Constant GTF2I L424H mutation in micronodular thymoma with lymphoid stroma: genetic evidence supporting its close relationship with type A and AB thymomas

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Constant GTF2I L424H mutation in micronodular thymoma with lymphoid stroma: genetic evidence supporting its close relationship with type A and AB thymomas

https://doi.org/10.21203/rs.3.rs-1747099/v1

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Micronodular thymoma with lymphoid stroma is a rare thymic neoplasm characterized by discrete nodules of epithelial tumor cells separated by abundant lymphoid stroma. The genetic features of micronodular thymoma with lymphoid stroma remain largely unexplored. Owing to the interference of abundant intra-tumoral non-neoplastic lymphoid cells, a highly sensitive approach is necessary to study the genetic changes in these tumors. In this study, we applied a highly sensitive next-generation sequencing assay using molecular barcoding Ion AmpliSeq HD technology to study the most commonly mutated genes in thymomas, including GTF2I, HRAS, NRAS, KRAS, and TP53. A total of 12 cases of micronodular thymomas with lymphoid stroma were tested, and 2 cases also had areas of type A thymoma in their tumor bed. Two micronodular thymic carcinomas with lymphoid stroma, a histological mimic of micronodular thymoma, were also included for comparison. Recurrent GTF2I p.L424H mutations were found in all cases of micronodular thymoma with lymphoid stroma but not in micronodular thymic carcinomas. In addition, 3 cases of micronodular thymoma with lymphoid stroma also had concomitant HRAS and/or KRAS mutations. Our study shows that GTF2I p.L424H mutation is a constant genetic feature in micronodular thymoma with lymphoid stroma. This finding strongly suggests micronodular thymoma with lymphoid stroma is closely related to type A and type AB thymomas as they all share GTF2I p.L424H mutations.

Micronodular thymoma with lymphoid stroma is a rare neoplasm that accounts for 1–5% of all thymic tumors. (1) It was first described by Suster and Moran in 1999 and is now recognized as a distinct subtype of thymoma according to the WHO classification. (2, 3) Histologically, micronodular thymoma with lymphoid stroma is characterized by spindle-shaped epithelial cells forming small tumor nodules separated by abundant lymphoid stroma with or without germinal centers. The prognosis is excellent. This is the only type of thymoma with an International Classification of Diseases for Oncology behavior code of "1 - low or uncertain malignant potential or borderline malignancy", while all other thymomas are regarded as malignancy. (2)

A close mimic of micronodular thymoma with lymphoid stroma is micronodular thymic carcinoma with lymphoid stroma, which was recently described by Weissferdt and Moran. (4) Micronodular thymic carcinoma with lymphoid stroma also comprises epithelial tumor cells arranged in a micronodular pattern with abundant lymphoid stroma which might be indistinguishable from micronodular thymoma at low power. Nevertheless, the epithelial tumor cells in micronodular thymic carcinoma show overt cytological atypia and brisk mitotic activity, which are absent in micronodular thymoma. Micronodular thymic carcinoma is rare, and its prognosis seems to be more favorable than that of conventional thymic carcinoma. (4–6) It was proposed that micronodular thymic carcinoma represents the malignant counterpart of micronodular thymoma with lymphoid hyperplasia. (4) However, the relationship between these two disease entities remains elusive.

Genetic alterations in micronodular thymomas and micronodular thymic carcinomas with lymphoid stroma are largely unexplored. The low tumor percentage due to the presence of abundant non-neoplastic lymphoid cells is a unique challenge in exploring molecular alterations in micronodular thymic neoplasms. A highly sensitive approach is required to detect low-frequency somatic variants in these low tumor purity samples. GTF2I is the most commonly mutated gene in thymoma, with all mutations reported in the literature occurring in a single codon (L424H). (7, 8) This mutation is especially enriched in type A and AB thymomas, and rarely present in type B thymomas. (2, 7, 8) A few cases of micronodular thymoma with lymphoid stroma harboring the GTF2I p.L424H mutation have been reported. The Cancer Genome Atlas (TCGA) study included two micronodular thymomas with lymphoid stroma, and the GTF2I p.L424H mutation was found in one of them. (8) Very recently, Wells et al. used Sanger sequencing and identified GTF2I p.L424H mutation in three of four micronodular thymomas with lymphoid stroma. (9) However, these studies did not utilize a highly sensitive method, and the possibility of false negativity was a serious concern. In addition, the number of cases investigated was quite limited; therefore, the results were not conclusive.

