Lung Cancer 81 (2013) 27–31
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Lung Cancer
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Review
Identification of novel mutations of TP53, ALK and RET gene in metastatic thymic
squamous cell carcinoma and its therapeutic implication
Zhenli Hu a , Jinghan Wang b , Tony Yao c , Ruey-Long Hong d , Keqiang Zhang c , Hanlin Gao e , Xiwei Wu f ,
Jie Li c , Chong Bai a , Yun Yen c,g,∗
a
Department of Respiratory Medicine, Changhai Hospital, The Second Military Medical University, Shanghai 200433, China
The First Department of Biliary Surgery, Eastern Hepatobiliary Surgical Hospital, The Second Military Medical University, Shanghai 200438, China
c
Department of Molecular Pharmacology, Beckman Research Institute of The City of Hope National Medical Center, Duarte, CA 91010, USA
d
Department of Oncology, National Taiwan University Hospital, Taipei 110, Taiwan
e
Solexa Core Lab, Beckman Research Institute of The City of Hope National Medical Center, Duarte, CA 91010, USA
f
Division of Information Sciences of Department of Molecular Medicine, Beckman Research Institute of The City of Hope National Medical Center, Duarte, CA 91010, USA
g
Taipei Medical University, Taipei 110, Taiwan
b
a r t i c l e
i n f o
Article history:
Received 31 January 2013
Received in revised form 2 April 2013
Accepted 6 April 2013
Keywords:
Thymic tumor
Gene mutation
Targeted therapy
a b s t r a c t
Thymic tumors are epithelial tumors of the thymus for which multimodal therapies are often ineffective
because of a lack of standardized regimens. Due to the low incidence, the molecular pathology and
genomic abnormalities of thymic epithelial tumors are largely unknown. In this study, we report our
comprehensively genomic study on a case of metastatic thymic tumor. Using next generation deep DNA
sequencing technology, we sequenced 190 segments of 46 cancer genes of the cancer genome to cover
739 COSMIC mutations in 604 loci. Among these sequenced cancer genes, we identified that three low
frequency (∼10% of cells) mutations in the TP53 gene (c.782+1G > T), ALK gene (c.3551C > T), and RET
gene (c.2651A > T). To the best of our knowledge, this is the first study to show those mutations in thymic
tumor. Of note, our study further indicates comprehensive molecular analysis may facilitate development
of novel diagnostic and therapeutic strategies for thymic tumors.
© 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Thymic tumors are rare intrathoracic neoplasms, with 3.2 incident cases per 1 million person-years [1]. They occur more often in
men than in women and more often in Asians/Pacific Islanders and
Blacks than in Whites [2]. The current World Health Organization
histological classification makes the distinction between thymomas (types A, AB, B1, B2, and B3) and thymic carcinoma (type C)
[3]. The thymic squamous cell carcinoma (TSCC) belongs to type
C thymic tumors. It has been reported that chemotherapy and
radiation therapy play an important role in treating this kind of
malignant tumor [4,5].
In the past decades, there have been dramatic progressions in
the diagnostic and therapeutic marker investigation of the thymic
tumor. Current molecular characterization of thymic tumors consists of identifying important oncogenes (EGFR, HER2, c-KIT, RAS,
and Bcl-2), tumor suppressor genes (TP53 and P16), chromosomal
∗ Corresponding author at: Department of Molecular Pharmacology, Beckman
Research Institute, City of Hope Comprehensive Cancer Center, 1500 E. Duarte Road,
Duarte, CA 91010, USA. Tel.: +1 626 256 4673x65707; fax: +1 626 301 8233.
E-mail address: yyen@coh.org (Y. Yen).
0169-5002/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.lungcan.2013.04.006
aberrations (loss of heterozygosity [6] 3p, 6p, 6q, 7p, and 8p),
and tumor invasion factors (matrix metalloproteinases and tissue
inhibitor of metalloproteinases) [7,8].
