Frequent amplification of receptor tyrosine kinase genes in welldifferentiated/ dedifferentiated liposarcoma

Abstract

Well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) are closely related tumors commonly characterized by MDM2/CDK4 gene amplification, and lack clinically effective treatment options when inoperable. To identify novel therapeutic targets, we performed targeted genomic sequencing analysis of 19 WDLPS and 37 DDLPS tumor samples using a panel of 104 cancer-related genes (NCC oncopanel v3) developed specifically for genomic testing to select suitable molecular targeted therapies. The results of this analysis indicated that these sarcomas had very few gene mutations and a high frequency of amplifications of not only MDM2 and CDK4 but also other genes. Potential driver mutations were found in only six (11%) samples; however, gene amplification events (other than MDM2 and CDK4 amplification) were identified in 30 (54%) samples. Receptor tyrosine kinase (RTK) genes in particular were amplified in 18 (32%) samples. In addition, growth of a WDLPS cell line with IGF1R amplification was suppressed by simultaneous inhibition of CDK4 and IGF1R, using palbociclib and NVP-AEW541, respectively. Combination therapy with CDK4 and RTK inhibitors may be an effective therapeutic option for WDLPS/DDLPS patients with RTK gene amplification.

Keywords: dedifferentiated liposarcoma; liposarcoma; next-generation sequencing; receptor tyrosine kinase; well-differentiated liposarcoma.

Figure 1. Summary of genetic alterations identified in 19 WDLPS and 37 DDLPS samples by targeted sequencing analysis using a panel of 104 cancer-related genes

Each column represents a patient sample. The top section indicates clinical variables of each patient. The following three sections indicate genetic alterations found in each sample.

Figure 2. Quantitative PCR and RT-PCR analyses of recurrently amplified RTK genes

(A) Comparison of NGS-estimated and quantitative PCR (qPCR)-estimated relative copy numbers of amplified RTK genes. (B) mRNA expression of amplified RTK genes estimated by quantitative RT-PCR (qRT-PCR), and normalized to GAPDH expression. Relative expression levels are expressed as ratios of the median expression in non-amplified samples.

Figure 3. Intratumoral heterogeneity of RTK gene amplification in FISH

Multiple FGFR3 signals (green) are observed in the majority of cells in a certain area (A) but not in any cells in another area (B) of the DD component of DDLPS_08T. Multiple ERBB3 signals (red) are observed in the majority of cells in the DD component (A) and only a few cells in the WD component (B) of DDLPS_27T.

Figure 4. Effects on 93T449 and SW872 cells of combined treatment with CDK4 and IGF1R inhibitors

(A and B) IGF1R amplification and expression in 93T449 and SW872 cells, as well as an IGF1R-amplified tumor (DDLPS_25T). Relative copy number was estimated by NGS and quantitative PCR (qPCR) (A). mRNA expression was estimated by quantitative RT-PCR (qRT-PCR) and normalized to GAPDH expression (B). Relative expression levels are expressed as ratios of the median expression in non-amplified tumor samples, as in Figure 2B. (C and D) Growth inhibitory effects of CDK4 and IGF1R inhibitors on 93T449 (C) and SW872 (D) cells. Palbociclib (CDK4 inhibitor) and NVP-AEW541 (IGF1R inhibitor) were added at various concentrations, and cell metabolic activities were assayed after 6 days of culture. In this assay, synergism of these inhibitors was evaluated using CompuSyn (http://www.combosyn.com) [41]. Their effects were synergistic in 93T449 cells (average combination index score = 0.42 ± 0.19), but not in SW872 cells (average combination index score = 6.28 ± 3.22). (E) Effect of IGF1R inhibitor on IGF1R phosphorylation in 93T449 and SW872 cells. Cells were treated with NVP-AEW541 (1 μM) for 12 or 24 h and harvested. Expression and Y1135 phosphorylation of IGF1R were evaluated by western blotting analysis.