Semi-comprehensive analysis of gene amplification in thymic malignant tumors using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization

Abstract

Research on the amplification of oncogenes in thymic malignant tumor is limited. In this study, we aimed to determine the gene amplification status of receptor tyrosine kinases and other cell regulator genes in thymic malignant tumors, with a view toward the future introduction of molecular targeted therapy. In addition, we examined the usefulness of multiplex, ligation-dependent probe amplification (MLPA) in the semi-comprehensive detection of these gene amplifications. The participants of this study were nine patients with thymic carcinoma and one patient with atypical carcinoid who underwent resection at our department from 1999 to 2016. Twenty-four oncogenes (MDM4, MYCN, ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, BRAF, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2, TOP2A, AURKA, AR) were analyzed for amplification by MLPA. In cases where amplification by MLPA was suspected, confirmation was performed by fluorescence in situ hybridization (FISH). Immunostaining for detected oncoproteins and p53 were performed in cases with confirmed oncogene amplification. MYC (2/10, 20%) and MDM2 (1/10, 10%) amplifications were detected using MLPA and FISH. Immunostaining in both cases was positive. The MDM2-amplified tumor relapsed and spread rapidly after operation despite the use of post-operative chemo-radiotherapy. MYC amplification may be involved in the carcinogenesis of thymic malignant tumors. In addition, MDM2 amplification may be a concern in the increased malignancy.

Keywords: MDM2; MYC; Thymic carcinoma; fluorescence in situ hybridization; gene amplification; multiplex ligation-dependent probe amplification.

Figure 1

Case 1 with MYC amplification was diagnosed histopathologically as basaloid carcinoma (A: hematoxylin and eosin stain, original magnification 10×). MYC amplification was confirmed by FISH (B: orange fluorescence, MYC; green fluorescence, centromere probe for chromosome 8). And the protein overexpression of MYC and p53(2+) were detected by immunohistochemistry (C: MYC, original magnification 400×; D: p53, original magnification 400×).

Figure 2

Case 2 with MYC and MDM2 amplifications was diagnosed histopathologically as keratinizing squamous cell carcinoma (A: hematoxylin and eosin stain, original magnification 200×). MYC and MDM2 amplifications were confirmed by FISH (B: orange fluorescence, MYC; green fluorescence, centromere probe for chromosome 8, C: orange fluorescence, MDM2; green fluorescence centromere probe for chromosome 12). And the protein overexpression of MYC, MDM2, and p53(3+) were detected by immunohistochemistry (D: MYC, original magnification 400×; E: MDM2, original magnification 400×; F: p53, original magnification 200×).