Genetic profile of thymic epithelial tumors in the Japanese population: an exploratory study examining potential therapeutic targets

Introduction

Thymic epithelial tumors (TETs), consisting of thymomas and thymic carcinomas (TC), are rare tumors with heterogeneous histological and clinical features. Surgical resection is the cornerstone of therapy for early-stage TETs (1), whereas systemic treatment is required for locally advanced or metastatic TETs (2). Although cytotoxic chemotherapy can reduce tumor volume, it is usually not curative for locally advanced or metastatic TETs. Molecular targeted drugs have become available as effective treatments for various malignancies in clinical settings. Everolimus (3), sunitinib (4), and lenvatinib (5) have been explored for patients with refractory or recurrent TETs in clinical trials. In addition, the efficacy of MCL-1 and BCL-xL inhibition and PI3K pathway-targeted therapy has been reported (6,7). However, the effects of these treatments remain modest because of the lack of obvious driver mutations. Target-specific drugs for TETs have not been developed because the genomic aberrations in TETs are poorly understood (2,8,9). Since TETs are rare thoracic tumors, their genomic profiles have not been investigated to the extent of other cancers, such as lung cancer and malignant melanoma. Therefore, there is an urgent need for patients with TETs to explore genomic profiles and abnormalities of TETs.

Next-generation sequencing (NGS) technologies have revealed new perspectives in molecular targeted cancer therapy and the molecular mechanisms of carcinogenesis (10). According to the population-based cancer registry of the USA, the incidence of thymoma is high in East Asian populations; in particular, there is a higher prevalence in Japan than in other East Asian populations (11). A comprehensive analysis of 117 TETs, including 105 thymomas, 10 TCs, and two micronodular thymomas, as part of The Cancer Genome Atlas (TCGA) project, revealed the integrated genomic landscape of TETs. This study showed that the most frequently mutated gene in thymomas is general transcription factor 2-I (GTF2I), followed by [Harvey RAS (HRAS) neuroblastoma RAS (NRAS)]. However, most of the patients in this study were Caucasian, and only 12 (10%) were Asians (12). Additionally, a recent comprehensive genetic analysis of TETs reported that GTF2I mutations were not identified in thymomas (13). Therefore, we hypothesized that the genetic profile of TETs may vary by race or country, leading to differences in their occurrence and genetic profiles in previous reports. Since the analysis of genetic profiles of TETs in the Japanese population is still insufficient, we explored the genetic aberration in a Japanese population to identify driver mutations in TETs as potential therapeutic targets and to elucidate the molecular mechanisms of TETs. We present the following article in accordance with the STROBE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-794/rc).

Methods

Study design

This prospective, single-institute cohort study was conducted at Nagasaki University Hospital, an officially authorized regional core cancer center. Patients who were diagnosed with an anterior mediastinal tumor and underwent thoracic surgery between January 2013 and March 2019 were included in this study. The diagnosis and tissue classification of TETs were determined according to the World Health Organization (WHO) classification (14). Thymomas are divided into five categories: type A, consisting of spindle-shaped or oval cells; type B1, in which polygonal cells are found in abundant lymphocytes; type B2, in which lymphocyte components are less than those in type B1; type B3, in which lymphocytes are less than those in type B2; and a mixture of types A and B, classified as type AB. In TCs, large polygonal cells proliferate solidly, accompanied by keratinization and intercellular bridging. The tumor cells show distinct nuclear atypia. Moreover, the stage of TETs was determined according to the Masaoka-Koga staging (15). Two independent pathologists evaluated the diagnoses and pathological classifications of TETs. If the assessments differed between the two pathologists, they re-evaluated the specimen and reached a consensus. Chest MRI and whole-body CT scans were used to evaluate the dissemination or metastasis of the TETs. Patients were excluded from the genetic analysis if no specimen was obtained during surgery and if they had a diagnosis other than TET after surgery. Clinical information was extracted from patients’ medical charts.

This study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by institutional ethics board of Nagasaki University Hospital (registration No. 13072237) and informed consent was taken from all individual participants. The protocol of this study was registered with the University Hospital Medical Information Network in Japan (registry number UMIN000039065).

