Genomic Aberrations of Thymic Epithelial Tumors

Thymic epithelial tumors (TETs) are rare neoplasms classified in thymoma and thymic carcinoma. According to the 2004 WHO classification, thymomas are further subdivided into five subcategories (A, AB, B1, B2, B3), depending on cancer cell shape, degree of atypia and number of intratumoral thymocytes. Surgical resection is recommended for localized tumors, but systemic therapy is the choice for metastatic and not resectable neoplasms. Usually, TETs respond to chemotherapeutic drugs but their effect lasts only for a limited time, thus, new targeted-molecules are currently under evaluation. However, the efficacy of biologic therapies in TET is scant, to date. The main limitation is the lack of a robust rationale to target specific molecules, mainly because the aberrations driving TET growth are obscure. The aim of this project is to clarify the molecular aberration driving the TET growth. Firstly, the frequency of aberrations described in TET case-reports has been evaluated. The presence of BRD4-NUT fusion gene was tested in 148 TETs, but resulted in a rare event, observed in only one thymic carcinoma. Similarly, no KIT mutations were detected in the 13 TETs sequenced. Reviewing literature data, KIT was mutated in only 9% of thymic carcinomas. Therefore, a systematic screening for new genomic aberrations in TET was started using whole genome sequencing to evaluate the presence of mutations, translocation and copy number (CN) aberrations in a B3 thymoma. The translocation t(11;X)(q14.2;q25), the presence of 7 single nucleotide mutations and 1 INDEL were observed together with CN gain of chromosome 1q, 5, 7, X and CN loss of 3p, 6, 13 and part of chromosome 11q. In order to identify which one of these genomic aberrations was recurrent and therefore candidate to drive TET growth, whole exome sequencing and array comparative genomic hybridization (CGH) were adopted. 26 tumors were screened by array CGH and 5 selected to be sequenced using tumor and normal DNA. The identified mutations were confirmed and tested for expression using transcriptome sequencing. The sequencing demonstrated a remarkably heterogeneous pattern of mutations. Recurrent were the amplification of BCL2, observed in 4 thymic carcinomas, and the inactivation of CDKN2A caused by a frame-shift INDEL and 2 CN losses. Because BCL2 and CDKN2A are more likely to be mutated by CN aberrations, 59 formalin fixed paraffin embedded tumors were evaluated using array CGH. Data confirmed the presence of focal CN loss of CDKN2A and amplification of BCL2 as recurrent events in TETs and suggest a possible prognostic role for these CN aberrations. Immunohistochemistry confirmed the lack of P16INK4 expression in tumors with CDKN2A CN loss and showed a poor prognosis for patients with negative staining. P16INK4 is a well-known tumor suppressor gene involved in the control of cell cycle through the binding of CDK4 and 6. The role of anti apoptotic BCL2 family proteins was elucidated using 3 TET cell lines because these molecules could represent targets for therapy. A siRNA approach demonstrated that the expression of BCL2 and MCL1, two anti-apoptotic proteins, was necessary for TET cell line growth. GX15-070, a BH3 mimetic inhibitor of anti-apoptotic BCL2 family members, reduced proliferation of TET cell-lines in vitro, through the induction of autophagy and necroptosis. Gx15-070 inhibited tumor growth in the xenograft model established using TY82 thymic carcinoma cell line; therefore this drug may be tested for patients’ treatment.