Lung Cancer 81 (2013) 27–31 Contents lists available at SciVerse ScienceDirect Lung Cancer journal homepage: www.elsevier.com/locate/lungcan 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 Z. Hu et al. / Lung Cancer 81 (2013) 27–31 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. References [1] de Jong WK, Blaauwgeers JL, Schaapveld M, Timens W, Klinkenberg TJ, Groen HJ. Thymic epithelial tumours: a population-based study of the incidence, diagnostic procedures and therapy. Eur J Cancer 2008;44:123–30. [2] Engels EA. Epidemiology of thymoma and associated malignancies. J Thorac Oncol 2010;5:S260–5. [3] Marchevsky AM, Gupta R, Casadio C, Hiroshima K, Jambhekar NA, Kim DJ, et al. World Health Organization classification of thymomas provides significant prognostic information for selected stage III patients: evidence from an international thymoma study group. Hum Pathol 2010;41:1413–21. [4] Korst RJ, Kansler AL, Christos PJ, Mandal S. Adjuvant radiotherapy for thymic epithelial tumors: a systematic review and meta-analysis. Ann Thorac Surg 2009;87:1641–7. [5] Evans TL, Lynch TJ. Role of chemotherapy in the management of advanced thymic tumors. Semin Thorac Cardiovasc Surg 2005;17:41–50. [6] Begemann M, Rosenblum MK, Loh J, Kraus D, Raizer JJ. Leptomeningeal metastases from recurrent squamous cell cancer of the skin. J Neurooncol 2003;63:295–8. [7] Kelly RJ, Petrini I, Rajan A, Wang Y, Giaccone G. Thymic malignancies: from clinical management to targeted therapies. J Clin Oncol 2011;29: 4820–7. 31 [8] Girard N. Thymic tumors: relevant molecular data in the clinic. J Thorac Oncol 2010;5:S291–5. [9] Bedini AV, Andreani SM, Tavecchio L, Fabbri A, Giardini R, Camerini T, et al. Proposal of a novel system for the staging of thymic epithelial tumors. Ann Thorac Surg 2005;80:1994–2000. [10] Chalabreysse L, Roy P, Cordier JF, Loire R, Gamondes JP, Thivolet-Bejui F. Correlation of the WHO schema for the classification of thymic epithelial neoplasms with prognosis: a retrospective study of 90 tumors. Am J Surg Pathol 2002;26:1605–11. [11] Okumura M, Miyoshi S, Fujii Y, Takeuchi Y, Shiono H, Inoue M, et al. Clinical and functional significance of WHO classification on human thymic epithelial neoplasms: a study of 146 consecutive tumors. Am J Surg Pathol 2001;25:103–10. [12] Loehrer Sr PJ, Jiroutek M, Aisner S, Aisner J, Green M, Thomas Jr CR, et al. Combined etoposide, ifosfamide, and cisplatin in the treatment of patients with advanced thymoma and thymic carcinoma: an intergroup trial. Cancer 2001;91:2010–5. [13] Grassin F, Paleiron N, Andre M, Caliandro R, Bretel JJ, Terrier P, et al. Combined etoposide, ifosfamide, and cisplatin in the treatment of patients with advanced thymoma and thymic carcinoma. A French experience. J Thorac Oncol 2010;5:893–7. [14] Disel U, Oztuzcu S, Besen AA, Karadeniz C, Kose F, Sumbul AT, et al. Promising efficacy of sorafenib in a relapsed thymic carcinoma with C-KIT exon 11 deletion mutation. Lung Cancer 2011;71:109–12. [15] Pich A, Chiarle R, Chiusa L, Ponti R, Geuna M, Palestro G. p53 expression and proliferative activity predict survival in non-invasive thymomas. Int J Cancer 1996;69:180–3. [16] O’Neill JP, Rogan PK, Cariello N, Nicklas JA. Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum. Mutat Res 1998;411:179–214. [17] Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN, Saltman DL, et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in nonHodgkin’s lymphoma. Science 1994;263:1281–4. [18] McDermott U, Iafrate AJ, Gray NS, Shioda T, Classon M, Maheswaran S, et al. Genomic alterations of anaplastic lymphoma kinase may sensitize tumors to anaplastic lymphoma kinase inhibitors. Cancer Res 2008;68:3389–95. [19] Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010;363:1693–703. [20] Galkin AV, Melnick JS, Kim S, Hood TL, Li N, Li L, et al. Identification of NVPTAE684, a potent, selective, and efficacious inhibitor of NPM-ALK. Proc Natl Acad Sci USA 2007;104:270–5. [21] Heuckmann JM, Holzel M, Sos ML, Heynck S, Balke-Want H, Koker M, et al. ALK mutations conferring differential resistance to structurally diverse ALK inhibitors. Clin Cancer Res 2011;17:7394–401. [22] Takahashi M, Ritz J, Cooper GM. Activation of a novel human transforming gene, ret, by DNA rearrangement. Cell 1985;42:581–8. [23] Bongarzone I, Pierotti MA, Monzini N, Mondellini P, Manenti G, Donghi R, et al. High frequency of activation of tyrosine kinase oncogenes in human papillary thyroid carcinoma. Oncogene 1989;4:1457–62. [24] de Groot JW, Links TP, Plukker JT, Lips CJ, Hofstra RM. RET as a diagnostic and therapeutic target in sporadic and hereditary endocrine tumors. Endocr Rev 2006;27:535–60. [25] Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ, et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 2002;347:472–80. [26] Sawyers CL, Imatinib GIST. keeps finding new indications: successful treatment of dermatofibrosarcoma protuberans by targeted inhibition of the plateletderived growth factor receptor. J Clin Oncol 2002;20:3568–9. [27] Strobel P, Bargou R, Wolff A, Spitzer D, Manegold C, Dimitrakopoulou-Strauss A, et al. Sunitinib in metastatic thymic carcinomas: laboratory findings and initial clinical experience. Br J Cancer 2010;103:196–200. [28] Faivre S, Demetri G, Sargent W, Raymond E. Molecular basis for sunitinib efficacy and future clinical development. Nat Rev Drug Discov 2007;6:734–45. [29] Chow LQ, Eckhardt SG. Sunitinib: from rational design to clinical efficacy. J Clin Oncol 2007;25:884–96. [30] Carlomagno F, Anaganti S, Guida T, Salvatore G, Troncone G, Wilhelm SM, et al. BAY 43-9006 inhibition of oncogenic RET mutants. J Natl Cancer Inst 2006;98:326–34. [31] Dejean E, Renalier MH, Foisseau M, Agirre X, Joseph N, de Paiva GR, et al. Hypoxia-microRNA-16 downregulation induces VEGF expression in anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphomas. Leukemia 2011;25:1882–90.