Massively parallel sequencing identifies recurrent mutations in TP53 in thymic carcinoma associated with poor prognosis.

Original Article

Massively Parallel Sequencing Identifies Recurrent Mutations in TP53 in Thymic Carcinoma Associated with Poor Prognosis Andre L. Moreira, MD, PhD,* Helen H. Won, MS,* Robert McMillan, MD,† James Huang, MD,† Gregory J. Riely, MD, PhD,‡ Marc Ladanyi, MD,*§ and Michael F. Berger, PhD*§

Background: The characterization of the molecular alterations in thymic epithelial tumors may lead to a better understanding of tumorigenesis, new therapeutic targets, and biomarkers in these tumors. Methods: Paired tissue (tumor and matched normal) from 15 thymic carcinomas (TCA) and six B3 thymomas were evaluated by exon capture of 275 cancer-related genes, followed by deep coverage nextgeneration sequencing, which identifies somatic sequence variants, small insertions and deletions, and copy number alterations involving all exons of the captured genes. Results: Non-silent somatic mutations were identified in 12 of 15 (80%) TCA with a median of one mutation per tumor (range 0–26). Recurrent mutations were identified in tumor suppressor genes TP53 (n = 4), SMAD4 (n = 2), and CYLD (n = 2); and chromatin remodeling genes KDM6A (n = 3), SETD2 (n = 2), MLL3 (n = 2), and MLL2 (n = 2). Tumors with TP53 mutation appeared to exhibit more aggressive behavior. Therefore, the role of P53 was evaluated by immunohistochemistry in an additional ten cases. P53 overexpression correlated with TP53 mutation. These tumors had a higher rate of recurrence and death of disease compared to carcinoma with normal p53 expression (p = 0.02 for disease-free survival and p = 0.05 for overall survival). Among the B3 thymomas, mutations were identified in four of six tumors. Mutations in BCOR (BCL6 co-repressor) were seen in three thymomas and MLL3 (involved in histone methylation) in one tumor. Conclusions: Next-generation sequencing of cancer genes in thymic epithelial tumors revealed a low frequency of mutation, with different patterns between TCA and B3 thymomas. TP53 and BCOR were the most frequently mutated genes in TCA and B3 thymomas, respectively. Alterations in p53 are associated with worse prognosis in TCA. Key Words: Next-generation sequencing, Thymoma, Thymic carcinoma. (J Thorac Oncol. 2015;10: 373–380) Departments of *Pathology, †Surgery, ‡Medicine, and §Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY. Disclosure: The authors declare no conflict of interest. Address for correspondence: Andre L. Moreira, MD, PhD, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065. E-mail: [email protected] DOI: 10.1097/JTO.0000000000000397 Copyright © 2014 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/15/1002-0373

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hymoma is a rare tumor of thymic epithelial cells with an estimated incidence of 0.13 per 100,000 persons a year.1 Thymomas represent a group of tumors with heterogeneous histological patterns,2 but a relatively homogeneous clinical history. In general, patients with thymoma have an indolent course of disease with a tendency for multiple local recurrences with later systemic metastases. Thymic carcinomas (TCA) are rarer than thymomas and have a more aggressive behavior. TCA are characterized histologically by overtly cytological malignant tumor cells. TCA are also heterogeneous histologically with squamous cell carcinoma as the most common histological presentation. Outcomes for patients with thymic tumors depend largely upon stage and histologic type. For thymomas, WHO Type A and B1 thymomas are considered to be low-grade tumors, whereas WHO Type B3 thymoma has a more aggressive behavior.3–5 However, measurements of outcome have not been standardized and are particularly problematic in thymic neoplasms due to the rarity of these tumors and pace of disease progression.6 Ten-year disease-specific survival is approximately 20% for stage 4 thymomas.6 TCA are typically invasive and more aggressive with a high risk of recurrence, distant metastasis, and short survival.6,7 Surgery is the mainstay of treatment for thymoma and TCA, whereas chemotherapy is offered to patients with more advanced disease either pre-operatively with intent for optimization of surgical resection or as palliative therapy for patients with unresectable disease.8 Despite reproducible response rates, complete response (radiographic or pathologic) to chemotherapy is rare. Therefore, new therapeutic strategies for the management of thymoma and TCA are needed. There have been very few studies dedicated to the understanding of the biology of thymomas and TCA, partially due to the rarity of these tumors. An improved appreciation of the molecular characteristics of thymic epithelial tumors may lead to identification of molecules that can be targeted therapeutically, thus opening new treatment options for the patients who suffer from these diseases. In addition, these studies may also identify biomarkers that can be potentially used for the clinical management of these patients. Earlier studies have demonstrated that TCA are molecularly different from thymomas. Molecular differences between thymomas type A from type B are also seen. Thymomas type