In the present study, written informed consent was obtained from 43 consecutive patients with anterior mediastinal tumors suspected of being TETs from January 2013 to March 2019 (Figure 1). Twelve patients who were not diagnosed with thymoma or TC were excluded, and the remaining 31 patients with a pathological proof of thymoma or TC were selected for analysis.

Figure 1 Patient flow diagram.

DNA extraction

The tumor and normal tissues were obtained in a cubic shape, with each side measuring approximately 5 mm on the day of surgery. They were further divided into 2-mm cubes on each side and stored at −80 ℃. The specimen was thawed and ≤25 mg was used for DNA extraction. Thereafter, 20 µL of proteinase K was added, and the mixture was incubated at 56 ℃ until the tissue was completely lysed. DNA was extracted using the QIAamp DNA Mini Kit (Catalog #51304, Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

Next-generation sequencing (NGS) and gene expression analysis

First, a custom panel was created with a gene list, which included major oncogenes and tumor suppressor genes, based on Ion AmpliSeq™ Cancer Hotspot Panel v2 and several previously reported genes for TETs, such as ENOX1 (whole exon), POSTN (whole exon), and GTF2I (whole exon) (Table 1). We did not test for any germline mutations but tested for somatic mutations in TETs in this study. Second, a library was created by placing 10 ng of DNA into a tube with custom panel primers and amplifying the target regions with the Ion AmpliSeq™ Library Kit 2.0 (Catalog #4480441, Thermo Fisher Scientific, Waltham, MA, USA). The adapters that recognized the sequence were ligated, and emulsion polymerase chain reaction (PCR) was performed. Finally, the library was loaded onto the chip and sequenced using paired-end runs on an Ion PGM™ (Catalog #4462921, Thermo Fisher Scientific). Genetic alterations were identified by comparing tumor samples with normal samples. The run was considered successful, and the sequencing quality was adequate when the following quality metrics were met: mapped reads ≥300,000, average base coverage depth ≥1,000, amplicons with at least 500 reads ≥90%, strand bias ≥90%, and amplicons read end-to-end ≥85% (16).

Analysis was performed using Ion Reporter™ Software 5.12 (Thermo Fisher Scientific) and CLC genomics workbench 11.0 (Filgen Inc., Nagoya, Japan). DNA reads were mapped to the human reference genome hg19. For Ion Reporter™, the parameters were a minimum amplicon coverage of 100 and a minimum allele frequency of 5%. For CLC genomics workbench 11.0, the parameters were a minimum amplicon coverage of 100 and a minimum allele frequency of 5%.

Sanger sequencing, digital droplet PCR (ddPCR), and TA cloning for validation

The gene mutations recognized by Ion Reporter™ and CLC Genomics Workbench 11.0 were further confirmed using Sanger sequencing. Sanger sequencing primers were generated using Primer3 Input (ELIXIREstonia, Tartu, Estonia). The primers, GoTaq^® Green Master Mix (Catalog # M7122, Promega, Madison, WI, USA), water, and each DNA sample were thoroughly mixed, and subsequent amplification was performed in a thermal cycler (Bio-Rad, Tokyo, Japan). The PCR product, one primer, and water were thoroughly mixed, and Sanger sequencing was performed using Eurofins Genomics according to the manufacturer’s instructions (Tokyo, Japan).

For HRAS c.182A>G (Q61R) and HRAS c.37G>C (G13R), ddPCR was performed to confirm the mutations. DNA, ddPCR Supermix for Probes with no dUTP (Catalog #186-3023, BIO-RAD, Tokyo, Japan), and two ddPCR Mutation Assays (Catalog #10049550 and #10049047, BIO-RAD) were thoroughly mixed and transferred to a DG8 cartridge for a QX100™/QX200 Droplet Generator (Catalog #186-4002JA, BIO-RAD). Droplet generation oil for the probes was added to the cartridge, which was placed into the QX200 Droplet Generator™. After droplet generation, the droplets were carefully transferred to a twin-tec, semi-skirted, 96-well PCR plate (Catalog #12001925, Bio-Rad), after which the plate was sealed two times for 5 s at 180 °C using a PX1 PCR Plate Sealer. Subsequent amplification was performed using a thermal cycler. Droplets were read in a QX200 Droplet reader (Catalog #186-4003JA, BIO-RAD), and ddPCR data were then analyzed using Quantasoft (Catalog #186-4011JA, BIO-RAD).

