Whole Genome Analyses of a Well-Differentiated Liposarcoma Reveals Novel <i>SYT1</i> and <i>DDR2</i> Rearrangements

<div><p>Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including <i>MDM2</i>, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (<i>NAV3</i>, <i>SYT1</i>, <i>PAWR</i>) where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified <i>DDR2</i>, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as <i>MDM2</i> and <i>DDR2</i>.</p></div

Depiction of genomic rearrangement hotspot on chromosome 12.

<p>We identified and further characterized a putative transposable element (LOC100507498) located on the (-) strand, within the PAWR-SYT1-NAV3 gene cluster (<b>3A</b>). The LOC100507498 and closely related sequences were characterized by comparing both nucleotide (<b>3B</b>,top) and translated (<b>3B</b>,bottom) sequences to known families of repetitive elements (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087113#s2" target="_blank">Methods</a>). Highly conserved sequence domains/motifs are color coded by known families of repetitive elements (Legend). Overall, these sequences exhibited the highest similarity to the L1 retrotransposon and Alu repeat elements (domain hit counts and similarity score). Sequence alignments of LOC100507498 (*) with known L1 elements <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087113#pone.0087113-Pickeral1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087113#pone.0087113-Goodier1" target="_blank">[33]</a> exhibited the highest overall homology to Class 3 L1 elements as described by Pickeral et al. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087113#pone-0087113-t001" target="_blank">Table 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087113#pone.0087113-Pickeral1" target="_blank">[32]</a>) and in addition to the 5′-GGAG and 3′-AATA signature motifs, LOC100507498 carries several ‘AATGTTTA’ motifs that suggest multiple rounds of L1-mediated transduction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087113#pone.0087113-Goodier1" target="_blank">[33]</a>. The LOC100507498 locus resides within a genomic region that is deleted in the Tumor (T) sample, but present in the Normal (N) genome (<b>3C</b>).</p

DNA validated fusion genes.

<p>DNA validated fusion genes.</p

GeneGo pathway analysis of genes containing SNV, gene fusions or amplifications.

<p>GeneGo pathway analysis of genes containing SNV, gene fusions or amplifications.</p

Metrics and summary statistics.

<p>Metrics and summary statistics.</p

IPA pathway analysis of genes containing SNV, gene fusions or amplifications.

<p>IPA pathway analysis of genes containing SNV, gene fusions or amplifications.</p

aCGH and Fluorescent <i>in-situ</i> hybridization of <i>DDR2</i>, <i>MDM2</i>, and <i>SYT1</i>.

<p>(A) aCGH dot plots of the chromosomal region of each gene. Tan colored shading indicates regions of significant copy number change. Red dots indicate copy number gain and green dots indicate copy number loss. (B) FISH images of cells probed for the indicated genes with SpectrumOrange probe and the chromosome control indicated with the SpectrumGreen probe.</p

Circos plot of validated genetic variation in a well-differentiated liposarcoma.

<p>Inner-most circle contains validated structural rearrangements of fusion genes with translocations indicated in purple, and intra-chromosomal rearrangements indicated in orange. The middle ring contains the aCGH plot with copy number loss indicated in green and copy number gain in red; each orange ring corresponds to a log2 value of 1. The outer-most ring indicates validated, damaging single nucleotide variants.</p

Validated functionally damaging SNV.

<p>Validated functionally damaging SNV.</p