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A show low frequency of allelic imbalances when compared to types B2–B3.9–12 The most comprehensive molecular analyses of thymic tumors have focused on the epidermal growth factor receptor (EGFR) and tyrosine protein kinase kit pathway, which can be targeted therapeutically with specific drugs. EGFR mutations have been reported in thymomas with a low frequency and a case report demonstrating response to gefitinib stimulated interest in this pathway. However, in other series, no somatic mutations in the EGFR gene were identified.9,12–17 Moreover, a previous study from our center involving profiling at mutational hotspots in seven genes mediating EGFR signaling (KRAS, HRAS, BRAF, PIK3CA, ERBB2, MEK1, and EGFR) identified somatic mutations in just 7% of tumors (two KRAS and one HRAS mutation) suggesting that analysis of additional pathways are required to identify driver oncogenes in thymic tumors.9 Expression of KIT can be identified by immunohistochemistry in approximately 60% of TCA but not in thymomas, suggesting that activation of this pathway is important for TCA pathogenesis.13,16,18,19 An objective response to a KIT inhibitor (imatinib) in a patient with TCA harboring an activating KIT mutation has been described in a case report.20 However, in broader analyses, KIT mutations appear to be relatively uncommon in TCA and absent in thymomas.9,14,15,17,21 Therefore, a better understanding of the biology and the molecular mechanisms in these tumors is essential for the formulation of new rationales for therapeutic intervention.22 Recent advances in DNA sequencing have enabled large-scale genomic characterization of tumor samples at increased throughput and sensitivity and decreased cost compared to more traditional methods. Through exon capture by hybridization, one can isolate and sequence dozens of genes up to the entire exome. We applied this technology to analyze 275 key cancer-associated genes, encompassing all known targetable “driver” alterations. This approach can identify mutations, gene amplifications, and other molecular alterations in all target cancer genes simultaneously with a small amount of input DNA, and it has been validated for use with DNA obtained from frozen and paraffin-embedded tissue.23,24

MATERIALS AND METHODS Tumors The study had the approval from the MSKCC Institutional Review Board Committee. Fifteen TCA and six thymomas WHO type B3 that had been resected in the hospital between 2004 and 2010 were identified retrospectively from a database prospectively maintained by the Thoracic Service of the Department of Surgery of Memorial Hospital. Fourteen TCA were classified as squamous cell carcinoma and one tumor was classified as an undifferentiated TCA. Patients were staged according to the Masaoka-Koga staging criteria.25

DNA Extraction Fresh frozen tissue or paraffin-embedded tissue from tumor and normal paired tissue were evaluated, confirmed, and marked by a pathologist (ALM), and samples underwent microdissection followed by DNA extraction. DNA extraction

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for all samples was performed using the Qiagen DNeasy Blood and Tissue Kit standard protocol. Sample concentration and purity were confirmed with a Qubit Fluorometer.

Sequencing Genomic alterations in key cancer-associated genes were profiled using a custom capture, deep sequencing assay termed integrated mutation profiling of actionable cancer targets (IMPACT). Hybridization capture of Illumina-compatible sequence libraries (Kapa Biosystems, Wilmington, MA) was performed using custom oligonucleotides (Nimblegen SeqCap, Madison, WI) designed to target all protein-coding exons and selected introns of 275 commonly implicated oncogenes, tumor suppressor genes, and members of pathways considered actionable by targeted therapies. DNA was subsequently sequenced on an Illumina HiSeq 2000 as paired-end 75-base pair reads to an average read depth of 435-fold per tumor (277fold per normal). Sequence reads were aligned to the reference human genome (hg19) using the Burrows–Wheeler Alignment tool26 and post-processed using the Genome Analysis Toolkit (GATK).27 Single nucleotide variants, small insertions and deletions, and copy number alterations were called as described elsewhere.24 A list of the 275 genes included in the assay is presented in Supplementary Table 1 (Supplemental Digital Content 1, http://links.lww.com/JTO/A740). Two out of 15 TCA samples were captured using a smaller probe set comprised of only 230 of the 275 genes. A TP53 mutation was found in one tumor using the 230 gene panel. Immunohistochemical stain. To evaluate a possible correlation between p53 mutation and aggressive behavior, an additional 10 cases of TCA were evaluated for p53 expression by immunohistochemical stain. Histological sections of TCA and B3 thymomas were evaluated for p53 expression (monoclonal antibody clone DO-7, Ventana, USA) in an automated stainer (BenchMark XT, Ventana, USA). Although there is no perfect correlation between p53 expression by immunohistochemistry (IHC) and mutation, we used the criteria set forth by Yemelyanova et al.28 Tumor that showed diffuse overexpression of p53 defined by strong expression in more than 60% of tumor cells or lack of staining with the anti-p53 antibody are considered to be consistent with p53 mutation. Tumors with scattered positive cells or low level expression (less than 50%) are considered to be negative for mutation.

