Next generation sequencing based detection of 15 target genes mutations in papillary thyroid carcinoma

Next generation sequencing (NGS) method provided a valuable tool for a comprehensive understanding of papillary thyroid carcinoma (PTC) biology. We explored the application of NGS based detection for a panel of 15 target genes mutations and analyzed the gene spectrum in PTC.

In this study, we focused on the application of NGS based detection of 15 target gene mutations and analyzed the mutation profiles in PTC.

| DNA extraction
DNA extraction used the QIAamp FFPE Tissue Kit (Qiagen, Hilden, Germany). DNA concentration was measured using a Qubit assay.

| Bioinformatics
The raw data were mapped to the human genome (hg19) by BWA

| Mutation allele frequency
In the present study, all point mutations found in PTC samples were heterozygous mutations with allelic frequency ranged from 1.12% to 48.04% of alleles (which corresponds to 2.24% to 96.08% of cells with a heterozygous mutation). The fusion mutations showed the percent of reads ranged from 2.30% to 55.54%. For samples with carried two mutations, the allelic frequency of mutations was similar.
Interestingly, for the sample harboured triple gene mutations, the allelic frequency of NRAS mutation was similar to TERT mutation (33.62%-39.67%) and TP53 mutation showed much lower abundance (4.13%).

| DISCUSSION
A complete understanding of the molecular mechanisms of tumor formation is essential for providing accurate diagnoses and personalized treatments. In this study, NGS-based detection method was used for detecting 15 target gene mutations, which offered a valuable tool for a comprehensive understanding of PTC biology.
Due to sample size limitations and patients from specific geographic locations, some mutations such as TSHR, AKT1, PETN, KRAS, and PAX8 mutations were negative in the present study, which varied from previous studies. 7,10,19,20 PTC samples were dominated by BRAF V600E mutations which were consistent with the previous studies showed the BRAF V600E mutation rate was 29% to 90%. 4,16,[20][21][22] Ke et al, showed the mutation types were point mutations, indel (insert and deletion) mutations and gene fusions in PTC. 20 However, only point mutations and gene fusions were detected in current study.
A recent study reported TP53 and PTEN mutations showed allelic frequency of more than 50%, suggesting either amplification of the mutant allele in tumor cells or a loss of the wild-type allele. 23 However, in the present study, no point mutations showed allelic frequency of more than 50%. Two CCDC6/RET fusion mutations showed the percent of reads more than 50%. For samples with carried two mutations, the allelic frequency of mutations was similar, indicating that these mutations were in the same clonal population of cells.
Interestingly, for sample harboured TERT, NRAS, and TP53 mutations simultaneously, the allelic frequency of TP53 mutation showed much lower than the others. This finding may be due to TP53 mutation was late events in tumor clone progression.
In conclusion, NGS-based detection method showed advantages in detecting multiple gene mutations simultaneously, economically and efficiently, and providing quantitative assessment of mutation frequency using targeted sequencing panel. It can improve the accuracy of diagnosis and prognostication and is useful for providing personalized treatments for PTC patients.