2. Case report
A 34-year-old male smoker was admitted to the National Taiwan
University Hospital after a mediastinal mass was discovered in a
routine health exam on August 2011. A computed tomography (CT)
scan of the chest during one of the visits revealed an anterior mediastinal mass (69.3 mm × 33 mm) with encasement of pulmonary
trunk and ascending aorta; also discovered were bilateral lung
metastases with enlarged mediastinal LNs at the right pulmonary
hilum, right paratracheal and prevascular area (Fig. 1A–C). Bone
scan reported a focal bone lesion at left parito-temporal skull
(Fig. 1D). Biopsy and aspiration of this lesion were performed and
indicated that the lesions consisted of thymic squamous cell carcinoma. The patient was diagnosed as T4N2M1, stage IVb thymic
carcinoma with lung and skull bone metastases, according to the
TNM-type staging system [9].
Subsequently, the patient received cisplatin-based combination chemotherapy. The chemotherapy regimens were listed in
Table 1. The VIP regimen, consisting of cisplatin (60 mg/m2 , D1),
28
Z. Hu et al. / Lung Cancer 81 (2013) 27–31
Fig. 1. Chest CT and bone examinations. The CT scan was performed before the treatment (A) and (B). The bone scan reported a focal bone lesion at skull (C). After three
courses of avastin + VIP regimens, the CT scan shows the disease of partial remission (D).
ifosfamide (3 g/m2 , D1–D2), and etoposide (75 mg/m2 , D1–D2), was
given as first-line treatment on September, 2011. Subsequently,
after four courses of chemotherapy, the patient underwent CT
scan, which revealed a progress disease. Instead of continuing with
the other combination chemotherapy regimens, patient received
Endoxan oral therapy from December, 2011 to August, 2012. Then,
it was found that the disease had progressed yet again. He received
Avastin plus VIP regiment afterwards, and, up to the present
(01/10/2013), the patient has demonstrated stable disease state.
3. Genomic analysis
Ten sections (5 m thickness) of FFPE (formalin fixed, paraffin embedded) thymic carcinoma collected from primary tumor
biopsy tissue were submitted for testing. Genetic analysis of 46
cancer-related and clinically actionable genes was undertaken.
DNA was isolated from the FFPE tumor tissues (block XX–YY–ZZ)
and amplified in 190 segments to cover 739 COSMIC mutations in
604 loci from 46 cancer genes. The amplified segments were then
sequenced using a next generation sequencing platform. 96.57%
of covered mutation spots had at least 100× coverage and all the
regions were sequence at least 20 times (20×).
The sequence analysis found that 9.9% of cancer cells from
the tumor tissue harbored c.782+1G > T at the splicing site ahead
of base 782G of p53 gene (Fig. 2A, shown as C > A on antisense
strand), while 5.84% and 8.4% of cancer cells harbored missense
mutation of c.3551C > T of ALK gene (Fig. 2B) causing the 1184th
amino acid glycine replaced by glutamine and missense mutation of
c.2651A > T of RET gene (Fig. 2C) leading the 884th amino acid glutamic acid converted to valine respectively. The potential clinical
Table 1
Combination therapeutic regimens.
Regiments
Number of courses
Treatment results
VIP
Cisplatin (60 mg/m2 , D1)
Ifosfomid (3 g/m2 , D1–D2)
Etoposide (75 mg/m2 , D1–D2)
1
Stable disease
9/26/2011–10/17/2011
Avastin + VIP
Avastin (200 mg, D0)
Cisplatin (60 mg/m2 , D1)
Ifosfomid (3 g/m2 , D1–D2)
Etoposide (75 mg/m2 , D1–D2)
3
Partial remission
10/18/2011–12/19/2011
Endoxan
50 mg/tab 1 tab PO QD
Avastin + VIP
Avastin (200 mg, D0)
Cisplatin (60 mg/m2 , D1)
Ifosfomid (3 g/m2 , D1–D2)
Etoposide (75 mg/m2 , D1–D2)
36
Progressive disease
12/20/2011–8/17/2012
6
Stable disease
8/18/2012–01/10/2013
Toxicity, GI, leukopenia
Z. Hu et al. / Lung Cancer 81 (2013) 27–31
29
Fig. 2. Identification of mutations of Tp53, ALK and RET genes in the cancer genome by next generation DNA sequencing technology. (A) The low frequent mutation of
c.782 + 1G > T at the splicing site ahead of base 782G of p53 gene (shown as C > A on antisense strand), (B) the low frequent missense mutation of c.3551C > T of ALK gene
causing the amino acid 1184th glycine replaced by Glutamine, (C) the low frequent missense mutation of c.2651A > T of RET gene causing the amino acid 884th glutamine
converted to valine.
implications of these genomic alterations were also summarized in
Table 2. We also sequenced 43 other genes; including EGFR, RAS,
and KIT genes, we found no mutation of these genes in the cancer
genome.