Statistical Analysis We used descriptive measures (χ2 test) to compare the association between quantity and quality of mutations within groups. Disease-free survival (DFS) was defined as the time from resection to recurrence or lung cancer-related death. Cause of death was determined from the death certificate or physician correspondence. Last follow-up, death from other causes, and unknown cause of death were censored. Overall survival (OS) is defined as time of death after surgery. DFS and OS were estimated using the Kaplan–Meier method. Associations between p53 expression, DFS, and OS were evaluated using Cox proportional hazards models. All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA).

Copyright © 2014 by the International Association for the Study of Lung Cancer


RESULTS Study Population The clinicopathological characteristics of the patients enrolled in the study are illustrated in Table 1. There were a similar distribution of man and woman among the groups of TCA and thymoma. Patients with thymoma were younger than patients with TCA. All patients with thymoma type B3 presented with advanced stage and received neoadjuvant therapy before surgery in contrast to eight patients in the TCA group that received neoadjuvant therapy.

Detection of Mutation in Thymic Epithelial Tumors All samples passed quality assurance tests, with mean sequence coverage of 435× per tumor. Two TCA had both frozen tissue and corresponding paraffin-embedded tissue available for analysis, and no discrepancies were detected, similar to what has been previously reported.23 Fourteen TCA were classified as squamous cell carcinoma and one classified as undifferentiated carcinoma (Table 2). Somatic non-synonymous protein-coding mutations were identified in 12 of 15 (80%) TCA. The rate of mutation per tumor was low with a median of one mutation per tumor (range 0–26) (Fig. 1). The most frequent mutations were seen in the tumor suppressor genes TP53 (n = 4), SMAD4 (n = 2), and CYLD (n = 2); and chromatin remodeling genes KDM6A (n = 3), SETD2 (n = 2), MLL3 (n = 2), and MLL2 (n = 2) (Fig. 2). In this small sample, it appears that there were no significantly co-occurring or mutually exclusive mutations in TCA. There were no mutations identified in KIT, and one tumor had a Q61K mutation in neuroblastoma RAS viral (v-ras)oncogene (NRAS). Table 2 shows the complete list of mutations identified in this cohort. The undifferentiated TCA had 26 identifiable mutations. This tumor came from a patient who presented with stage 4b, and received neoadjuvant therapy before surgery. A thymic origin was confirmed by immunohistochemistry expression of TABLE 1. Clinicopathological Characteristics of Study Population Sex Male Female Age Mean ± SD Range Tumor size Mean ± SD Range Masaoka stage 1 2 3 4 Neoadjuvant

Thymoma B3

Thymic Carcinoma

2 4

5 10

45 ± 8 34–59

61 ± 11 43–79

6.3 ± 2.5 cm 3.6–10 cm 0 0 0 6 6

6 ± 2.2 cm 2–9 cm 2 3 3 7 8

Massively Parallel Sequencing

CD5 and KIT in tumor cells. This tumor also had two separate mutations in KDM6A and MLL2 (Table 2). Overall there was no significant difference in the number of mutations in the group that received neoadjuvant therapy and the untreated group (p = 0.59). Among the B3 thymomas, mutations were identified in four of six tumors. Mutations in BCOR (BCL6 co-repressor) were seen in three thymomas and MLL3 (involved in histone methylation) was seen in one tumor. It is interesting to note that there were three TCA and two B3 thymomas that harbored no mutations in the 275 genes studied, though it is possible that mutations were present and not detected due to sub clonality. All specimens were adequate for tumor cellularity. Among the three TCA with no detectable mutations, two patients were in the treated group and one in the untreated group. TCA showed areas of chromosomal instability characterized by gains and losses. The most frequent gains involved chromosomes 1q and 5p, and the most frequent losses involved chromosomes 3p and 6. No chromosomal gains and losses were detected in B3 thymomas.