In the present study, we developed a highly sensitive next-generation sequencing (NGS) assay using molecular barcoding Ion AmpliSeq HD technology. In addition to GTF2I mutations, we studied other commonly mutated genes in thymomas, including HRAS, NRAS, KRAS, and TP53. (8) A total of 12 micronodular thymomas and 2 micronodular thymic carcinomas with lymphoid stroma were tested in this study. Furthermore, two cases of micronodular thymoma also had areas of type A thymoma, and mutation analysis were separately performed in their different histologic components.

Patient selection and sample preparation

This study included 12 cases of micronodular thymoma with lymphoid stroma (including 2 cases with areas of type A thymoma) and 2 cases of micronodular thymic carcinoma with lymphoid stroma. The clinical data of these patients were retrieved from medical records. All tumors were removed completely by surgery. Pathological slides were reviewed by three pathologists (Y. C. Y., M. S. H., and H. L. K.). The tumors were diagnosed in accordance with the 2021 WHO classification, and staged using modified Masaoka or American Joint Committee on Cancer staging system. (2) Sections cut from formalin-fixed paraffin-embedded specimens were used for DNA extraction using a QIAamp DNA FFPE Tissue Kit (Qiagen, Inc.) according to the manufacturer's instructions. For the two cases with mixed areas of type A and micronodular thymomas with lymphoid stroma, the type A and micronodular thymoma areas were separated by manual microdissection and submitted to mutation analysis.

NGS analysis to detect mutations

To achieve highly sensitive and accurate detection of mutations at low variant allele frequencies (VAF), we designed a customized high-coverage deep NGS panel using Ion AmpliSeq HD technology, which leverages dual molecular tags to achieve ultra-high levels of sensitivity down to VAF below 1%. (10) The customized panel targeted frequently mutated genes previously reported in thymomas, including GTF2I, HRAS, NRAS, KRAS, and TP53. (8) The details of the target regions are summarized in Supplemental Table 1. A challenge in detecting the GTF2I p.L424H hotspot mutation is the presence of two pseudogenes, GTF2IP1 and GTF2IP4, which share high sequence homology with GTF2I in the region near the mutation locus. (7) To overcome this problem, we took advantage of the single base difference [cytosine (C) in GTF2I at chr7:74146870 and thymine (T) in the two pseudogenes] upstream of the p.L424H mutation site (chr7:74146970). (7, 11) The primers were carefully designed so that the amplicons could span the region covering both the single-base difference (chr7:74146870) between GTF2I and the pseudogenes and the p.L424H mutation site (chr7:74146970). Such a design enables unambiguous discrimination of reads from GTF2I and the pseudogenes. (Supplemental Figure 1)

The NGS experiment and data analysis workflow were performed in accordance with the manufacturer's instructions. Briefly, after DNA extraction, the sequencing library was prepared using Ion AmpliSeq HD primer, Ion AmpliSeq HD Dual-Barcode Kit, and Ion AmpliSeq HD Library Kit (Thermo Fisher Scientific), as previously described. (10) The libraries were purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA) and sequenced using an Ion GeneStudio S5 Prime System (Thermo Fisher Scientific). The mean depth of coverage was 39847×. Sequence data were processed using a standard pipeline with Torrent Suite software (version 5.16) (Thermo Fisher Scientific). The raw reads were aligned with respect to the human genome 19 reference. Variant calling and annotation were performed using Ion Reporter Software (version 5.18.2.0) (Thermo Fisher Scientific) with “AmpliSeq HD for Tumor - w2.4 - DNA” workflow. As a previous study showed that deep sequencing NGS assays had increasing proportions of false positives at a VAF of 1%, we rigorously excluded variants with a VAF of less than 2% to minimize false-positive calls. (12) Variants that were likely germline polymorphisms were filtered using the Taiwan Biobank population database. (13) Synonymous mutations were also excluded.

