Impact of different methodologies on the detection of point mutations in routine air-dried fine needle aspiration...

Currently the best method to select suspicious thyroid nodules for surgery is fine needle aspiration (FNA) cytology. However, FNA cytology has some inherent limitations, which can partly be overcome by molecular analysis. Therefore, molecular testing for somatic mutations has emerged as the most promising approach for molecular FNA diagnostics. The objective of this methodological study was to evaluate the feasibility of detecting BRAF, NRAS, HRAS, and KRAS mutations from routine air-dried thyroid FNA smears, and to find an optimal method for detecting these mutations in FNA samples. DNA was extracted from 110 routine air-dried FNA smears and the corresponding surgically obtained formalin-fixed paraffin-embedded tissues. The presence of BRAF, NRAS, HRAS, and KRAS mutations was assessed by real-time PCRs and high resolution melting analysis, and/or pyrosequencing in comparison to real-time PCRs using hybridization probes and fluorescence melting curve analysis. The high-resolution melting-PCRs revealed a significantly lower number of PCR failures and questionable results, and detected more mutations than the PCRs using hybridization probes. The number of PCR failures ranging from 14-16% by high-resolution melting-PCRs could be further reduced to 5-14% by adding pyrosequencing assays. Moreover, pyrosequencing increased the specificity of the assays, up to 98-100%, while the sensitivity ranged between 32-63%. In summary, the mutation detection, especially in air-dried FNA samples, improves when using PCR assays in combination with high resolution melting analysis. Additional improvement can be obtained by subsequent pyrosequencing in comparison to previously described real-time PCRs using hybridization probes and fluorescence melting curve analysis.