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Arun Rajan, MD,* Nicolas Girard, MD, PhD,† and Alexander Marx, MD‡
Abstract: The rapid advent of technology in recent years has resulted
in a substantial increase in our knowledge of the molecular underpin-
nings of thymic epithelial tumors. In addition to previously described
chromosomal aberrations and alterations in DNA methylation, genome
sequencing has helped unravel hitherto unknown mutations in these
tumors. Attempts are also being made to develop gene signatures to help
in the identification of patients likely to benefit from adjuvant therapy.
Some of the recently identified genetic alterations have the potential
to serve as targets for biological therapy, thus opening newer avenues
for treatment of thymic epithelial tumors and increasing the number of
effective options for treatment of recurrent or refractory disease.
Key Words: Thymoma, Thymic carcinoma, Methylation, Chromosomal
abnormalities, Gene expression, Sequencing, Gene signatures
(J Thorac Oncol. 2014;9: S131–S136)
T hymic epithelial tumors (TETs) are rare mediastinal
cancers that have traditionally been treated using a mul-
timodality approach consisting of surgery, radiotherapy, and
platinum-based chemotherapy.1 Effective treatment options
are limited for patients with recurrent disease. The World
Health Organization (WHO) classification is the most widely
used system for the histological classification of TETs.2 Based
on thymic epithelial cell morphology, the degree of thymocyte
involvement and the extent of epithelial atypia TETs are clas-
sified into the following subtypes: A, AB, B1, B2, B3, and C.
Efforts such as The Cancer Genome Atlas (TCGA)
research network have helped in providing a deep understanding
of the molecular alterations associated with a variety of tumors.3–7
In comparison, until recently, scant information was available
on genomic changes associated with TETs. Major challenges in
obtaining genetic data still exist, especially from the more com-
mon thymoma subtypes (AB, B1, and B2) due to the presence
of a significant number of non-neoplastic thymocytes. The latter
*Thoracic and Gastrointestinal Oncology Branch, National Cancer Institute,
Bethesda, MD; †Respiratory Medicine, Thoracic Oncology Service,
Hôpital Louis Pradel, Hospices Civils de Lyon, Lyon, France; and
‡Institute of Pathology, University Medical Centre Mannheim, University
of Heidelberg, Mannheim, Germany.
Disclosure: The authors declare no conflict of interest.
Address for correspondence: Arun Rajan, MD, Thoracic and Gastrointestinal
Oncology Branch, National Cancer Institute, 10 Center Drive, 10-CRC,
Room 4-5330, Bethesda, MD 20892. E-mail. rajana@mail.nih.gov
Copyright © 2014 by the International Association for the Study of Lung
Cancer
ISSN: 1556-0864/14/0909-S131
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typically outnumber and tightly intermingle with the neoplas-
tic epithelial cells and largely preclude classical or array-based
comparative genomic hybridization (CGH) approaches except
in rare cases in which epithelial content is unusually high or in
which thymocytes can be removed through short-term cell cul-
ture of neoplastic epithelial cells.8–11 Fluorescence in situ hybrid-
ization analyses have been sparse as well.8,12,13 However, with
the advent of newer and more sensitive techniques in molecular
biology (e.g., next generation sequencing) and bioinformatics,
there has been and will be an incremental increase in the avail-
ability of high-quality data that has helped in expanding our
understanding of the molecular pathogenesis of these diseases.
Development of newer therapies based on an understanding of
genomic alterations could potentially revolutionize treatment of
TETs and result in an improvement in clinical outcomes. This
review provides an overview of genetic alterations associated
with TETs with a focus on recent discoveries that could have an
impact on the management of thymic cancers.
CHROMOSOMAL ABNORMALITIES
Copy number (CN) gains and losses of various chromo-
somes have been well described in TETs, and the frequency
of these changes is higher in the more aggressive histological
subtypes. Based on the pattern of chromosomal changes, TETs
can be clustered into various groups, demonstrating a correla-
tion between genetic findings and WHO-based histology, with
overlapping alterations between type AB and B2 thymoma,
B2 and B3 thymoma, and B3 thymomas and thymic carcino-
mas.8–11,14 In short, 6q25.2–25.3 loss is observed in all sub-
types except B1 thymomas (a paucity of B1 thymomas among
cases analyzed might have had a bearing on this result); 1q
gains are common in type B2, B3 thymomas and thymic car-
