroid carcinomas found in pediatric and young adult patients might be explained by the predominance of RAS rather than BRAF mutations.
