Genomic profiling of thymoma using a targeted high-throughput approach

Abstract

Introduction: Thymomas and thymic carcinoma (TC) are the most common neoplasms localised in the thymus. These diseases are poorly understood, but progress made in next-generation sequencing (NGS) technology has provided novel data on their molecular pathology. Material and methods: Genomic DNA was isolated from formalin-fixed paraffinembedded tumour tissue. We investigated somatic variants in 35 thymoma patients using amplicon-based TruSeq Amplicon Cancer Panel (TSACP) that covers 48 cancer related genes. We also analysed three samples from healthy individuals by TSACP platform and 32 healthy controls using exome sequencing. Results: The total number of detected variants was 4447, out of which 2906 were in the coding region (median per patient 83, range: 2-300) and 1541 were in the non-coding area (median per patient 44, range: 0-172). We identified four genes, APC, ATM, ERBB4, and SMAD4, having more than 100 protein-changing variants. Additionally, more than 70% of the analysed cases harboured protein-changing variants in SMAD4, APC, ATM, PTEN, KDR, and TP53. Moreover, this study revealed 168 recurrent variants, out of which 15 were shown to be pathogenic. Comparison to controls revealed that the variants we reported in this study were somatic thymoma-specific variants. Additionally, we found that the presence of variants in SMAD4 gene predicted shorter overall survival in thymoma patients. Conclusions: The most frequently mutated genes in thymoma samples analysed in this study belong to the EGFR, ATM, and TP53 signalling pathways, regulating cell cycle check points, gene expression, and apoptosis. The results of our study complement the knowledge of thymoma molecular pathogenesis.

Targeted Next-Generation Sequencing of Thymic Epithelial Tumours Revealed Pathogenic Variants in KIT, ERBB2, KRAS, and TP53 in 30% of Thymic Carcinomas

“…Unlike other authors, we did not identify pathogenic variants of TP53 , KRAS , PIK3CA , AKT1 , EGFR , or RET in thymomas [ 30 , 35 , 38 , 49 , 50 , 67 ]. Significant enrichment of mutated genes of the RAS and PI3K-Akt signalling pathways was reported in thymomas ( AKT3 , ALK , CSF1R , FGFR4 , KRAS , NRAS , HRAS , PIK3CA ), and their role in thymoma development was suggested [ 30 , 31 , 35 , 38 , 44 , 49 ].…”

“…The ERBB2 and KIT variants were detected in B2B3 thymomas, and the FOXL2 variant in a micronodular thymoma with lymphoid stroma. Missense variants in ERBB family genes have only occasionally been reported in thymomas [ 30 , 49 , 50 ]. The missense ERBB2 p.(Ser703Arg) variant has not been referred to elsewhere; there is no information on its clinical significance in genetic databases and no entry in gnomAD.…”

“…Alterations in FOXL2 have not previously been associated with TETs. However, FOXL2 was not analysed in most studies of genetic alterations in thymomas or thymic carcinomas by targeted sequencing because it is absent from NGS panels [ 31 , 44 , 49 , 50 , 60 ]. In our analysis, this variant was heterozygous and had an allele frequency of 6% (4905 total reads), suggesting a somatic origin.…”

Correlation of genomic alterations and PD-L1 expression in thymoma

“…Additionally, 168 recurrent variants were detected. On the other hand, some of the genes selected using TSACP panel including FGFR1, MPL, NPM, and SRC had less than 5 NFM variants(20).…”

“…and RB1 were highly mutated harboring more than 40 NFM changes. TP53 and KDR contained more than 90 NFM variants, out of which the majority were well known polymorphisms (familiar one rs1042522, and rs1870377)(20).Analyzing genetic findings and clinical data, they found that only the presence of variants in SMAD4 gene predicted significantly shorter overall survival. Recurrent mutations in this gene previously have been already 27 patients, its alterations were associated with the aggressive subtypes B2 and B3, while "High risk alteration" at APC locus was noted in AB type that are not aggressive forms of thymoma, suggesting there is another tumor suppressor gene that have the opposite effect to APC(22).…”