Sanger sequencing

Sanger sequencing was also performed to investigate its ability to detect the GTF2I p.L424H mutation in our cohort. Primer design and experimental protocols were previously described. (7, 11) The sequences of forward and reverse primers are ATCCCGTACCCTCTTTTCC and AGACAAGAGTTCAACAGG, respectively. The forward primer was designed with its last 3' base covering the C nucleotide (chr7:74146870) specific for GTF2I to optimally amplify GTF2I but not the pseudogene sequences. PCR was carried out for 35 cycles at 95 °C for 40 sec, 59 °C for 40 sec, and 72 °C for 40 sec, followed by a 5 min extension at 72 °C. The amplicons were electrophoresed on 2% agarose gels. The PCR products were subjected to Sanger sequencing and analyzed using an automated DNA sequencer (ABI 3730xl DNA Analyzer; Applied Biosystems).

Clinicopathological characteristics

The clinicopathological data of 14 patients are summarized in Table 1. For 12 cases of micronodular thymoma with lymphoid stroma, the male to female ratio was 1 : 3, and the age ranged from 53 to 80 y (mean: 68.8 y). The tumor size ranged from 2 to 16.5 cm (mean: 5.8 cm). All patients were in stages I and II. For 2 cases of micronodular thymic carcinoma with lymphoid stroma, one had perithymic lymph node metastasis (stage IVA) and the other one was in stage I. None of the patients had disease recurrence or died from disease.

Table 1

Patient characteristics and the GTF2I p.L424H mutation status
Case no.	Sex	Age (year)	Tumor size (cm)	Stage^#	Pathological diagnosis	Follow-up	GTF2I p.L424H (NGS)	VAF (%) (NGS)	GTF2I p.L424H (Sanger)
1	F	72	6.5	IIA	Micronodular thymoma	AWD at 52 months	MUT	8.3	WT
2	M	68	2.6	I	Micronodular thymoma	AWD at 124 months	MUT	13.8	WT
3	M	80	6	I	Micronodular thymoma	AWD at 91 months	MUT	22.9	MUT
4	F	66	16.5	IIA	Micronodular thymoma	AWD at 7 months	MUT	12.3	WT
5	F	53	6.5	IIA	Micronodular thymoma	AWD at 68 months	MUT	7.9	WT
6	F	74	7.5	IIA	Micronodular thymoma	AWD at 66 months	MUT	5.9	WT
7	F	66	2.5	I	Micronodular thymoma	AWD at 14 months	MUT	8.8	WT
8	F	78	2.8	IIA	Micronodular thymoma	AWD at 6 months	MUT	10.9	WT
9	F	66	2	I	Micronodular thymoma	AWD at 9 months	MUT	7.8	WT
10	M	57	3	IIA	Micronodular thymoma	AWD at 3 months	MUT	4.9	WT
11	F	74	10	I	Micronodular thymoma + Type A	AWD at 110 months	MUT	17.6 (M) / 21.4 (A)	MUT
12	F	72	3.8	IIA	Micronodular thymoma + Type A	AWD at 4 months	MUT	17.0 (M) / 31.5 (A)	MUT
13	F	73	5	IVA	Micronodular thymic carcinoma	AWD at 50 months	WT	-	WT
14	M	49	2.4	I	Micronodular thymic carcinoma	AWD at 30 months	WT	-	WT
^#Modified Masaoka staging in patients with micronodular thymoma and AJCC stages in patients with micronodular thymic carcinoma.
NGS, next-generation sequencing; VAF, variant allele frequency; AWD, alive without disease recurrence; MUT, mutated; WT, wild-type; M, micronodular thymoma area; and A, type A thymoma area

Representative pathological images of tumors are shown in Figs. 1 and 2. All cases of micronodular thymoma with lymphoid stroma had characteristic features with discrete nodules and islands of epithelial tumor cells in a lymphoid stroma. The cytologically bland-looking tumor cells were short spindle or oval in shapes with inconspicuous nucleoli and minimal mitotic activities (Figs. 1A and 1B). In this cohort there were two cases containing mixed type A and micronodular thymoma elements, and in each case the borders between different histologic areas were distinct (Figs. 1C and 1D).

For micronodular thymic carcinoma with lymphoid stroma, the low-power appearance is almost indistinguishable from that of micronodular thymoma with lymphoid stroma as both entities have characteristic micronodular tumor nests in a lymphoid stroma (Fig. 2A). At high power, the tumor cells displayed apparent cytologic atypia, large vesicular nuclei, conspicuous nucleoli, and brisk mitotic figures (Fig. 2B). In addition, the tumor cells were more epithelioid in shapes rather than spindle-to oval. Positive immunoreactivity for CD5 and CD117 was observed only in micronodular thymic carcinoma with lymphoid stroma (Figs. 2C and 2D).