Sanger sequencing was performed for 12 cases with GTF2I mutations. TA cloning was performed in six cases in which the peaks of the Sanger sequence in the samples had low values. The DNA was amplified using Taq DNA polymerase, 2x Ligation Buffer, pTA2 Vector, and T4 DNA Ligase (Catalog #TAK-101, TOYOBO, Osaka, Japan). The amplification product was mixed, and the ligation solution was adjusted. The ligation reaction was used to transform chemically competent E. coli cells (Catalog #310-06231, NIPPON GENE, Toyama, Japan), which were plated on Luria-Bertani/ampicillin/X-gal plates. The mixtures were incubated overnight at 37 ℃. The following day, white colonies were selected by blue/white colony determination, and colony direct PCR and sequencing were performed.

Statistical analysis

Statistical analysis is not applicable since this study is not a comparative or controlled study requiring statistical analysis.

Results

Patients’ characteristics

Of 43 patients with anterior mediastinal tumors suspected of being TETs, 12 patients who were not diagnosed with thymoma or TC were excluded, and total 31 TETs were analyzed in this study (Figure 1). The clinical and pathological characteristics of the patients are presented in Table 2. The patients ranged in age from 34 to 84 years (median, 63 years), and 21 (67.7%) were female. According to the WHO classification, type AB is the most common, followed by type B2. Myasthenia gravis (MG) was diagnosed in seven patients and was distributed in the wide histological thymoma categories, except for type A thymoma and TC.

Detection of major oncogene mutations

As we aimed to investigate the driver oncogenes of TETs, we first checked the major oncogene mutations that were found in other cancers using NGS. As a result, HRAS was detected in two (6.5%) cases, while NRAS and additional sex combs like 1 (ASXL1) mutation were found in one (3.2%) case each using NGS and gene expression analysis (Table 3 and Table S1). NRAS c.181C>A (Q61K) and ASXL1 c.2077C>T (R632*) were confirmed by Sanger sequencing (Figure 2A,2B). Since the peaks of HRAS c.182A>G (Q61R) and HRAS c.37G>C (G13R) in the samples had low values in the Sanger sequence, ddPCR was performed to validate the mutations. The genotype assays yielded positive droplets for HRAS Q61R with channel amplitude signals between 1,000 and 5,000 (Figure 3A). The genotype assays also provided positive droplets for HRAS G13R with channel amplitude signals between 1,000 and 3,500 (Figure 3B). Thus, RAS mutations were confirmed in all three cases. Although some receptor tyrosine kinase (RTK) inhibitors have been explored in patients with refractory or recurrent TETs in clinical trials (3-5), we did not detect any gene mutation in RTKs.

Figure 2 Representative results of Sanger sequencing. (A) NRAS c.181 C>A (Q61K) (reverse), and (B) ASXL1 c.2077 C>T (R632*).

Figure 3 Results of digital droplet polymerase chain reaction (ddPCR). (A) HRAS Q61R; (B) HRAS G13R.

GTF2I mutations

Previous reports have revealed that the GTF2I mutation was most frequently detected in thymomas and was predominantly found in type A, AB, and B1 thymomas (12,17). Therefore, we evaluated the frequency of the GTF2I mutation in TETs in the Japanese population. GTF2I c.74146970T>A (L424H) was detected in 12 (38.7%) of the 31 TETs (Table 3 and Table S1) by NGS and gene expression analysis. The GTF2I mutation was mainly observed in type A and B1 thymomas, which is consistent with previous studies that investigated Caucasian populations (12,17). Six of the 12 GTF2I-mutated cases were confirmed by Sanger sequencing (Figure 4). Because the peaks of the remaining six samples had low values in the Sanger sequence, TA cloning was additionally performed in the samples. When 30–48 colonies were picked from each sample and sequenced by PCR, two to six colonies harboring mutations were observed (Figure S1). Taken together, the GTF2I mutation was confirmed in all 12 cases. In addition, the aforementioned RAS mutations were detected in GTF2I-mutated cases (Table 3).

Figure 4 Representative results of Sanger sequencing [GTF2I c.74146970T>A (L424H)].