Immunohistochemical Stains for p53 In this small sample, three out of four TCA with TP53 mutation appeared to exhibit more aggressive behavior. Three patients presented as Masaoka stage 4 (two patients had 4b and one 4a) and received neoadjuvant therapy. Two patients with TP53-mutated TCA died of disease (mean survival 2.2 years), and the third is alive with disease. In contrast, in the group of patients without TP53 mutations, there were four patients with stage 4a at presentation, two are alive with disease, and two died of unrelated causes (mean follow-up 8 years). All other TCA patients were Masaoka stage 1 to 3 at diagnosis and are alive since last follow-up. To evaluate a possible correlation between p53 mutation and aggressive behavior, an additional 10 cases of TCA were evaluated for p53 expression by immunohistochemical stain. Mutations in the TP53 have previously been shown to correlate strongly with upregulated p53 expression as measured by IHC.28 There was perfect correlation between TP53 mutation and p53 expression by IHC. All cases that had mutation showed over expression of p53 in more than 60% of tumor nuclei. All cases of TCA that were negative for TP53 mutation by next-generation sequencing had only focal positive staining in tumor nuclei (Fig. 3). From the additional 10 cases of TCA, the staining pattern in three tumors were considered to be consistent with p53 mutation; one tumor showed over expression of p53 and two showed complete absence of stain. Seven cases had focal positivity in tumor nuclei and therefore scored as negative. None of the six B3 thymomas evaluated showed alteration in the p53 by IHC, similar to what we observed from sequencing. All B3 thymomas showed focal reactivity for p53 in less than 10% of tumor nuclei.

Association of p53 Expression and Disease Progression To evaluate an association of TP53 mutation and disease progression, the expression pattern of p53 mutation by IHC was


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Sample Class

B3 Thymoma B3 Thymoma B3 Thymoma B3 Thymoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma

Sample

AM-23Ta AM-28Ta AM-29Ta AM-41Ta AM-16 AM-26T AM-26T AM-30T AM-30T AM-30T AM-30T AM-31T AM-31T AM-32T AM-33Ta AM-34Ta AM-34T AM-34T AM-34T AM-34T AM-36T AM-38Ta AM-39Tab AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T

X X X 7 17 1 1 1 3 16 22 16 X 10 X 3 7 17 18 19 21 19 1 1 1 3 3 3 3 3 4 5 6 7 7 8 11

Chrom

Reference allele

39922097 C 39916570 CTTC 39933697 AATCAACAGGA 152027747 G 7574034 C 27105610 C G 115256530 9780226 G C 89448564 50811826 C 41568668 G AAATG 50783884 70360647 CAG 104156502 T 44945137 C 47158121 C 151845846 AA 7578406 C 48573432 A 5215308 G 44514780 C 33793007 CGC 38197175 C 43804223 C 120480068 C 47098956 52440390 69988315 C 134920412 C 187446261 G 187517988 T 35871316 G C 106536260 106508239 C 151845523 A 68864736 G AAGA 118342507

Position T A A T T A T A A A G A A G A T T T T T C T T A C T T T T -

Alternate allele

TABLE 2. Mutations Identified in Thymoma B3 and Thymic Carcinomas Gene BCOR BCOR BCOR MLL3 TP53 ARID1A NRAS PIK3CD EPHA3 CYLD EP300 CYLD MED12 NFKB2 KDM6A SETD2 MLL3 TP53 SMAD4 PTPRS U2AF1 CEBPA EPHA10 MPL NOTCH2 SETD2 BAP1 MITF EPHB1 BCL6 FAT1 IL7R PRDM1 PIK3CG MLL3 PREX2 MLL

Variant Class Missense_Mutation Frame_Shift_Del Frame_Shift_Del Nonsense_Mutation Splice_Site Nonsense_Mutation Missense_Mutation Missense_Mutation Nonsense_Mutation Nonsense_Mutation Splice_Site Frame_Shift_Del In_Frame_Del Nonsense_Mutation Nonsense_Mutation Missense_Mutation Frame_Shift_Del Missense_Mutation Missense_Mutation Missense_Mutation Missense_Mutation In_Frame_Del Missense_Mutation Missense_Mutation Missense_Mutation Frame_Shift_Ins Frame_Shift_Ins Nonsense_Mutation Missense_Mutation Missense_Mutation Missense_Mutation Splice_Site Missense_Mutation Missense_Mutation Frame_Shift_Del Missense_Mutation Frame_Shift_Del

p.G1359R p.E1477fs p.P298fs p.R110a p.I332_splice p.Q1741a p.Q61K p.D466N p.R510a p.S371a p.D1539_splice p.I92fs p.2069_2070QQ>Q p.Y55a p.S1154a p.M1526I p.S4389fs p.R175L p.I6V p.P1437L p.R156H p.D105del p.R524H p.R75C p.C1120Y p.K2106fs p.E221fs p.R217a p.R743W p.T476M p.K4236E p.T179_splice p.S76L p.A78V p.K4497fs p.R36L p.K211fs