GTF2I mutation analysis

In this study, we used two approaches to investigate the GTF2I p.L424H mutation. First, we developed a highly sensitive deep NGS assay using Ion AmpliSeq HD technology that utilized dual molecular barcodes to detect rare somatic variants. (10) Second, we performed Sanger sequencing of GTF2I. The results of GTF2I mutation analysis are summarized in Table 1 and Fig. 3. Using Ion AmpliSeq HD assay, we detected GTF2I p.L424H mutations in all micronodular thymomas with lymphoid stroma, including two cases with combined type A thymoma areas. None of the micronodular thymic carcinomas with lymphoid stroma harbored GTF2I p.L424H mutations. In contrast, Sanger sequencing detected GTF2I p.L424H mutations in only 3 of the 12 (25%) micronodular thymomas with lymphoid stroma. The Sanger sequencing results reflected the VAF detected by the Ion AmpliSeq HD assay, as illustrated in Fig. 4. Those three tumors with GTF2I p.L424H mutations detected by Sanger sequencing had the highest VAF, with a discernible T > A mutant peak readily observed in the electropherogram (e.g., Case 3 in Fig. 4). For other tumors, the T > A mutant peak either had a height like other background noise peaks (e.g., Case 2 in Fig. 4) or was almost imperceptible (e.g., Case 10 in Fig. 4).

Mutation analysis in HRAS, KRAS, NRAS, and TP53

In addition to GTF2I mutations, we used the Ion AmpliSeq HD assay to analyze mutational hotspots in HRAS, KRAS, NRAS, and exons 2–11 of TP53. The results are summarized in Fig. 3 and Table 2. HRAS mutations were detected in 2 of the 12 micronodular thymomas with lymphoid stroma (p.A11T or p.Q61K). KRAS mutations were detected in 2 of the 12 micronodular thymomas with lymphoid stroma (p.G12V or p.G12R). No NRAS mutations were noticed in any patient. None of the micronodular thymic carcinomas with lymphoid stroma harbored HRAS, KRAS, or NRAS mutations. TP53 mutations were detected in one micronodular thymoma with lymphoid stroma and both cases of micronodular thymic carcinomas with lymphoid stroma.

Table 2

Variant details
Sample no.	Pathological diagnosis	GTF2I	HRAS	KRAS	TP53
1	Micronodular thymoma	p.L424H (8.3%)		p.G12V (27.6%)
2	Micronodular thymoma	p.L424H (13.8%)	p.A11T (2.4%)		p.E298K (2.8%)
3	Micronodular thymoma	p.L424H (22.9%)
4	Micronodular thymoma	p.L424H (12.3%)
5	Micronodular thymoma	p.L424H (7.9%)
6	Micronodular thymoma	p.L424H (5.9%)
7	Micronodular thymoma	p.L424H (8.8%) p.A425V (4.4%)
8	Micronodular thymoma	p.L424H (10.9%)
9	Micronodular thymoma	p.L424H (7.8%)
10	Micronodular thymoma	p.L424H (4.9%)
11_A	Micronodular thymoma + Type A (Type A area)	p.L424H (21.4%)	p.Q61K (27.9%)
11_M	Micronodular thymoma + Type A (Micronodular area)	p.L424H (17.6%)	p.Q61K (12.9%)	p.G12R (9.5%)
12_A	Micronodular thymoma + Type A (Type A area)	p.L424H (31.5%)
12_M	Micronodular thymoma + Type A (Micronodular area)	p.L424H (17.0%)
13	Micronodular thymic carcinoma				p.R342Q (2.9%)
14	Micronodular thymic carcinoma				p.V31I (27.5%)

Comparison of mutations between different histologic areas in cases having mixed type A and micronodular thymoma with lymphoid stroma

Mutation tests were separately performed in different histologic components of 2 cases with mixed type A and micronodular thymoma with lymphoid stroma. The results are shown in Fig. 5. In case 11, the GTF2I p.L424H and HRAS p.Q61K mutations were detected in both elements, with a comparatively high VAF in the type A area. Additionally, a KRAS p.G12R mutation was identified in the micronodular thymoma area. In case 12, the GTF2I p.L424H mutation was detected in both elements, with relatively high VAF in the type A area.

