MassARRAY-based simultaneous detection of hotspot somatic mutations and recurrent fusion genes in papillary thyroid carcinoma: the PTC-MA assay

Abstract

Purpose: We 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).

Methods: The 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.

Results: The 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.

Conclusions: The 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.

Keywords: BRAF; MassARRAY; NTRK1; Papillary thyroid carcinoma; RET; TERT.

Conflict of interest statement

Conflict of interest

The authors declare that they have no competing interests.

Informed consent

Informed consent was obtained from all participants included in the study.

Figures

Fig. 1

MA spectra for gene rearrangements and hotspot mutations. a Schematic representation of PCR and extension primers for the detection of fusion genes and representative MA spectra. The chromosomal regions involved in the formation of fusion genes are indicated in white (RET and NTRK1) and gray (CCDC6, NCOA4, TPM3, TPR, PRKAR1A, and TFG), and are reported on the top of the corresponding MA spectrum. The positions of the PCR and extension primers are indicated by dotted and black arrows, respectively. In each spectrum, the empty arrow indicates the position of the unextended primer (when the fusion gene is absent); the black arrow points to the position of the extension product, in presence of the fusion gene. The spectra of RET/PTC1 and RET/PTC3 were generated from the positive tissue samples, (#3 and #4, Table 1), and the spectra of RET/PTC2, TRK, TRK-T1 and TRK-T3 were generated from corresponding positive NIH 3T3 cell lines (#24–27, Table 1). b MA spectra of the coexistent BRAF V600E and TERT G228A mutations and the corresponding Sanger sequences in sample #2. On the top, the MA spectra for both BRAF V600E and TERT G228A. In each spectrum, the black arrow indicates the mutated allele, the gray arrow the wild-type allele, and the empty arrow indicates the position of the extension primer. The corresponding electropherograms obtained by Sanger sequencing are reported below. Black arrows point to the mutated bases. For TERT assays, MA and Sanger sequencing were designed to assess the reverse and forward strands of the gene, respectively