INTRODUCTION: Genomic landscape of primary thymic epithelial tumors has been explored previously. However, the molecular alterations contributing to recurrence and potential transformation remain largely unknown.

METHODS: We performed whole-exome sequencing on three primary and 11 recurrent thymoma samples from five patients using the novel EXaCT-2 platform. Genomic alterations, including somatic mutations and copy number variations, were analyzed to investigate clonal evolution and molecular changes during disease progression.

RESULTS: Relapsed tumors exhibited a higher tumor mutational burden than primary tumors. Shared mutations across primary and recurrent samples support a clonal relationship, whereas distinct alterations in later recurrences suggest ongoing tumor evolution. A total of 313 altered genes were identified across multiple samples in this cohort. Analysis of The Cancer Genome Atlas cohort revealed that 11 of these genes exhibited alteration frequencies at least 2.5-fold higher in the progressed group compared with the disease-free group. Histologic transformation from type AB to B2 and B3 thymoma was observed during the second recurrence in one case. Genes mutated in the transformed sample were altered more frequently in aggressive histologic subtypes in The Cancer Genome Atlas cohort. The driver mutation GTF2I L424H was detected in relapsed thymomas and was inversely correlated with chromosome 1 amplification/gain.

CONCLUSIONS: Our findings suggest a clonal evolution model in recurrent thymomas, with accumulation of genetic alterations and, in some cases, histologic progression. Longitudinal genomic analysis revealed clonal mutations which may inform risk stratification and identify potential therapeutic targets in recurrent thymic epithelial tumors.