Protein Change

YES

YES

YES

YES

In COSMIC

(Continued )

0.13 0.45 0.58 0.28 0.59 0.10 0.46 0.26 0.27 0.36 0.26 0.34 0.11 0.22 0.46 0.43 0.29 0.22 0.24 0.07 0.07 0.11 0.21 0.18 0.21 0.20 0.11 0.23 0.24 0.17 0.18 0.22 0.21 0.23 0.22 0.24 0.18

Mutation Freq

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YES

p.R4212W p.A221fs p.F299C p.Y126N p.H429fs p.T676fs p.R361C p.K84del p.V1025fs p.S38fs p.A974V p.G2182V p.Y220S

YES YES YES YES

0.14 0.15 0.21 0.25 0.17 0.15 0.25 0.15 0.16 0.17 0.28 0.35 0.57


FIGURE 1. Illustration of the number of somatic non-synonymous protein-coding mutations per thymic carcinoma. Each bar represents one tumor. Red bar The number of mutations seen in a random pulmonary adenocarcinoma. Note that in three thymic carcinomas, no mutations were identified in the panel of 275 cancer-related genes.

b

a

Indicate patients received neoadjuvant therapy before surgery. This carcinoma was classified as undifferentiated carcinoma. All others were classified as squamous cell carcinoma.

Missense_Mutation Frame_Shift_Ins Missense_Mutation Missense_Mutation Frame_Shift_Ins Frame_Shift_Ins Missense_Mutation In_Frame_Del Frame_Shift_Del Frame_Shift_Del Missense_Mutation Missense_Mutation Missense_Mutation MLL2 MLL2 TBK1 TP53 FLCN NF1 SMAD4 STK11 PTPRS KDM6A KDM6A NOTCH3 TP53 A C G T G C T T A G G T A C AAG G C C C T Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma Thymic carcinoma AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-39T AM-40Ta AM-42Ta

12 12 12 17 17 17 18 19 19 X X 19 17

49425854 49447772 64875705 7578554 17119708 29553477 48591918 1207152 5222730 44732909 44937733 15271894 7578190

Alternate allele Sample Class Sample

TABLE 2. (Continued)

Chrom

Position

Reference allele

Gene

Variant Class

Protein Change

In COSMIC

Mutation Freq


correlated with DFS and OS. As illustrated in Figure 4, survival analysis showed that TCA with staining patterns consistent with p53 mutation had a 5-year DFS of 12.5% compared with 54% in the group with wild-type p53 staining pattern (p = 0.02, HR: 0.2848, 95% CI of ratio 0.06 to 0.72). The median survival for patients with mutated p53 was 2 years (731 days), whereas median survival could not be defined for the p53 wild type group. Interestingly, there was also a trend of poor prognosis in OS for patients with TCA that overexpressed p53 by IHC (p = 0.05).

DISCUSSION This study represents, to the best of our knowledge, the most comprehensive mutation profiling analysis of TCA conducted to date. Our results are consistent with prior reports9,29 confirming a low rate of mutation in these tumors. This low rate of mutations is not related to inadequate sensitivity of the technique used, because we obtained deep sequence coverage with an average of 435× for the evaluated exons. It is possible that the analysis of larger numbers of tumors may reveal other significantly mutated genes. Furthermore, whole exome sequencing may reveal additional clinically or biologically relevant mutations in other genes outside of the 275 cancerrelated genes that were captured by our assay.

FIGURE 2. Illustration of the number of the most frequent mutations identified in thymic carcinoma.


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FIGURE 3. Expression of p53 by immunohistochemistry. A, The staining pattern of a thymic carcinoma with wild-type TP53. Note scattered positive nuclei representing less than 10% of the tumor cells. B, The staining pattern of p53 in a thymic carcinoma with TP53 mutation. Note positive nuclei in more than 60% of tumor cells. There was a perfect correlation between mutation and immunohistochemistry staining pattern in these cases.