cinomas; in addition, thymic carcinomas present with 1q, 4, 5,
7, 8, 9q, 12, 15, 17q, 18, 20 gains, and 3p, 6, 6p23, 9p, 13q,
14, 16q, 17p losses.9–11,14 The biological significance of these
changes is yet to be determined in the vast majority of cases.
However, it is known that the 6p23 region encompasses the
FOXC1 tumor suppressor gene, and chromosomal loss is cor-
related with lower protein expression. Patients with FOXC1-
negative tumors had a shorter time to progression and a trend
for a shorter disease-related survival.15
ALTERATIONS IN DNA METHYLATION
Silencing of tumor suppressor genes such as FHIT,
MLH1, and E-cad by promoter hypermethylation has been
described in TETs.16 These changes along with DNA hypo-
methylation have been correlated with Masaoka stage and
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WHO subtype of the TET. Other genes affected by aber-
rant methylation include DAP-K, p16, MGMT, and HPP1.17
MGMT methylation and loss of its protein expression has
been reported to be significantly more frequent in thymic car-
cinoma than in thymoma (74% versus 29%) and in advanced
TETs (stages III/IV) compared with early stage tumors (stages
I/II).18 Methylation of the promoter region of CDKN2 has
been observed in up to 14% of thymomas and 25% of thymic
carcinomas.19 Immunohistochemical analysis of 132 TETs
demonstrated CN loss of CDKN2A accompanied by lack
of expression of its related protein p16INK4 and identified
tumors with poor prognosis.20
A deeper understanding of alterations in DNA methyla-
tion can have important therapeutic implications. For example,
MGMT hypermethylation is associated with a greater degree
of sensitivity to alkylating agents.21 Dysregulation of the
cyclin-dependant kinase pathway can potentially be offset by
using cyclin-dependent kinase inhibitors such as milciclib. An
ongoing phase II study is evaluating the efficacy of milciclib
in thymic carcinoma (NCT01011439). Forty-three patients
have been enrolled and treated to date including 26 patients
with thymic carcinoma and nine patients with B3 thymoma.
Of 30 evaluable patients, 14 have met the prespecified primary
end point of progression-free survival at 3 months (PFS-3 rate
= 47%; 95% confidence interval [CI] 28.3–65.7%) including
one partial response.22
GENOMIC PROFILING
Gene Expression Analysis
In an effort to overcome the limitations associated with
traditional histological classification of TETs, efforts have
been made to correlate the results of gene expression deter-
mined by whole genome gene expression analysis with clinical
parameters such as stage of disease. Badve et al.23 conducted
a retrospective analysis in 34 patients with type AB and B thy-
moma using RNA extracted from fresh frozen tumors. Ten of
34 patients had received chemotherapy and eight patients had
received radiation therapy. Eight patients were known to have
metastatic disease either at diagnosis or developed it during the
follow-up period. In addition, although all patients underwent
surgery with curative intent, eight patients developed disease
relapse during the follow-up period. After gene expression
analysis using Partek Genomics Suite and Ingenuity Pathways
Analysis, four molecular clusters of tumors were identified
by unsupervised cluster analysis of 8260 genes that showed
unique gene expression profiles. Supervised cluster analysis
using 15 genes with the highest positive and negative fold
changes helped in the identification of three clusters of his-
tologically heterogeneous tumors. Analysis of clinical param-
eters such as stage of disease and presence of metastases did
not show a correlation with either histological subtype or the
newly identified gene expression clusters. However, ingenuity
pathway analysis resulted in the identification of differentially
expressed genes in patients with or without metastatic dis-
ease. In tumors obtained from the metastasis-positive group,
there was a sixfold and threefold increase in the expression
of AKR1B10 and JPH1, respectively, whereas expression
S132 Copyright © 2014 by the International Association for the Study of Lung Cancer
of COL1A1 was decreased 13-fold. This is the first study to
attempt a correlation between expression of individual genes
and clinical factors in TETs. Validation of data obtained from
this study has the potential to identify newer therapeutics tar-
gets such as AKR1B10.24
Gene Signatures
Traditional prognostic factors associated with TETs
have included Masaoka stage, histological subtype, and the
completeness of surgical resection.25 Most attempts to identify
prognostic and predictive biomarkers have focused on correla-
tions with the stage and histological subtype of disease.9,26–28
The first study to attempt a correlation between gene expres-
