Comprehensive genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic axis in fusion-positive and fusion-negative tumors

Abstract

Despite gains in survival, outcomes for patients with metastatic or recurrent rhabdomyosarcoma remain dismal. In a collaboration between the National Cancer Institute, Children's Oncology Group, and Broad Institute, we performed whole-genome, whole-exome, and transcriptome sequencing to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Two genotypes are evident in rhabdomyosarcoma tumors: those characterized by the PAX3 or PAX7 fusion and those that lack these fusions but harbor mutations in key signaling pathways. The overall burden of somatic mutations in rhabdomyosarcoma is relatively low, especially in tumors that harbor a PAX3/7 gene fusion. In addition to previously reported mutations in NRAS, KRAS, HRAS, FGFR4, PIK3CA, and CTNNB1, we found novel recurrent mutations in FBXW7 and BCOR, providing potential new avenues for therapeutic intervention. Furthermore, alteration of the receptor tyrosine kinase/RAS/PIK3CA axis affects 93% of cases, providing a framework for genomics-directed therapies that might improve outcomes for patients with rhabdomyosarcoma.

Significance: This is the most comprehensive genomic analysis of rhabdomyosarcoma to date. Despite a relatively low mutation rate, multiple genes were recurrently altered, including NRAS, KRAS, HRAS, FGFR4, PIK3CA, CTNNB1, FBXW7, and BCOR. In addition, a majority of rhabdomyosarcoma tumors alter the receptor tyrosine kinase/RAS/PIK3CA axis, providing an opportunity for genomics-guided intervention.

Figure 1

Circos Plots of RMS represenative tumors. Circos plot tracks representing verified somatic mutations, from outside circle; mutated genes missense mutations (Black), Nonsense and indel mutations (Red); genomic location, genome wide copy number alterations (Gray), lesser allele frequency (Green) LOH (dotted track), density of heterozygous SNPs (Orange) homozygous SNPs (Blue). Intrachromasomal rearrangements (inner circle gray) and Interchromasomal rearrangements (Inner circle red). a, NCI-40: A PAX7-FOXO1 translocation noting the associated high level copy number gain. This tumor also has high level copy number gain of MYCN on chromosome 2. b, RMS224: Represenative PAX3-FOXO1 fusion with no somatic point mutations. Note LOH on short arm of Chr11p. c, RMS2046: Multiple rearrangements on Chromosome 2 with corresponding junctions and copy number changes. This rearrangement produces a novel gene fusion of PAX3-INO80D. d, RMS216: Represenative PFN RMS. Note relative increase in point mutations including NRAS mutation on Chr1 and increase in aneuploidy including gain of Chr8. Complete loss of heterozygozity on Chr 10 and the short arm of Chr11 e, RMS2030: Multiple genome wide alterations in a tumor with TP53 mutation. Point mutation of FGFR4 on Chromosome 5.

Figure 2

Rearrangement of chromosome 2 in RMS2046 produces PAX3-INO80D fusion. a, WGS junctions. Purple lines represent tail-to-head junction, green lines show head-to-tail junction (possibly tandem duplication) and orange lines show tail-to-tail junction or head-to-head junction (inversion). LogR ratio and VAF (Variant Allele Frequency) demonstrate two distinct copy number patterns with multiple breakpoints. b, RNA sequencing discovered 22 high quality reads spanning the junction of the PAX3 and INO80D genes. The fusion joins exon 7 of PAX3 with exon 9 of INO80D. The predicted fusion protein maintains the paired box domain (labeled PAX in white) and homeobox DNA binding domain (labeled HBD in white) of the PAX3 protein. The two predicted N-terminal DNA binding domains of INO80D are lost in the fusion. c, Unsupervised clustering of transcriptome expression data demonstrates a clear definition between fusion positive (Red) and fusion negative (Blue) tumors including the alternate PAX gene fusions with NCOA1 and INO80D. “Fusion negative” alveolar histology tumors are underlined. RMS 2080 is a fusion negative embryonal tumor that appears to cluster amoung the fusion positive tumors however no rearrangement of the PAX gene was discovered by RT-PCR or transcriptome sequencing d, Histologic diagnosis of tumors evaluated by transcriptome sequencing reveals 10 tumors with “fusion negative” alveolar histology.

Figure 3

Fusion-positive and fusion-negative RMS have distinct genotypes. a, Number of protein coding mutations in fusion-positive tumors (red) and fusion negative tumors (blue). b, Significant difference in the number of aneuploid chromosomes between fusion-positive tumors (red) and fusion-negative (blue) tumors. c, Age at diagnosis versus genome-wide mutations in fusion-positive (red) versus fusion negative (blue).

Figure 4

The Genomic Landscape of pediatric RMS highlighting candidate alterations. Demographic characteristics, histologic subtypes and selected genes with copy number alterations or somatic mutations across 147 rhabdomyosarcoma cases. Unique sample identifier and sequencing platform. Sex, males in blue, females in pink. Age, years at diagnosis divided into less than 5 years and greater than 5 years. Histologic diagnosis, Red, Alveolar; Blue, Embryonal including Spindle and Botryoid subtypes; Gray, RMS not otherwise specified. Mixed alveolar and embryonal histology in green. Copy number gains and losses for selected genes. Blue, losses; red, gains; green, loss of heterozygozity. Selected genes with somatic mutations. Purple, fusion protein; black, missense; orange, nonsense/splice site/indel mutations.

Figure 5

Expressed mutations in 80 RMS tumors. a. Candidate somatic alterations found to be expressed in whole transcriptome sequencing and the discovered genes ranked by frequency. Top, the number of expressed mutations by sample; Blue, PAX gene fusion negative, Red, PAX gene fusion positive. The color (yellow to red) of the mark represents the variant allele frequency (VAF) with many mutations appearing to favor the mutant allele. The size of the circle is proportional to the fragments per kilobase of transcript per million mapped reads (FPKM). b. Gene Ontology analysis of the expressed mutations reveals multiple alterations of cell cycle, cellular response to stress, protein amino acid phosphorylation, response to DNA damage stimulus, microtubule-based movement, chromosome organization, muscle cell differentiation, regulation of MAP kinase activity, mitotic cell cycle checkpoint, chromatin modification, induction of apoptosis by intracellular signals, organelle localization, regulation of Rac protein signal transduction and regulation of transferase activity. Many tumors appear to accumulate multiple mutations in the same pathway (blue = 2, black = 3 or more).

Figure 6

a, Gene interaction map of Reactome pathway analysis that discovers alteration of FGFR signaling as the most altered pathway. 22/25 fusion negative tumors alter at least one gene in the pathway. b, GSEA enrichment plot of altered genes in fusion negative RMS tumors versus altered genes in the PAX3-FOXO1 expressing model cell line (7250_PF) with enrichment scores plotted for each gene moving down the ranked list of genes. Genes altered in the 7250_PF cell line show significant enrichment in the fusion negative tumors (FDR q-value = 0.004). GSEA enrichment plot of the altered genes in published mouse models of PAX3-FOXO1 in c, somite cells (FDR q-value = 0.009) and d, forelimb cells (FDR q-value 0.0009).

Figure 7

Model pathway altered in Rhabdomyosarcoma. Genes colored red are found in fusion positive tumors while genes colored blue are found in tumors without a PAX gene fusion. Alterations and their frequency in the population include mutations and small indels (M), copy number deletions and amplifications (C), or structural variations (S) that affect the gene.