Despite several publications in the field of TCA suggesting that KIT mutations are present in these tumors, we did not detect mutations in the gene in any of the 15 TCA evaluated. Previous studies have showed a low rate of KIT mutations in TCA,9,21,30–32 thus our sampling of only 15 cases may be in part responsible for our negative KIT results. Several studies have suggested that TCA and B3 thymomas have different molecular signatures.9,33,34 Our data further support these findings. In our series, none of the B3 thymomas evaluated had mutations in p53 gene or showed chromosomal gains or losses, which were commonly observed in TCA. In contrast, none of the TCA had mutation in BCOR, which we observed repeatedly in B3 thymomas. It is interesting to note that a recent study presenting a molecular analysis of one B3 thymoma also reported a mutation in BCOR.35 It is possible that BCOR (BCL6-corepressor) may be a marker of B3 thymoma. Further studies evaluating the role of this mutation in other thymoma subtypes are needed

FIGURE 4. Relationship of p53 expression by immunohistochemistry and disease-free survival Kaplan–Meier curve in 25 cases of thymic carcinoma (p = 0.0205). Similar observation was seen when overall survival was analyzed (p = 0.0545) (not shown).

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to confirm these observations. BCOR, a gene related to germinal center formation and prevention of apoptosis, is frequently mutated in acute myeloid leukemia (AML) and other myelodysplastic syndromes.36 In hematological diseases, mutations in BCOR are associated with poor prognosis. The role of this gene in thymomas is currently unclear. Mutations in TCA were frequently observed in tumor suppressor genes and chromatin remodeling genes. p53 is a tumor suppressor gene and the most commonly mutated gene in human malignant tumors.37 TCA is not an exception. Although data in TCA are sparse, it has been shown in smaller series by polymerase chain reaction and other techniques that mutations in the p53 gene can be seen in approximately 30% of TCA,38,39 similar to our study. Although there is no targeted therapy toward p53-mutated tumors, the mutation status and/ or overexpression of p53 may be used as prognostic marker in TCA. p53 mutant TCA seem to be associated with a more aggressive behavior, confirming earlier reports.38–40 Similar observations have also been made in other tumors with p53 mutation.41,42 As no mutations in p53 were observed in six cases of B3 thymoma, it is possible that mutation in this gene is a marker of TCA compared with thymomas. Other tumor suppressor genes mutated in TCA are SMAD4 and CYLD. The former is a gene involved in the transforming growth factor beta (TGF-β) pathway. Mutations in SMAD4 often lead to loss of expression and are associated with worse prognosis in colon and pancreatic cancers.44,45 CYLD is involved in another inflammatory pathway, the NF-ĸB pathway. Germline mutations in the gene are tumorigenic to epithelial cells, and are associated with familial cylindromatosis that leads to multiple benign skin adnexal tumors.46 Somatic mutations in CYLD are rare in solid tumors47 and have been described in TCA.48 Notably, both mutations we observed in CYLD are truncating mutations: one is a nonsense mutation (p.S371*) and the other is a frameshift deletion (p.I92fs). The possible role for these genes in the biology of TCA remains to be evaluated.



Chromatin remodeling genes are associated with epigenetic regulation, and mutations in this class of genes are commonly seen in solid tumors.49–52 Mutations in several chromatin remodeling genes were observed in our series, with multiple mutations in KDM6A, SETD2, MLL2, and MLL3 in TCA. All identifiable mutations in B3 thymomas were in this class, with recurrent mutations in BCOR and a single mutation in MLL3. There has been a renewed interest in the study of chromatin remodeling genes in carcinogenesis due to the potential of targeting these genes for therapy.53 All B3 thymomas and eight out of 15 TCA received neoadjuvant therapy before resection, a standard practice for thymic epithelial tumors that present with advanced clinical stage. Although chemotherapy and or radiation can induce somatic mutations,54 there was no difference in the number of observed mutations between treated and untreated TCA (p > 0.005). It is possible that chemotherapy-induced mutations were present but not detected due to subclonality in the evaluated tumors and the time required for their development. In summary, TCA show a relatively low rate of mutations compared with other solid tumors. Similar to most carcinomas, mutations in p53 are most frequent and seem to be associated with more aggressive behavior. Further studies involving more cases and the sequencing of an expanded panel of genes are warranted for a better understanding of the oncogenic mechanisms in these rare tumors.

ACKNOWLEDGMENTS

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