This paper discusses the development of an expanded mutation panel (ThyGeNEXT®). Additionally, it demonstrates the analytical validation of the expanded NGS panel test and compares analytical characteristics of ThyGeNEXT to the original (focused mutational panel), which it superseded, thereby providing the basis for clinical validation.
Included are descriptions of the development of the expanded NGS panel 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 CAP/CLIA certified laboratory. Analytical validation of ThyGeNEXT as a laboratory-developed test included the demonstration of assay reproducibility and lower limit of detection, as well as other fundamental quality parameters.
Data presented here firmly demonstrate that the qualitative and quantitative performance of ThyGeNEXT is similar to the original smaller, focused panel test, applicable to both FNA biopsies placed directly into RNA protectant and stained cytology smear slides. The expanded test was found not to generate false positive or false negative molecular results for the five genes (BRAF, HRAS, KRAS, NRAS, PIK3CA) and six fusions (PAX8-PPARγ and RET-PTC) previously reported by the focused test. At the same time, expanded testing clearly provided additional molecular information on gene alterations potentially associated with aggressive forms of differentiated thyroid cancer for poor outcome (eg, TERT, RET, and ALK genes).
ThyGeNEXT is designed to be used in conjunction with a 10-miRNA classifier (ThyraMIR®). Classifiers based upon microRNA expression are advantageous in identifying malignancy in nodules without the determination of the occurrence of various oncogenic mutations. Although neither approach is perfect, with limitations preventing complete separation of cancer from non-cancer states, they can complement each other and thereby improve test performance.