Abstract
PurposeWe exploited the MassARRAY (MA) genotyping platform to develop the “PTC-MA assay”, which allows the simultaneous detection of 13 hotspot mutations, in the BRAF, KRAS, NRAS, HRAS, TERT, AKT1, PIK3CA, and EIF1AX genes, and six recurrent genetic rearrangements, involving the RET and TRK genes in papillary thyroid cancer (PTC).MethodsThe assay was developed using DNA and cDNA from 12 frozen and 11 formalin-fixed paraffin embedded samples from 23 PTC cases, together with positive and negative controls.ResultsThe PTC-MA assay displays high sensitivity towards point mutations and gene rearrangements, detecting their presence at frequencies as low as 5%. Moreover, this technique allows quantification of the mutated alleles identified at each tested locus.ConclusionsThe PTC-MA assay is a novel MA test, which is able to detect fusion genes generated by genomic rearrangements concomitantly with the analysis of hotspot point mutations, thus allowing the evaluation of key diagnostic, prognostic, and therapeutic markers of PTC in a single experiment without any informatics analysis. As the assay is sensitive, robust, easily achievable, and affordable, it is suitable for the diagnostic practice. Finally, the PTC-MA assay can be easily implemented and updated by adding novel genetic markers, according to clinical requirements.
Highlights
Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Laura Fugazzola and Silvia Tabano contributed to this work.Papillary thyroid cancer (PTC) accounts for approximately 80% of thyroid cancers with an increasing prevalence over the last decade [1]
The papillary thyroid cancer (PTC)-MA assay is a novel MA test, which is able to detect fusion genes generated by genomic rearrangements concomitantly with the analysis of hotspot point mutations, allowing the evaluation of key diagnostic, prognostic, and therapeutic markers of PTC in a single experiment without any informatics analysis
To optimize the multiplex polymerase chain reaction (PCR) panel, primers were designed to detect 4 out of 9 exonic point mutations on cDNA (Supplemental Table 1). This was possible, since we previously verified that in two PTC samples, harboring BRAF V600E and NRAS Q61R respectively, the percentage of mutated alleles found in DNA or cDNA were similar (Supplemental Fig. 1), demonstrating that the reaction template (DNA or cDNA) did not influence the point mutations detection
Summary
Papillary thyroid cancer (PTC) accounts for approximately 80% of thyroid cancers with an increasing prevalence over the last decade [1]. The extensive characterization of PTC reported in The Cancer Genome Atlas allowed the reclassification of PTC cases into molecular subtypes that better reflect their differential properties improving the pathological classification and clinical surveillance [2]. Given the established relevance of molecular characterization, clinical diagnosis is facing the need for cost and time-effective techniques to investigate multiple molecular markers. We propose the mass spectrometry array (MassARRAY, MA) as a rapid, cost-effective, and sensitive method for the analysis of multiple hotspot point mutations concomitantly with fusion genes typical of PTC, in a single experiment. The procedure requires minimal amounts of material, representing an advantage for routine
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