sion and the risk of development of metastases was reported
in 2013.29 In a retrospective study using archival samples of
thymomas from multiple institutions, gene expression levels
were used to categorize tumors into two groups based on the
probability of developing metastases. This study included
111 cases of thymoma (36 in the training set and 75 in the
validation set). Based on the prediction analysis of microar-
rays (PAMs), 19 differentially expressed genes and four refer-
ence genes were selected for quantitative reverse transcriptase
polymerase chain reaction analysis. The primary end point of
the study was to determine whether a gene signature could
predict 5- and 10-year metastasis-free survival (MFS). A nine-
gene signature was identified that could identify patients at
low- and high-risk for metastasis with 5- and 10-year MFS of
77% and 26% (p = 0.0047) and 97% and 30% (p = 0.0004)
in the training and validation sets, respectively. In univariate
Cox regression analysis, the nine-gene signature was a strong
predictor of the development of metastasis with a hazard ratio
(HR) of 8.33 (95% CI = 1.99–34.9; p = 0.004) compared with
a HR of 2.51 (95% CI = 1.22–5.15; p = 0.012) for presence
of residual disease and a HR of 2.09 (95% CI = 0.95–4.59;
p = 0.067) for Masaoka stage. In multivariate analysis, the
nine-gene signature was the only independent predictor of the
risk of developing metastasis (HR 5.26, 95% CI = 1.12–24.8;
p = 0.036). Genes with increased expression in tumors with
a higher metastatic potential included AKR1B10, JPH1, and
NGB. Conversely, genes with decreased expression in tumors
with high metastatic potential included DACT3, SLC9A2,
PDGFRL, FCGBP, PRRX1, and SERPINF1.
The practical implications of development of prognos-
tic biomarkers such as the gene signature illustrated above
are the identification of patients at high risk for recurrence
who might benefit from administration of appropriate adju-
vant therapy and the potential to identify novel drug targets
for patients at higher risk for development of unresectable,
metastatic disease. However, significant challenges need to be
overcome before widespread adoption of gene signatures in
clinical practice, especially for rare diseases like TETs. These
include the need for validation of results in a prospective set-
ting, a clear demonstration of benefit over widely used clini-
cal parameters such as Masaoka stage, histological subtype
and completeness of resection, and the ease of performing the
gene expression assay. Some of the limitations that might oth-
erwise preclude mass validation of a gene signature for TETs
can be surmounted by performing prospective trials involving
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Genetic Alterations in Thymic Epithelial Tumors
multiple institutions or in a cooperative group setting to maxi-
mize accrual of cases.
Whole Genome and Transcriptome Sequencing
The first reported TET to be analyzed using whole
genome and transcriptome sequencing involved a surgi-
cally resected stage IVA, B3 thymoma.30 In addition to
well-described changes in gene CN, a number of previously
unreported observations were made including a translocation
involving chromosome 11 and the X chromosome, t(11;X),
and mutations in BCOR, FBN3, PCNXL3, PHF15, SFXN3,
SRGAP1, VN1R5, and WDR70. The pathogenic role of FBN3,
PCNXL3, SFXN3, SRGAP1, VN1R5, and WDR70 is unclear.
However, PHF15 belongs to the PHF protein family, several
members of which affect chromatin-mediated gene regulation
and BCOR is a corepressor of several transcription factors.
The mutation rate of 0.72/Mb seen in this case (including sin-
gle nucleotide variations and insertions/deletions) was com-
parable with that observed in more aggressive malignancies
such as breast cancer and myelogenous leukemia. However,
no mutations were observed in well-characterized cancer
genes. This report highlights the fact that TETs have a unique
mutational profile that is not detectable by gene panels used
for molecular profiling of common solid tumors, and strate-
gies such as whole exome sequencing might be better suited
to identify recurrent mutations in this rare tumor type, some of
which may represent targets for treatment.
IDENTIFICATION OF DRUG TARGETS
Initial experiences of the use of targeted therapy in
TETs have been disappointing. The reasons for these negative
results have become clear with the advent of technology and
the relative ease of performing mutational analysis in recent
years. However, a subset of patients with TETs do benefit from
targeted therapy, highlighting the need to gain greater insight
into TET biology to help select patients for targeted therapy.
These concepts are illustrated by the following examples.
