Targeted resequencing designed specifically for thymic epithelial tumours confirmed the high prevalence of GTF2I mutations

BackgroundThymic epithelial tumors (TETs) are rare neoplasms and the mutations driving their growth have been partially elucidated. We designed a targeted resequencing assay specific for TETs.MethodsReviewing the literature, we identified frequently mutated genes in TETs and designed a custom panel of 77 genes for targeted resequencing specific for thymic tumors. Between 2021 and 2024, we collected tumor samples during surgical resections. We sequenced tumor and blood DNA to identify somatic mutations. Libraries were prepared using DNA Prep with enrichment and sequencing was conducted using NextSeq 550 (Illumina).ResultsWe enrolled 70 TETs: 3 A, 21 AB, 6 B1, 4 B1/B2, 11 B2, 11 B2/B3, 6 B3, 1 micronodular, 1 metaplastic thymoma and 6 thymic carcinomas. For tumors, the average coverage was 758 reads (SD± 179) and 97% (SD± 12%) of the target sequences was covered with at least 50 reads. There were 150 somatic mutations in 53 genes: 84 were missense mutations, 5 nonsense, 54 synonymous and 7 insertion/deletions. At least one somatic mutation was observed in 43 tumors and 20 patients harbor multiple mutations. A B2 thymoma had the higher number of mutations: 12 synonymous and 12 missense including a GTF2I mutation. GTF2I mutation was the most common being present in 29 tumors (41%). Somatic GTF2I mutations determined the substitution of a leucine with a histidine on position 404. The frequency of GTF2I mutation was 67% in A thymoma, 76% in AB, 17% in B1, 50% in B1/B2, 27% in B2, 9% in B2/B3. 17% in B3, 33% in thymic carcinomas and in 1 micronodular thymoma. A and AB thymomas had more GTF2I mutations (75%) than thymic carcinomas and B histotypes (29%, p<0.0001). GTF2I mutation was more common in stage I-II (52%), than III-IV tumors (21%, p= 0.0115). Four out of 5 HRAS mutations cooccurred with GTF2I in an A(K117T), AB(G13R), B2 (K117T) and 1 thymic carcinoma (A146T), only the L133H mutation of HRAS did not occur in a GTF2I mutant tumor.ConclusionsTETs exhibit a low mutational burden. GTF2I mutation was the most prevalent, particularly in early stages and within histotypes exhibiting a more indolent behavior. Recent studies have demonstrated that transgenic mice expressing mutated GTF2I in the thymus develop thymomas, thus confirming its oncogenic potential.Legal entity responsible for the studyUniversity of Pisa.FundingTuscany Region Ail Pisa: Grant in memory of Dr. Guido Arzilla.DisclosureAll authors have declared no conflicts of interest.