Novel genes implicated in embryonal, alveolar, and pleomorphic rhabdomyosarcoma: a cytogenetic and molecular analysis of primary tumors

Abstract

Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, likely results from deregulation of the skeletal myogenesis program. Although associations between PAX3, PAX7, FOXO1A, and RMS tumorigenesis are well recognized, the entire spectrum of genetic factors underlying RMS development and progression is unclear. Using a combined approach of spectral karyotyping, array-based comparative genomic hybridization (CGH), and expression analysis, we examined 10 primary RMS tumors, including embryonal, alveolar, and the rare adult pleomorphic variant, to explore the involvement of different genes and genetic pathways in RMS tumorigenesis. A complete karyotype established for each tumor revealed a high aneuploidy level, mostly tetraploidy, with double minutes and additional structural aberrations. Quantitative expression analysis detected the overexpression of the AURKA gene in all tumors tested, suggesting a role for this mitotic regulator in the aneuploidy and chromosomal instability observed in RMS. Array-based CGH analysis in primary RMS tumors detected copy number changes of genes involved in multiple genetic pathways, including transcription factors such as MYC-related gene from lung cancer and the cytoskeleton and cell adhesion-encoding genes laminin gamma-2 and p21-activated kinase-1. Our data suggest the involvement of genes encoding cell adhesion, cytoskeletal signaling, and transcriptional and cell cycle components in RMS tumorigenesis.

Figure 1

SKY analyses of primary RMS tumors. (A) ARMS2 sample demonstrated tetraploidy and dmins classified as chromosome 13. (B) ARMS6 cells displayed dmins originating from chromosome 1 in a hypertetraploid karyotype. (C) ARMS5 presented a novel rearrangement between 13q14 and 20q13 and dmins classified as originating from chromosome 13. The trisomies of chromosomes 10 and 21 shown here are not clonal. (D) ARMS1 tumor cells display a structural rearrangement at the 13q14 region and two dmins from chromosome 3 in a complex hypotetraploid karyotype. (E) Representative SKY image of the pleomorphic RMS tumor (PRMS1) demonstrating multiple numerical and structural alterations in a hypertriploid karyotype involving all chromosomes (see also Table 1).

Figure 2

Interphase FISH analyses of primary RMS tumor cells with specific BAC and PAC clones confirmed the increased copy number change detected by array-based CGH analysis: ARMS5 tumor cells hybridized with SAS/CDK4 (A) and GLI (B) probes. LAMC2 (C; red) and PAK1 (C; green) signals were detected in ARMS1 cells. (D) Hybridization of ARMS6 cells with the MYCL1 probe.

Figure 3

Quantitative RT-PCR expression analysis of primary RMS tumors and cell lines relative to average expression in normal skeletal muscle controls: (A) PAX3, (B) PAX7 (C), FOXO1A, and (D) AURKA genes. Relative expression values are calculated in log scale and are presented above each bar.