The results of the target sequences used in the current study are summarized in Figure 5.

Figure 5 Overview of characteristics and somatic mutations in TETs. SNV, single nucleotide variant; TETs, thymic epithelial tumors.

Discussion

This study investigated TETs in a Japanese cohort and revealed that GTF2I mutations were predominant in indolent types of thymomas, consistent with the trend in a previous report in the USA. HRAS and NRAS mutations were detected at low frequencies but interestingly coexisted only among the GTF2I-mutated cases.

GTF2I is a multifunctional transcription factor, and its mutations are relatively specific to TETs (18). Although the germline GTF2I mutation was reported to be associated with Williams-Beuren syndrome, no patient in our study had a phenotype or family history of Williams-Beuren syndrome (12,19). Recent preclinical studies have reported that the GTF2I-L424H missense mutation, which was detected in this Japanese cohort, may be responsible for the transformation of thymic epithelial cells and have tumor-promoting properties (17,20). As previously reported, GTF2I mutations are predominant in indolent type A, AB, and B1 thymomas (12,17). While a recent Chinese report showed that no GTF2I mutations were detected in 40 patients (13) and there might be a racial difference in the frequency of GTF2I mutations between Asian and Caucasian people in TETs, our data indicated that GTF2I mutations were also present in Japanese TETs. We have summarized the molecular profiles of Caucasian patients using data from Radovich et al. (12), in Table 4. The similar points between our study and that of Radovich et al. are as follows: the frequency of GTF2I mutation in our patients and Caucasian patients (Radovich et al.) [38.7% (12/31) and 39.3% (46/117), respectively], frequency of HRAS mutations [6.5% (2/31) and 8.5% (10/117), respectively], and frequency of NRAS mutations [3.2% (1/31) and 2.6% (3/117), respectively]. The differing points are as follows: types of TETs harboring HRAS mutations {HRAS mutations were detected only in type AB [100% (2/2)] in our study, but were detected in type A [80% (8/10)], followed by type AB [20% (2/10)] in the study by Radovich et al. in Caucasian patients}, types of TETs harboring NRAS mutations (NRAS mutations were detected in type B1 in our study, but were detected in types AB, B2, and TC in the study by Radovich et al. in Caucasian patients), types of TETs harboring TP53 mutations (TP53 mutations were detected in types B2, B3, and TC in the study by Radovich et al. in Caucasian patients, but not detected in our study), and types of TETs harboring ASXL1 mutations (ASXL1 mutation was detected in TC in our study, but not detected in the study by Radovich et al. in Caucasian patients.

In our study, myasthenia gravis (MG) occurred only in GTF2I wild-type patients. Yasumizu et al. reported that they could not find any significant somatic mutations associated with MG, whereas missense mutations in GTF2I were observed in 49% of patients with thymoma (21). MG occurred only in GTF2I wild-type patients in our study; contrarily, Liang et al. reported that GTF2I mutations were detected in some TET cases with MG (19). GTF2I has been reported to be associated with autoimmune diseases. However, L424H is a somatic mutation variant that exists in TETs, not a germline mutation, and MG-related gene was not observed in either GTF2I wild-type or GTF2I L424H mutation (21). The association between GTF2I status and MG has not yet been observed; therefore, several antibodies for MG that have been approved recently do not appear to be a potential treatment for TETs regardless of GTF2I status. Since there is currently no therapeutic approach for GTF2I mutations at present, further studies are needed to identify the therapeutic potential of targeting them. Recent study has reported that activation of cell cycle-related pathways, such as Myc- and E2f-mediated targets, initiate the tumorigenesis in the Gtf2i-mutant thymus, which may enable targeted therapies (22). In addition, the wild-type GTF2I is associated with a relatively poor prognosis (20,23). Therefore, the existence of GTF2I mutations could be one of the biomarkers that predict the prognosis of patients. Furthermore, it is necessary to consider novel therapeutic strategies for patients with TETs and wild-type GTF2I.