KIT overexpression is frequently observed in thy-
mic carcinomas (75–80%), whereas KIT gene mutations are
found in less than 10% of cases.9,31 Based on the type of KIT
mutation, TETs demonstrate varying degrees of sensitivity to
tyrosine kinase inhibitors. In order of sensitivity to sunitinib
and imatinib, these mutations include: W557R, V559A, and
V560del (all on exon 11 and highly sensitive to sunitinib and
imatinib); Y553N and D579del (both on exon 11 and highly
sensitive to imatinib); E490K on exon 9 (highly sensitive to
sunitinib and moderately sensitive to imatinib); H697Y on
exon 14 (highly sensitive to sunitinib and low sensitivity to
imatinib); L576P on exon 11 (moderate sensitivity to sunitinib
and low sensitivity to imatinib); and D820E on exon 17 (resis-
tant to sunitinib and imatinib).1 The rarity and spectrum of KIT
mutations in thymic carcinoma explains the lack of activity of
imatinib in phase II trials where patient selection was based
on histology (B3 thymomas and thymic carcinomas) or KIT
staining by immunohistochemistry and not genotyping.32,33 A
similar pattern of overexpression of epidermal growth factor
receptor (EGFR) and a paucity of sensitizing mutations in the
EGFR gene has been observed in TETs.31 Not surprisingly,
Copyright © 2014 by the International Association for the Study of Lung Cancer
clinical trials evaluating EGFR inhibitors have yielded disap-
pointing results.34,35
Despite these observations, a few recently completed
clinical trials of targeted therapy in TETs have yielded prom-
ising results. The multikinase inhibitor, sunitinib, has been
evaluated in a phase II clinical trial in TETs. Among 23 evalu-
able patients with thymic carcinoma, sunitinib demonstrated
a response rate of 26% and disease control rate of 91%.36
The response to sunitinib was independent of the mutational
status of KIT and seems to be partly related to an antiangio-
genic effect. Cixutumumab, a monoclonal antibody–directed
against the insulin-like growth factor-1 receptor, has been
evaluated in a phase II trial and demonstrated a 89% disease
control rate in patients with relapsed or refractory thymoma.37
Results from selected clinical trials of targeted therapy for
TETs are presented in Table 1.34–41
In the aforementioned trials, patient selection was not
based on the presence of targetable mutations. Expression of
potential drug targets such as the vascular endothelial growth
factor receptor and the insulin-like growth factor 1-recep-
tor that could potentially explain the activity of drugs like
sunitinib and cixutumumab are being evaluated in TETs
(Table 2).12,13,42–50 Greater use of genomic analysis to optimize
patient selection based on the presence of targetable mutations
has the potential to improve upon the results obtained so far
with targeted therapy.
Role of Newer Methods of Genomic and
Integrative Analysis to Identify Drug Targets
Because strategies to identify genetic mutations often
seen in other solid tumors have not yielded promising results
for TETs, newer panels of tests need to be developed that
focus on identifying genetic alterations that might be unique
to TETs.
Recently, differentially expressed miRNAs have been
identified in TETs. Upregulation of miR-21-5p, miR-34b-5p,
and miR-141-3p, as well as down-regulation of miR-486-5p
and miR-145-5p has been observed in TETs in comparison
with normal tissue.51 miR-145-5p down-regulation is thought
to lead to EGFR signaling pathway overexpression. miR-
128, miR-142-5p, and miR-181c-5p are also differentially
expressed in thymoma versus thymic carcinoma. Interestingly,
among the miRNAs down-regulated in thymic carcinoma,
some putatively target BIRC3, SCYA20, and MYC.52
Differential expression of antiapoptotic genes has also been
identified in B3 thymoma and squamous cell carcinoma of the
thymus. These results have raised the possibility of therapeu-
tic intervention with drugs targeting inhibitors of apoptosis
and BIRC3.52 In another study, miR-515 overexpression was
observed in type A-AB thymomas; this may result in PTEN
inhibition, which could potentially be targeted by PIK3CA/
AKT inhibitors.53
Array CGH in 59 TETs reported recurrent CN gain of
BCL2, providing further evidence for the role of an antiapop-
totic strategy as a major hallmark of TETs.20 Moreover, TET
cell lines were reported to be sensitive to siRNA knockdown
of the antiapoptotic molecules BCL2 and MCL1. Gx15-070, a
pan-BCL2 inhibitor, induced autophagy-dependent necrosis in
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TABLE 1.
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Time to Progression Overall Survival
Intervention Evaluable Patients Responses (months) (months) Reference
Gefitinib 26 1 4 NR 34

