Molecular profiling of rare thymoma using next-generation sequencing: meta-analysis

Thymic epithelial tumors (TETs) are localized in the anterior mediastinum and comprise several forms of thymomas with different malignant potentials, aggressive forms of thymic carcinoma (TC), and thymic neuroendocrine thymoma.¹ Thymomas originate from thymic epithelial tissue. Thymoma and TC are similar and overlapping in many characteristics, but despite these histopathological and cytological features, thymomas could be considered a more benign form of TETs in comparison with TC with aggressive forms. That is the reason why thymomas and TC, exhibit main differences in therapy approach.^2,3 Here we analyze thymoma including the following subtypes A, AB, B1, B2, and B3.⁴ A and AB subtypes belong to GTF2I, B1, and B2 are the T-cell signaling group, B2 is as well chromosomal stability group, and B3 (atypical thymoma because is the most aggressive, if we exclude TC) belongs to the chromosomal instability group. Thymomas are classified as rare thoracic tumors with an overall incidence of 0.15 per 100 000 persons per year.^5,6 The majority of thymomas are less aggressive forms and their treatment is based on surgery, while in the case of more aggressive TC treatment approach includes multimodal therapy. This tumor characterizes high heterogeneity which is the reason for precise molecular profiling to choose an adequate precision therapy approach.^7,8 Due to the rarity of the disease, there is still a lack of information about external factors that cause diseases, such as smoke or alcohol. Moreover, epidemiological studies suggest that it is poorly known if various environmental impacts have any specific impact on disease development.⁸ The role of mutated genes is one of the crucial factors for thymoma formation and development. Identification of potentially pathogenic variants is obligatory to better explain the molecular milieu of the disease. Our latest study published in January 2020 related to this pathology exhibited variants that play an important role in thymoma.⁹

To that end, advances in high-throughput technology (next-generation sequencing-NGS) have enabled assess of the mutational profiling of various types of diseases including cancer. NGS in genomics includes whole-genome sequencing (WGS), whole-exome sequencing (WES), and targeted sequencing (TS).¹⁰ WGS covers all genomes but provides lower sequencing coverage in comparison to TS. Advances in NGS have contributed to a better understanding of molecular events that lead to disease origin as well as the development of various genetic tests and potential targeted therapy. TS approach targets specific regions of interest and is more cost-effective and often suitable for diagnostic panels which include a specific set of genes characteristic of the disease. Thus, TS provides a patient-specific mutational landscape for effective targeted therapy and better patient management.^11,12 Different approaches are used depending on the need required by the type of study. WGS represents the most comprehensive method for genome analysis and covers the entire DNA of interest. The main limitation of WGS is low sequencing coverage (25 - 30x), high cost per sample (which is lower with the increasing role of methodology), and complex and demanding computation analyzed genome, in comparison to WGS or TS.¹³

This study aimed to identify all relevant articles that evaluate the frequency of SNVs and InDels in thymomas using NGS technology through PubMed, Web of Science, and SCOPUS databases, and to perform a meta-analysis of the prevalence to get better insight into their possible involvement in thymomas. The introduction should summarize the rationale for the study or observation, citing only the essential references and stating the aim of the study.

This systematic review was performed by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses.¹⁴

The aim of this study was to explore genomic background of indolent forms of thymoma using genomic high-throughput approach. Thymoma epithelial tumors (TETs) comprise thymoma, thymic carcinoma (TC), and thymic neuroendocrine thymoma, as we said previously. In this meta-analysis, we take into consideration several forms of thymomas, including A, AB, B1, B2, and B3 subtypes, which have diverse invasive (malignant) potential. Thymoma is a tumor type that belongs to rare types of thoracic tumors, which is the reason for a lower number of studies that we have finally selected as eligible for analysis, along with the relatively recently developed method of analysis that we have applied.

As well, keywords used in this research are limited to the NGS genomics approach to analyzing genes included in thymoma. The potential of high-throughput sequencing or NGS enables the detection of the molecular profile, typical for specific tumors or a variety of diseases. All genomic sequencing methods, including WGS, WES, and TS have been applied in the research or in diagnostics. The selection of methodology depends on all advantages or disadvantages suitable for appropriate use. Applying previously explained inclusion/exclusion criteria we were able to select and analyze articles related to this topic, and we were able to find genes associated with thymoma.

Meta-analysis of the prevalence of the gene variants that we performed using selected studies, has indicated that the majority of patients exhibited variants in GTF2I, TP53, and HRAS genes. The number of analyzed articles was relatively low, due to the rarity of the disease. Moreover, most of the studies have been published from 2014 to 2022.