HRAS and NRAS mutations were detected in only three cases (two cases of type AB thymomas and one case of type B1 thymoma) in this study, and the low frequency was consistent with that of previous studies (12,24). HRAS and NRAS mutations were likely to be sporadically found in all types of thymoma and thymic cancer (12,24-26). HRAS and NRAS activate the proliferation and survival of cancer cells through the RAF-MEK-ERK downstream pathway (27,28) and are considered candidate therapeutic targets. Although several inhibitors have been established for downstream RAF-MEK mediators (29-31), targeted therapy for RAS mutations has not been developed until recently. Several recent clinical trials have shown that HRAS and NRAS inhibitors have promising antitumor activities (32,33). Furthermore, sotorasib, a KRAS GTPase family inhibitor, has demonstrated clinical benefits in patients with solid tumors and non-small cell lung cancer (NSCLC) harboring the KRAS G12C mutation (34). It has been approved by the Food and Drug Administration (FDA), European Commission, and other countries, including Japan. In the future, HRAS and NRAS inhibitors may benefit patients with TETs.

Moreover, all RAS mutations were found in GTF2I-mutated cases in the present study, whereas RAS mutations did not always correlate with GTF2I mutations in previous studies (12,17). Recently, Hsieh et al. reported that HRAS and KRAS mutations were detected in two of the 12 micronodular thymomas, in addition to the GTF2I mutation (35). Since micronodular thymoma with lymphoid stroma is genetically close to type A and AB thymomas, their results supported our results that GTF2I mutations were observed in indolent types of thymoma. Further investigations are required to elucidate the biological role of the coexistence of RAS and GTF2I mutations. To the best of our knowledge, no treatment has thus far been reported for TETs harboring RAS and GTF2I co-mutations. However, a recent preclinical study showed that the activation of cell cycle-related pathways, such as Myc- and E2f-mediated targets, initiate tumorigenesis in the Gtf2i-mutant thymus, which may enable targeted therapies (22). Moreover, compounds targeting RAS mutations are being developed for patients with RAS mutations. Future studies are required to clarify whether these treatments show antitumor effects in TETs harboring RAS and GTF2I co-mutations as monotherapy or combination therapy, which will be valuable for further clinical translational strategies.

In the current study, ASXL1 mutations were detected in 3.2% of tumors (one of 31 TETs). The ASXL1 mutation in the current study was also found in TC. Generally, ASXL1 is frequently mutated in hematological malignancies such as acute myeloid leukemia and myelodysplastic syndrome (36), and its mutations are associated with poor prognosis (37,38). It is an epigenetic regulatory gene, and loss of ASXL1 function is reported to compromise tumor suppressor activity (39). There were several reports that showed the low frequency of ASXL1 mutations in TETs; however, the cases with ASXL1 mutations were more aggressive types (40,41). Since ASXL1 mutations were found in aggressive TETs, treatments targeting epigenetic reprogramming should be considered in the future, despite their rare incidence.

This study has several limitations. First, the reason for the higher incidence of TETs in East Asian populations remains unknown. Since our custom multigene panel searched for a limited number of mutations in 53 genes, other pathogenic gene mutations were not investigated. Whole exome or whole genome sequences may be used to clarify unknown genetic abnormalities in TETs. Second, only resected specimens from operable cases were examined in this study. The genome profile in operable cases may differ from that in advanced unresectable cases. Although the results from the resected specimens were reliable in the current study, biopsy specimens of unresectable cases could confirm genetic evolution during the progression of TETs. Third, the present study was conducted at a single institute and was potentially subject to selection bias. For example, the small number of TC may be due to the limited number of resected cases; however, the composition of the types of TETs was similar to that in a previous study (42).

Conclusions

Dominant GTF2I mutations in specific types of TETs and low frequencies of HRAS and NRAS mutations were detected in a Japanese population. In addition to developing RAS pathway inhibitors for TETs, clarifying the significance of co-mutations with RAS and GTF2I mutations may provide a new molecular perspective in TETs treatment.

Acknowledgments

The authors thank the patients and their families, and the authors would like to thank Emi Saito (Department of Respiratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan) for technical assistance with the experiments.

Funding: This work was partially funded by a Japan Society for the Promotion of Science KAKENHI (#17K16049), Non-Profit Organization aimed to support Community Medicine Research in NAGASAKI.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-794/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-794/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-794/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-794/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by institutional ethics board of Nagasaki University Hospital (registration No. 13072237) and informed consent was taken from all individual participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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