According to the Cancer Genome Atlas (TCGA) study, PTC demonstrates a relatively low mutation rate (11 non- synonymous mutations per tumour; 0.41 mutations per Mb on average), compared to other solid tumours. This may explain the indolent behaviour of most PTCs, although it should be recognized that as aggressive malignancies as leukaemias also carry a reduced mutation burden.
The TCGA study revealed a number of new somatic point mutations and other genetic alterations, which reduced the fraction of PTCs with unknown genetic background approximately to 3.5%. Among commonly mutated genes are those encoding proteins of the MAPK pathway: BRAF, KRAS, HRAS, and NRAS, whose mutations were present in 74.6% of PTCs, but also EIF1AX, PPM1D, and CHEK2. The most frequent molecular event in PTC is the BRAFV600E (valine- to- glutamate) point mutation (36– 83% PTCs). The BRAF gene encodes a serine- threonine kinase located downstream to RAS in the MAPK pathway, and its activation leads to the phosphorylation of MEK, which, in turn, mediates phosphorylation of MAPKs (ERK) kinases and finally transcription of specific genes. The BRAFV600E mutation is responsible for the constitutive RAS- independent and dimerization- independent activation of BRAF kinase and consequently of the MAPK pathway. This genetic alteration is capable to initiate PTC, as it was demonstrated in vivo in mouse models, and was described as an early molecular event. However, some studies showed the presence of BRAFV600E mutation in metastatic lymph nodes but not in matched primary thyroid tumour, thus suggesting that the mutation may have arisen de novo during progression. Some studies have reported the presence of subclonal BRAF mutations in PTC, thus raising the possibility they can be a progression rather than initiating events. However, the TCGA and other studies indicated the clonal nature of BRAFV600E mutation in PTC. Theoretically, there can be a third possibility that BRAFV600E mutation initiates PTC, however, powerful secondary alterations occur during the tumorigenesis and take over cancer progression as drivers, and the BRAF mutation is removed by DNA repair mechanisms. BRAFV600E shows a strong association with PTC phenotype being most frequent in its CV- PTC and TCV- PTC (more than 90% of cases) variants. Moreover, many studies demonstrated that the presence of BRAFV600E was associated with poorer PTC outcomes, including aggressive pathological features, loss of sensitivity to radioiodine, or increased recurrence risk. Other BRAF point mutations (including K601E) are rather uncommon, less potent MAPK drivers, and usually present in encapsulated FV- PTC.
RAS somatic point mutations are mainly restricted to FV- PTC with a frequency of 10% to 21%. RAS genes (NRAS, HRAS, KRAS) encode members of small GTPase superfamily, which act as molecular transmitters of signals from tyrosine kinase and non- tyrosine kinase receptors to MAPK and PI3K- AKT pathways. NRAS is predominantly mutated in TC, mostly in codons 12 and 61.
The EIF1AX gene, encoding an essential eukaryotic translation initiation factor, was among the mutated genes in PTC in the TCGA study. Although the frequency of its mutations did not exceed 2%, it was classified as a cancer gene associated with PTC. Further studies indicated the presence of EIF1AX mutations, mainly in encapsulated FV- PTC, and also in ATC. The presence of EIF1AX mutations in FTA and their absence in FTC may suggest that EIF1AX- mutated adenomas may progress to FV- PTC rather than to FTC.
Recently TERT promoter mutations were detected in a number of cancers, including TCs. The TERT gene encodes the reverse transcriptase component of telomerase with the main function, together with its RNA component, of telomere elongation by adding TTAGGG at the end of chromosomes. In addition to unlimited replication, telomerase activation leads to increased proliferation, angiogenesis, resistance to apoptosis, and metastatic potential of cancer cells. Two TERT promoter mutations, 1,295,228 C >T and 1,295,250 C >T (– 124 and – 146 bp from the ATG; termed C228T and C250T, respectively) are common. Both alterations create the 11- nucleotide fragment (5’- CCCCTTCCGGG- 3’), which contains a consensus binding site, GGAA, for ETS (E26 transformation specific) transcription factors. It has been demonstrated that both C228T and C250T increase the transcriptional activity of the TERT promoter. TERT promoter mutations are present in 11.3% of PTCs, with C228T being more frequent than C250T. The frequency is correlated with aggressiveness of the tumour, and it is higher in TCV- PTC, showing an aggressive nature. Recent studies indicated the association of TERT promoter mutations with the presence of BRAFV600E mutation and their coexistence with poorer outcomes compared to the impact of each of these alterations alone. Similar consequences were observed in the coexistence of TERT promoter and RAS mutations.
RET rearrangements are characteristic of radiation- induced PTCs. These chromosomal aberrations concern mainly young patients since thyroid cells of children are particularly sensitive to radiation. RET encodes a transmembrane receptor with tyrosine kinase activity, and normally, in contrast to C cells, it is not expressed in follicular thyroid cells. Activation of RET in these cells results from a fusion of RET fragment encoding domain with tyrosine kinase activity with 5’ end of other normally expressed genes. More than 16 different RET rearrangements have been described so far with RET/ PTC1 (CCDC6- RET), RET/ PTC3 (NCOA4- RET), and RET/ PTC2 (PRKAR1A- RET) being the most frequent and accounting for 60%, 30% and 5% of all RET/ PTC translocations, respectively. The overall frequency of RET rearrangements varies between studies, from 3% to 85% in the adult population, an average of 35%. Although RET rearrangements are the hallmark of radiation- induced PTCs, these alterations are also present in benign lesions, like in Hashimoto’s disease. Currently, it is obvious that RET rearrangements are characteristic of childhood PTC, also sporadic one. In addition to RET/ PTC rearrangements, other chromosomal aberrations are observed in PTCs, however, in a significantly lower number of cases. NTRK rearrangements concern mainly NTRK1 and NTRK3 genes with ETV6/ NTRK3 being the most frequent one in radiation- induced TC. There are also BRAF gene rearrangements (AKAP9/ BRAF; AGK/ BRAF), detected in about 10% of radiation- induced PTC and ALK rearrangements described in 1– 5% cases.
Depending on the underlying driver mutation, two molecular subtypes of PTCs have been proposed: BRAF- like (BRL) and RAS- like (RL). BRL tumours include not only carcinomas with BRAFV600E mutation but also tumours with TERT promoter mutations, RET/ PTC and BRAF gene rearrangements, whereas RL tumours include all cases with BRAF point mutations other than V600E, EIF1AX, RAS point mutations, and PAX8/ PPARgamma rearrangements. BRL tumours are most typically CV- PTC, while RL tumours have FV- PTC features. Moreover, the two molecular sub types differ in terms of genomic, epigenetic, and proteomic profiles, however, the BRL tumours displayed a more heterogeneous nature. BRL PTCs activated mainly the MAPK signaling pathway, whereas in the RL tumours activation of MAPK, as well as PI3K pathway was observed.
As far as the epigenetic modifications, increased expression of miR- 146b, miR- 221 and miR- 222 has been documented in PTC. Moreover, BRAFV600E mutation seems to be associated with hypermethylation of several tumour suppressor genes, including TIMP3, DAPK1, or RARB, and with hypomethylation of other genes.