4. Discussion
The TSCC is the most common type of thymic carcinoma, with
a higher incidence in Asia (90%) as compared with that in Western
countries (30%) [10,11]. Patients with TSCC usually present with
symptoms of chest pain, shortness of breath, fatigue, weight loss,
and most of them are only being diagnosed at advanced stage with
only 14.5% 5-year survival rate [10]. The TSCCs is difficult to treat
in advanced stages because there are no established therapeutic
protocols. At present, cisplatin-based combination chemotherapy
can be beneficial; unfortunately, the responses often short-lived.
The combined VIP regimen has moderate efficacy in patients with
thymic carcinoma [12,13]. Targeted molecular therapy may serve
as the second-line treatment for TSCCs, which would provide new
therapeutic options for this kind of malignant diseases.
Many molecular abnormalities have been reported the past
years such as EGFR, Her2, and c-Kit, and their corresponding medications (imatinib, sunitinib, and sorafenib) have demonstrated
promising effect in TSCCs [14]. Here we report a case which has
novel gene mutations (found in the genes p53, ALK, and RET) which
may lead to development of new molecular therapy strategies for
treating TSCCs.
The first gene we identified, p53, codes for the P53 (17p13.1)
protein. It is a transcription factor that functions as a tumor suppressor by inducing expression of genes that facilitate cell cycle
arrest, DNA repair, and apoptosis. Mutation of the p53 gene is the
most common genetic feature observed in human tumors [15].
Indeed, p53 mutation can be found in up to 38% of thymic carcinomas, which suggests that this protein has potential to be a
Table 2
Sequence analysis of the patient’s thymic carcinoma tissue.
Mutation
% Alternative allele frequency
Clinical relevance
Details
71
9.9%
Poor prognosis,
Avoid radiotherapy
ALK:c.3551C > T,
p.Gly1184Glu
606
5.8%
Unknown
RET:c.2651A > T,
p.Glu884Val
227
8.4%
Unknown
This mutation suggests a poor prognosis for this patient
and radio-therapy should be avoided if possible as the
p53-dependent apoptosis is affected in the tumor cells.
The presence of -ALK fusions is associated with EGFR
tyrosine kinase inhibitor (TKI) resistance. However,
whether this missense mutation acts like the ALK gene
fusion is not known.
Tumor with RET arrangement may be sensitive to
multi-targeted kinase inhibitors such as imatinib,
sorafenib, and sunitinib. If this misense mutation acts like
the RET gene arrangement is still not known.
TP53:c.782 + 1G > T,
Total coverage
30
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Fig. 3. Targeting of thymic tumor by VEGF inhibitor and the tyrosine kinase domain inhibitors of the ALK and RET. Imatinib, sunitinib and sorafenib block the tyrosine kinase
domain of the RET. TAE684 and crizotinib inactivate ALK kinase activity, disengaging oncogenic signaling pathways like ERK and AKT signaling. Those signaling pathways
can lead to malignant transformation. Avastin recognizes and binds VEGF to disrupt angiogenesis and metastasis.
pathological background for the thymic tumor. Specifically, we
found c.782 + 1G > T (exon 7) mutation in p53. This splicing mutation may have interfered with the identification of the consensus
sequences by small nuclear RNA (snRNA) and therefore blocked
the removal of intron 7 [16]. In addition, recent reports suggested
that the mutation of p53 in thymoma probably play a key role in
a stage of malignant progression that precedes invasion [15]. The
tumor cells bearing the p53 mutation is not dominant in whole
cell populations, however, tumor cells are dynamically changing; these cells with inactivated p53 tumor suppressor may have
growth advantage, they may finally become dominant under special conditions such as chemotherapy, and gain drug resistance.