In this study, we demonstrated that recurrent GTF2I p.L424H mutations can be identified in all cases of micronodular thymomas with lymphoid stroma, and none of the tested micronodular thymic carcinomas with lymphoid stroma harbored this mutation. To date, this is the largest study investigating genetic changes in these micronodular thymic neoplasms and is the first study to utilize highly sensitive NGS assays with molecular barcode technology.

A high frequency of GTF2I p.L424H mutations in thymomas was first discovered in a landmark study by Petrini et al. in 2014 and subsequently verified by several studies, including the TCGA study. (7–9, 11) This mutation seems to be unique to thymoma and highly associated with the WHO histological classification. (7, 8) In the TCGA study, the GTF2I p.L424H mutation was observed in 100% of type A and 70% of type AB thymomas. In contrast, only 1 of 10 B3 thymomas contained this mutation, and none of the B1 or B2 thymoma or thymic carcinoma showed the GTF2I p.L424H mutation. (8)

Data regarding GTF2I p.L424H mutations in micronodular thymoma with lymphoid stroma are scanty. In the TCGA study, only two cases were investigated, and one of them showed the GTF2I p.L424H mutation. (8) Wells et al. recently found the GTF2I p.L424H mutation in three of four cases. (9) A highly sensitive method is required to accurately assess GTF2I p.L424H mutations because the high content of non-neoplastic lymphoid cells in these tumors. Previous studies used either whole-exome sequencing (TCGA study) or Sanger sequencing (study by Wells et al.), which may not be sufficiently sensitive to detect mutations with low VAF. Using a highly sensitive NGS assay, we successfully detected recurrent GTF2I p.L424H mutations in all 12 cases of micronodular thymoma with lymphoid stroma. The VAF of GTF2I p.L424H mutations detected in this study ranged from 4.9–22.9%. Since the limit of detection for Sanger sequencing is approximately 15–20% mutant alleles, (14) this method may not be sensitive enough to detect GTF2I mutations in micronodular thymomas with lymphoid stroma. This is also confirmed in our study as GTF2I p.L424H mutations were only detected in three cases with VAF > 15% by Sanger sequencing. Sanger sequencing is not appropriate to study genetic mutations in micronodular thymomas with lymphoid stroma and may give significant numbers of false negatives. This methodological issue should also be taken into consideration when studying other types of thymoma rich in lymphocytes, such as type B1, B2, AB, and the recently described “lymphocyte-rich spindle cell thymoma”. (15) A sensitive method such as high-depth NGS, or single nucleotide extension and mass spectrometry is required to accurately detect genetic mutations in these tumors.

In addition to GTF2I, enrichment of HRAS, KRAS, NRAS, and TP53 mutations have been found in thymomas. (7, 8, 16) Whether mutations in these genes are also enriched in micronodular thymomas with lymphoid stroma is unknown. We found recurrent mutations in HRAS (codon A11 or Q61) and KRAS (codon G12) in micronodular thymomas with lymphoid stroma. These mutations are oncogenic gain-of-function mutations. No NRAS mutation was detected in our study. Only 1 case of micronodular thymoma with lymphoid stroma had TP53 mutation.

The presence of recurrent GTF2I p.L424H mutations in micronodular thymoma with lymphoid stroma strongly suggests that this tumor might be closely related to type A and type AB thymomas, as they all have a high frequency of GTF2I p.L424H mutations. In the initial report of micronodular thymoma with lymphoid stroma by Suster and Moran in 1999, the tumor was proposed to represent an unusual but distinctive morphological variant of spindle cell thymoma (type A thymoma). (3) This hypothesis was primarily based on the observation that tumor cells in micronodular thymoma with lymphoid stroma showed substantial morphological resemblance to those in type A thymoma. This belief was further strengthened by subsequent reports showing that micronodular thymoma with lymphoid stroma can occasionally combine with type A thymoma in the same tumor. (1, 17–19) In this study there were two cases with mixed type A and micronodular thymoma with lymphoid stroma, and GTF2I p.L424H mutations could be detected in both type A and micronodular thymoma elements. Furthermore, an identical HRAS p.Q61K mutation was found in both type A and micronodular thymoma elements in one case (case 11), suggesting that these two elements were clonally related. Besides, an additional KRAS p.G12R mutation was identified in micronodular thymoma element but not in areas of type A thymoma. To our knowledge, this is the first time that KRAS p.G12R was detected in thymoma. It is worth noting that the types of substitution at G12 are highly dependent on cancer types. (20) KRAS p.G12R mutations are especially enriched in pancreas cancer and are very rare in other cancers that frequently harbor KRAS mutations. Besides, a recent study showed that KRAS p.G12R may be functionally distinct from other KRAS driver mutations, and it is impaired in PI3K signaling and macropinocytosis. (21) Further studies are needed to explore the role of KRAS p.G12R mutation in the development of micronodular thymoma with lymphoid stroma.

