PO-249 Phylogenetic relationships between paired primary papillary thyroid carcinomas and distant metastasis: intra-tumour molecular heterogeneity and clonal evolution

Abstract

IntroductionThough the vast majority of Papillary Thyroid carcinomas (PTC) are indolent tumours, around 5%–15% behave aggressively, developing distant metastases (DM), which cause patient´s death. The molecular events underlying metastatic spread are poorly understood. Little it is known about the contribution of intratumor molecular heterogeneity to DM.Material and methodsIn this study, by genotyping 13 cases of matched primary tumours (PrT) and DMs, we sought to determine the prevalence of mutations in genes that have been associated with progression and aggressiveness in thyroid cancer (TERTp, BRAF, NRAS, KRAS, HRAS, PIK3CA). To asses the contribution of intratumor heterogeneity and clonal evolution to DMs, 54 tumour areas, including different areas across space and time within the PrTs and DMs, were characterised. Mutational analysis was done by means of PCR and SSCP or direct sequencing.Results and discussionsTwelve cases (92%) were mutated in at least one of the genes screened [TERTp=9 cases (69%), BRAF=7 cases (54%), KRAS=3 cases (23%), NRAS=3 cases (23%), HRAS=2 cases (15.4%) and PIK3CA=2 cases (15.4%). Among the mutated cases 67% exhibited more than 1 gene activated. Three mutated genes coexisted in 62.5% of the cases bearing several mutations [3 cases (60%) TERTp +RAS + BRAF and 2 cases (40%) TERTp +BRAF+PIK3CA]. Concurrent activation of TERTp +RAS or TERTp +BRAF was seen in 5 cases each combination(62.5%). Parallel activation of BRAF and RAS was found in 4 of the cases (50%) with genetic heterogeneity. In all of the cases in which more than 1 area of DM, across space and time, was analysed, the mutations at TERTp, KRAS and HRAS resulted clonal. ’De novo’ mutations at DM, not present in the PrT, were seen in 3 cases mutated at TERTp, 2 cases mutated at NRAS and 1 case mutated at KRAS. Among the mutated cases, in which more than one area of PrT was analysed, TERTp mutations were clonal in 80% of the cases and BRAF mutations subclonal in 80% of the cases.The activation of RAS within the PrT tended to be a clonal event.ConclusionThe number of mutational events in PTC with DM is strikingly higher than in in PTC without DM. While TERTp and RAS mutations tend to be clonal within the PrT and the DMs, BRAF mutations tend to be subconal. TERTp and RAS mutations may appear the novo at DM. PTC with DM display a much higher rate of genetic heterogeneity (67%). The coexistence of mutations in different genes is in agreement with the hypothesis that tumour progression relies on progressive accumulation of genetic alterations.