Recurrent somatic structural variations contribute to tumorigenesis in pediatric osteosarcoma

Abstract

Pediatric osteosarcoma is characterized by multiple somatic chromosomal lesions, including structural variations (SVs) and copy number alterations (CNAs). To define the landscape of somatic mutations in pediatric osteosarcoma, we performed whole-genome sequencing of DNA from 20 osteosarcoma tumor samples and matched normal tissue in a discovery cohort, as well as 14 samples in a validation cohort. Single-nucleotide variations (SNVs) exhibited a pattern of localized hypermutation called kataegis in 50% of the tumors. We identified p53 pathway lesions in all tumors in the discovery cohort, nine of which were translocations in the first intron of the TP53 gene. Beyond TP53, the RB1, ATRX, and DLG2 genes showed recurrent somatic alterations in 29%-53% of the tumors. These data highlight the power of whole-genome sequencing for identifying recurrent somatic alterations in cancer genomes that may be missed using other methods.

Figure 1. Whole genome sequencing of osteosarcoma

a) Representative CIRCOS plots of validated mutations and chromosomal lesions in a diagnostic and metastatic osteosarcoma tumor from different patients. Loss of heterozygosity (orange), gain (red), and losses (blue) are shown. Intrachromosomal translocations (green lines) and interchromosomal translocations (purple lines) are indicated. Sequence mutations in Refseq genes included silent single nucleotide variants (SNVs, green), nonsense and missense SNVs (brown), splice-site mutations (dark blue), and insertion/deletion mutations (red). An additional track was added to the innermost ring of the plot showing the density of SNVs to highlight regions adjacent to SVs characteristic of kataegis. b) Boxplots of validated basal mutation rate (BMR), number of non-silent single nucleotide variations (SNVs), total structural variations (SVs) and number of total copy number variations (CNVs) in the ERMS and ARMS tumors in the discovery cohort. The (*) represents statistical significance of p<0.001 as compared to the osteosarcoma genomes. c) Representative plot of sequence reads on chromosome 12 for the matched germline (green) and tumor (red) sample. Several distinct regions of copy number change are identified (arrows) spanning the MDM2 gene consistent with sequential chromosomal lesions. d) MDM2 FISH of SJOSO18 showing amplification (red) relative to the probe for chromosome 12 (green). Abbreviations: ERMS, embryonal rhabdomyosarcoma; TALL, T-cell acute lymphoblastic leukemia; MB, medulloblastoma.

Figure 2. Kataegis in osteosarcoma

a) Rainfall plot showing the Log₁₀ of the intermutation distance versus genomic position for a representative osteosarcoma sample (SJOS005) with evidence of Kataegis. The chromosomes are demarcated by gray shading and the number of SVs in each chromosome is shown in brown at the bottom. The validated SNVs are plotted and color coded by the type of mutation. b) The proportion of each type of validated SNV in osteosarcomas with evidence of kataegis versus those without kataegis. c) The distribution of each nucleotide sequence 5′ to the C mutations in tumors with kataegis and those without kataegis. d) A rainfall plot in a representative regions of chromosome 3 in SJOS005 with kataegis showing the strand of the hypermutation based on the C>T or G>A sequence clusters. f) A macrocluster of hypermutation with evidence of kataegis on chromosome 3 of SJOS005 with two sequential magnifications (boxes) showing the existence of microclusters within a single macrocluster.

Figure 3. Validated mutations in TP53

a) Structure of the TP53 gene showing the transactivation, proline, DNA binding and oligomerization domains with splice site, frameshift and missense mutations in the 19 patient’s tumors in the discovery cohort. b) Structure of the genomic locus of the TP53 gene showing the exon boundaries color coded in accordance with the protein domains shown in (a). Sites of interchromosomal translocations are shown with black arrowheads between exons 9 and 10. The sizes of the introns and exons are scaled proportionally except for intron 1, which is much larger than the other introns in human TP53. c) A magnified view of intron 1 of TP53 showing the deletions (blue arrowheads), intrachromosomal (red arrowheads) and interchromosomal translocations (black arrowheads).

Figure 4. FISH and IHC for TP53 in Osteosarcoma

a) Genomic location of the 5′ (green) and 3′ (red) break-apart FISH probes showing their position relative to the TP53 gene. The full-length probe used to identify deletions at the TP53 locus is shown in black. b–d) Images of H&E, TP53 IHC and break apart FISH for SJOS001 with biallelic rearrangement of the TP53 gene. e–g) Images of H&E, TP53 IHC and break apart FISH for SJOS002 with monoallelic rearrangement of the TP53 gene. h–j) Images of H&E, TP53 IHC and break apart FISH for SJOS010 with polysomy and a missense mutation leading to elevated accumulation of nuclear TP53 protein. k–m) Images of H&E, TP53 IHC and FISH using the full length probe (green) for SJOS008 with monoallelic deletion of TP53.

Figure 5. ATRX mutations correlate with ALT in osteosarcoma

a) Diagram of the 5 single-nucleotide variations, 4 deletions and 1 inter-chromosome SV found in the ATRX genes of the osteosarcoma cohort. 3 of the samples (SJOS006, SJOS018 and SJOS011) with ATRX SVs had matching RNAseq data. SJ006 has a short deletion at exon 23 and the RNASeq data confirmed a read-through event that would result in a T1885 frameshift. For SJOS011, the RNAseq and WGS supported a junction connecting exon 1 to exon 28 creating a nonsense mutation (M6R*). For SJOS018, the RNASeq and WGS data supported a deletion connecting exon 1 to exon 13 thereby creating an in frame fusion protein (M6RS1406). The WGS for SJOS016 predicts a deletion that connects exon 1 to exon 16 producing a frameshift (M6fs). b) Representative IHC for ATRX showing nuclear ATRX in a sample with intense staining and wild type ATRX (SJOS012), a sample with fainter nuclear localized ATRX (SJOS014) and a sample with a nonsense mutation (SJOS001). Arrows indicate representative nuclei stained positive for ATRX. Asterisks indicate ATRX immunopositive vascular endothelial cells among the tumor cells that are negative for ATRX IHC. c,d) Relative telomere length in the osteosarcomas compared to that in the matched germline DNA, as analyzed by WGS and qPCR. e) Representative telomere FISH showing characteristics of ALT (arrow) in an osteosarcoma with an ATRX deletion.