They may cause failure of therapy and or recurrence. The second
mutated gene we found in our case study codes for the protein ALK
(2q23), which was originally implicated in the carcinogenesis of
anaplastic large cell lymphoma as a fusion partner of nucleophosmin after a chromosomal rearrangement [17]. Mutation of the ALK
gene is infrequent in thymic tumor; the mutation we did identify is located in codon 1184 (Gly to Glu). However, studies using
the diaminopyrimidine scaffolds ALK kinase inhibitor, TAE684,
have revealed that a subset of human cancer-derived cell lines
(namely the anaplastic large cell lymphomas, non-small cell lung
cancers and neuroblastomas) harboring ALK gene rearrangements
and/or amplifications are exquisitely sensitive to ALK kinase inhibition [18,19]. Another aminopyridine ALK inhibitor, crizotinib, is
a potent, selective, ATP-competitive, small molecule ALK inhibitor
[20]. Those two inhibitors can target different mutations (L1196M,
G1269S) in kinase domain of the protein in lung cancer cells [21].
Knowing these, we suggest that an ALK inhibitor would also have
good effect in treating this case.
The third gene we would like to discuss, the RET (10q11.2) protooncogene, was first identified in vitro in 1985 by transfection of
NIH3T3 cells with lymphoma DNA [22], and first recognized in
human diseases in papillary thyroid carcinoma [23]. RET is a singlepass transmembrane protein and plays a central role in several
intracellular signaling cascades that regulate cellular survival, differentiation, proliferation, migration, and chemotaxis [24]. In this
study, we found a mutation of RET exon 15 GAG-to-GTG (Glu to
Val) at codon 884 in this case of TSCC. In the past several years,
some tyrosine kinase inhibitors have been proven to inhibit RET
activity. Emergence of these inhibitors, including imatinib, demonstrated that the tyrosine kinase inhibition could be rather specific
and effective [25,26]. Sunitinib is another highly effective inhibitor
of RET for metastatic thymic carcinoma [27]; it is a multi-targeted
tyrosine kinase inhibitor that was designed to block intracellular
receptor binding sites of the RET receptor [28]. From current publication, it have provided very strong circumstantial evidence that
sunitinib may be able to block tumor escape through antiangiogenesis in thymic carcinoma [27,29]. It is reported that sorafenib is yet
another known potent inhibitor of RET kinase [30].
Since ALK and RET are both members of RTKs, it is necessary to
take into consideration this type of kinase inhibitors while treating
this case. In recent years, researches for clinical application of RTKbased cancer therapies have reached new heights. Currently, there
are several strategies involving RTK as a therapeutic target, such
as inhibiting RKT signaling, ligand binding, and gene therapeutic
Z. Hu et al. / Lung Cancer 81 (2013) 27–31
approaches. Molecular target-based therapy directing against the
RTK family members has received much attention; among the drugs
in physician’s current arsenal are imatinib, sunitinib, sorafenib,
gefitinib, Avastin, and so on.
Our initial experience shows that Avastin, a monoclonal antibody, seems promising for the patient. Our rationale for choosing
this drug came from analyzing the signaling pathways of the ALK
and RET (Fig. 3). It has been reported that those two genes have
the same downstream pathway (ERK and AKT signaling) [28]. We
hypothesized that the mechanism of the curative effects of Avastin
is likely brought about through prevention of VEGF from binding to the VEGFR, which disrupts angiogenesis and thus halting
tumor growth and metastasis. It is already reported that mutation of ALK and RET in large-cell lymphoma and thyroid carcinoma
both can upregulate VEGF expression level [31]. Therefore, when
the disease progressed after the beginning of oral treatment with
Endoxan, patient resumed Avastin and VIP regimen, and he once
again achieved stable disease state.
5. Conclusion
Thymic carcinoma is a rare neoplasm that portends poor prognosis as this tumor entity is often advanced at its discovery. Because
of its low incidence rates, the most optimal treatment is, as of
yet, uncertain. Therefore, the concept of personalized molecular medicine, which consists of selecting patients for available
systemic therapies, is applicable to rare tumors such as thymic
carcinomas. With advancements of chemotherapy and application
of molecular therapeutic strategies, the survival rates of patients
with TSCCs should improve. The new clinical protocol may be further refined base on the information presented here to improve the
overall therapeutic outcome.
Conflict of interest statement
None declared.
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