The molecular background of this pathology is still poorly understood, including all types of A, AB, B1, B2, and B3 thymoma. We have analyzed only NGS genomics-based articles. Further in the discussion will also be considered other methodological approaches to better explain obtained results. The newest data that appeared 2022, have indicated that KIT gene is an important factor in different processes related to thymoma disease. Unfortunately, we have not had enough data to demonstrate the statistical significance of this gene to put it in a group of prevalent genes for thymoma.^17,18

GTF2I (General Transcription Factor IIi) encodes a phosphoprotein that binds to the initiator element (Inr) and Inbox element in promoters regulating transcription.²⁰ GTF2I gene is a member of a B cell receptor signaling pathway, the AKT signaling pathway (genecards.org), and is involved in crucial processes particularly, cell proliferation, cell cycle, and development.¹⁹ Finally, the advances in next-generation sequencing characterized GTF2I as a master gene in TETs pathology.²⁰ The frequency of GTF2I variants is typical for less aggressive forms of thymoma.²¹ GTF2I is a gene that embryonic aberration could lead to lethality, which indicates its essential role in embryo development.

The most pathogenic variants in this gene are Chr 7 c.1211T > A and c.1271 (COSM5095139) which could be used in targeted therapy that could lead to successful clinical application.^21,22 Despite GTF2I pathogenicity in thymomas, GTF2I mutations are very rare (< 1%) in other types of cancer, according to TCGA. Moreover, many types of molecular events lead to thymoma genesis. TCGA’s studies of thymoma have shown recurrent mutations in the GTF2I gene and suggested this gene as a potential drug target for this disease.^{23, 24, 25} Our findings confirm the prevalence of the SNVs/InDels variants in GTF2I in thymoma. Results have been reported in several articles which have been presented in the Forest and Funnel plots. Petrini and colleagues, using WES have discovered missense mutation Chr 7 c.1211T > A and subsequent protein change p. Leu404His in all types of thymoma A, AB, B1, B2, B3, and TC.^19,26 In the publications that we selected for analysis, GTF2I harbored p. Leu404His and p. Leu424His (COSM5095139) pathogenic variants, recognized as a disease-marker gene in thymoma.^{22,26, 27, 28}

TP53 is a crucial tumor suppressor gene regulating the most important processes in the cell including cell cycle regulation, DNA repair, senescence, and apoptosis.²⁹ TP53 has been described as the gene responsible for disease pathogenesis. A recent study has demonstrated common loss-of-function in TP53 in type B thymomas and TCs.³⁰ In a study by Peric et al., two variants with stop codon have been identified R213* and V91*, as well as four pathogenic missense changes namely, D281N, G244S, R158C, E221K.^22,31,32 One of the newest studies by Xu S et al., included in our meta-analysis, has analyzed the genomic profile of TETs that has indicated TP53 as the most mutated gene especially in both B3 and C thymoma causing worse prognosis.³³ HRAS gene belongs to the Ras signaling pathway, commonly mutated in many tumors harboring pathogenic variants, for instance, G13V in thymoma. One study suggests gain-off function mutations in HRAS, especially in type A and AB thymomas.³⁰ A recent study by Jovanovic D et al. has identified recurrent mutations in HRAS, marked as a thymoma-specific oncogene.³⁴ TCGA’s studies of thymoma observed enrichment in the HRAS gene, as well as NRAS and TP53.^24,25 Our meta-analysis of prevalence has reported the HRAS gene to be involved in the pathogenesis of thymoma.^22,33,35,36 Genes that have shown the prevalence in less aggressive forms of thymoma, especially thymomaspecific oncogene GTF2I (transcription factor), TP53, and members of Ras family HRAS have been involved in crucial processes including cell cycle regulation, cell proliferation, cell differentiation, apoptosis, and cell survival. Therefore, pathogenic variants within these genes could be important disease markers and potential therapeutic targets for thymoma.

Our meta-analysis of articles that analyze a mutational portrait using NGS of thymoma has pointed out GTF2I, HRAS, and TP53 genes as thymomaspecific oncogenes. These genes harbor variants SNVs/InDels which contribute to disease development. Moreover, this study indicated the highest prevalence of the GTF2I gene (58%). Some of the identified variants are driver mutations, for instance, GTF2I (Chr7 c.1211T > A, p. Leu404His and c.1271T > A, p. Leu424His), associated with thymoma pathology. In addition, the majority of detected molecular changes are classified as passenger variants, unable to cause disease and further progression, without the presence of other molecular events. However, the thymoma molecular landscape is still insufficiently understood and explored. Therefore, there is a need for additional analysis and information to get a comprehensive genomic picture for better precision treatment of the patients.