In this study, micronodular thymic carcinoma with lymphoid stroma showed a different mutation profile as all cases had TP53 mutations but no GTF2I, HRAS, KRAS, or NRAS mutations. Despite the limited case number, this finding suggests that micronodular thymic carcinoma with lymphoid stroma is genetically distinct from micronodular thymoma with lymphoid stroma. Using a highly sensitive assay to detect the GTF2I p.L424H mutation can be helpful in the differential diagnosis between these two tumor types.

In conclusion, using a highly sensitive molecular barcoding NGS assay, we showed recurrent GTF2I p.L424H mutations were present in all micronodular thymoma with lymphoid stroma (12/12) but not in micronodular thymic carcinoma with lymphoid stroma. A subset of micronodular thymoma with lymphoid stroma also had concomitant HRAS (2/12) and KRAS (2/12) mutations. Our study demonstrates GTF2I p.L424H mutations is a constant genetic feature of micronodular thymomas with lymphoid stroma and provides compelling molecular evidence that this tumor is closely related to type A and type AB thymomas.

Acknowledgments

The authors are grateful to the Biobank, Taipei Veterans General Hospital for assistance with sample preparation.

Conflict of interest

The authors declare no competing financial interests.

Ethics approval and consent

This study was approved by the Taipei Veterans General Hospital institutional review board, and the requirement for informed consent was waived. The study was performed in accordance with the Declaration of Helsinki.

Author contributions

Min-Shu Hsieh: Conceptualization, Investigation, Data Curation, Writing- original draft preparation

Hua-Lin Kao: Conceptualization, Investigation, Data Curation

Wen-Chang Huang: Investigation, Data Curation

Shu-Ying Wang: Methodology, Investigation, Experimental procedure

Shin-Ying Lin: Methodology, Investigation, Experimental procedure

Ping-Yuan Chu: Methodology, Investigation, Experimental procedure

Chin-Chen Pan: Investigation, Data Curation

Teh-Ying Chou: Project administration, Funding acquisition, Supervision

Hsiang-Ling Ho: Methodology, Investigation, Supervision, Project administration

Yi-Chen Yeh: Conceptualization, Methodology, Investigation, Data Curation, Writing- original draft preparation, Funding acquisition

All authors read and approved the final paper.

Funding

This work was funded by the Taipei Veterans General Hospital, Taiwan [grant numbers V111C-063 and V111E-004-3] and the Ministry of Science and Technology, Taiwan [grant number MOST 109-2320-B-075-004-MY3].

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Supplementary information

Supplementary information is available on the website of Modern Pathology.

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There is NO conflict of interest to disclose.

SuppFigure1GTF2IAmpliconDesignOK.tif
(A) Schematic amplicon design. GTF2I and its two pseudogenes share high sequence homology, with a single base difference. The NGS amplicon was designed to span the region containing both the single base differences (chr7:74146870) between GTF2I and the pseudogenes and the p.L424H mutation site (chr7:74146970). (B) Representative screenshot showing alignments at GTF2I in a sample with the p.L424H mutation using the Integrative Genomics Viewer. All short reads of NGS in this screenshot have cytosine (C) at chr7:74146870, and therefore were derived from GTF2I, instead of the pseudogenes. NGS: next-generation sequencing
SuppTable1NGSTargetRegions.xlsx

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Constant GTF2I L424H mutation in micronodular thymoma with lymphoid stroma: genetic evidence supporting its close relationship with type A and AB thymomas

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