Thymoma 19 NR NR NR
Thymic carcinoma 7 NR NR NR
Erlotinib + bevacizumab 18 0 NR Not reached 35

Thymoma 11 0 NR Not reached

Thymic carcinoma 7 0 NR Not reached
Sunitinib 39 7 NR NR 36

Thymoma 16 1 8.5a Not reached

Thymic carcinoma 23 6 6.7a 16.3
Cixutumumab 49 5 8.2 16.2 37

Thymoma 37 5 9.9 27.5

Thymic carcinoma 12 0 1.7 8.4
Imatinib 15 0 3a Nor reached 38

Thymoma 12 0 NRa NR
Thymic carcinoma 3 0 NRa NR
Saracatinib 21 0 NRa NR 39

Thymoma 14 NR 3.4a Not reached

Thymic carcinoma 7 NR 1.4a Not reached
Belinostat 40 2 NR NR 40

Thymoma 24 2 11.4 Not reached

Thymic carcinoma 16 0 2.7 12.4
Everolimus 35 4 12.1a 24 41

Thymoma 23 1 NRa NR
Thymic carcinoma 12 3 NRa NR
Progression-free survival.
a

NR, Not reported.

TABLE 2.
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IMPACT OF THE GENOMICS REVOLUTION AND
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In view of the rarity of TETs, translation of preclinical
Thymic
Gene Thymoma Carcinoma References observations into newer therapies poses unique challenges.
One approach to validate the concept of personalized medi-
EGFR 13, 42, 43
cine in thymic malignancies is the development of open-label,
Amplification 20% 25% multicenter phase II trials using high-throughput genome anal-
Overexpression 23% 67–100% ysis (multiplex mutation sequencing, CGH arrays, next gen-
HER2 12, 44
eration sequencing) as a therapeutic decision tool to identify
Amplification 6% 0–4% patients for targeted therapy directed against specific molecu-
Overexpression 53–58% lar alterations identified in their tumors. TETs have been inte-
KIT 45–47
grated into a few clinical trials using this approach such as
Overexpression 0–4% 46–86% the recently completed CUSTOM trial (NCT01306045), the
IGF-1R 48, 49
first prospective study of molecular profiling to determine eli-
Overexpression 14–43% 86–88% gibility for targeted therapy in patients with TETs.56 Tumors
VEGF 50 were analyzed for molecular changes including mutations in
Overexpression 32% 63% EGFR, KRAS, HRAS, NRAS, BRAF, PIK3CA, AKT, PTEN,
ERBB2, KIT, and PDGFRA, amplification of HER2 or ALK
IGF-1R, insulin-like growth factor-1 receptor.
translocations in a CLIA-certified laboratory, and patients
were assigned to one of five treatment groups on the basis
TET cells through a mechanism involving mammalian target of these changes. This study showed that the genomic altera-
of rapamycin (mTOR) pathways, and inhibited TET xenograft tions described above were uncommon in TETs and included
growth. Hence, mTOR targeting may represent a clinically HER2 amplification in 7.7% of samples analyzed, and a
relevant approach for treatment of advanced TETs.54,55 Table 3 mutational frequency of 4.7% in HRAS and 1.4% in PIK3CA
summarizes selected molecular alterations in TETs. and EGFR genes. This trial confirms the uniqueness of the

