Lakatos, Péter1, Kocsis-Deák, Barbara1, Árvai, Kristóf 2, Balla, Bernadett 2, Tóbiás, Bálint2, Kövesdi, Andrea2, Járay, Balázs3, Székely, Tamás3, Horányi, János4, Takács, István1, Kósa, János1,2
1 Department of Medicine, Semmelweis University, Budapest, Hungary
2 PentaCore Laboratory, Budapest, Hungary
3 Department of Pathology, Semmelweis University, Budapest, Hungary
4 Department of Surgery, Semmelweis University, Budapest, Hungary
Background: Cancer progresses through accumulation of genetic alterations, which can serve as important diagnostic, prognostic, and predictive biological markers.
Objectives: We aimed to develop a targeted sequencing panel for parallel testing of multiple mutations in thyroid cancer genes utilizing next-generation sequencing (NGS) for high-throughput.
Methods: A custom-made multiplex PCR-based hot spot panel was designed to target 23 cancer genes (NRAS, MET, CTNNB1, PIK3CA, DICER1, VHL, BRAF, PTEN, LPAR4, EIF1AX, HRAS, RET, GAS8-AS1, KRAS, TSHR, AKT1, GNAS, TERT, TP53, AXIN1, APC, IDH1, SMAD4) that contain 357 known COSMIC mutations related to thyroid carcinogenesis. Sequencing was carried out on an Ion Torrent PGM instrument. Variant calling was performed on 56 papillary thyroid carcinomas with matched normal fresh frozen samples. The average coverage was 400X.
Results: The most frequently mutated genes were BRAF (p.Val600Glu), TSHR (p.Tyr466Phe), APC (p.Thr930Pro), LPAR4 (p.Val83Ala), TP53 (p.Val217fs), AXIN1 (p.Gln634Ter) and SMAD4 (p.Tyr322Asn) in our papillary thyroid cancer samples. Altogether, mutations with at least 5% variant coverage and less than 1% minor allele frequency in general population could be shown in all of the cancer samples, while mutations could be seen in 7 control samples as well. The distribution of genetic alterations was similar to the published data. The sensitivity and the specificity of the method were 100% and 90%, respectively, with a positive predictive value of 87%.
Conclusion: Our multi-gene testing approach allows simultaneous analysis of multiple mutations with high accuracy and sensitivity.