S134 Copyright © 2014 by the International Association for the Study of Lung Cancer
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Genetic Alterations in Thymic Epithelial Tumors

TABLE 3.
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Chromosomal
WHO Arm-Level Chromosomal Mutations and Copy Number Changes RNAs
Type Aberrations Translocations Affecting Selected Genes Methylation MicroRNAs Reference
A 6q25 loss HRAS (G13V) miR-515 upregulation 9–11, 14, 15, 53

6p23 loss (FOXC1) (targets PTEN)

AB 6q25 loss
6p23 loss (FOXC1)
7p15 loss
B1 1p, 2q, 3q, 6q losses 9–11, 14

B2 6q25 loss KRAS (G12A) 9–11, 14, 15

6p23 loss (FOXC1)

1q gain
B3 6q25 loss t(11;X) BCL2 copy number gains (18q21.33) 9–11, 14, 15, 20, 30

6p23 loss (FOXC1) MCL1 copy number gain

11q4 losses CDKN2A/B copy number losses (9p21.3)
1q gain BCOR
PHF15
TSCC 6q25 loss KIT mutations: E490K (exon 9), Y553N, MGMT miR-142-5p 1, 9–11, 14, 15, 17–19, 20, 51

6p23 loss (FOXC1) W557R, V559A, V560del, L576P, CDKN2 down-regulation

9p, 13q, 16q, losses P577-D579del (exon 11), H697Y (targets MYC)
1q, 9q, 17q gains (exon 14), D820E (exon 17)
KRAS (G12V)
BCL2 copy number gains (18q21.33)
MCL1 copy number gain
CDKN2A/B copy number losses (9p21.3)
TSCC, Thymic squamous cell carcinoma.

mutational profile of TETs and the inability of gene panels
designed for other solid tumors to detect mutations in TETs
that can help identify patients for targeted therapy. Another
approach is to promote the enrolment of patients with refrac-
tory TETs in appropriate phase I trials, which may lead to
the identification of new molecular pathways of therapeutic
interest. For example, the mTOR pathway has emerged as
a potential area of interest after tumor responses observed
in phase I trials reported by several groups.54,55 In addition,
disease stabilization as a result of treatment with the mTOR
inhibitor, everolimus, in combination with somatostatin ana-
logs has been described in a case series of two patients with
thymoma.57 An ongoing phase II clinical trial is evaluating
the role of the mTOR inhibitor, everolimus, in patients with
previously treated TETs (NCT02049047). Preliminary results
show a disease control rate of 71%, median PFS of 12 months,
and overall survival of 24 months.41 These novel approaches
have the potential to help in the successful translation of the
increasing volume of data generated by newer genomic tech-
nologies and help in the development of newer therapies for
patients with TETs.
CONCLUSIONS
Rapid advances in technology have helped uncover
novel genetic alterations in TETs and resulted in an increas-
ing recognition that TETs have a unique molecular profile
compared with other solid tumors. These new discoveries
have helped in gaining greater insight into thymic tumor biol-
ogy and have resulted in a steady increase in the identifica-
tion of molecular alterations that have the potential to serve
as biomarkers or drug targets. Attempts should be made to
incorporate these data into future clinical trials to help select
patients for appropriate interventions with an aim to improve
response to treatment and survival.
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