The genomics of desmoplastic small round cell tumor reveals the deregulation of genes related to DNA damage response, epithelial-mesenchymal transition, and immune response

Abstract

Background: Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive, and poorly investigated simple sarcoma with a low frequency of genetic deregulation other than an Ewing sarcoma RNA binding protein 1 (EWSR1)-Wilm's tumor suppressor (WT1) translocation. We used whole-exome sequencing to interrogate six consecutive pre-treated DSRCTs whose gene expression was previously investigated.

Methods: DNA libraries were prepared from formalin-fixed, paraffin-embedded archival tissue specimens following the Agilent SureSelectXT2 target enrichment protocol and sequenced on Illumina NextSeq 500. Raw sequence data were aligned to the reference genome with Burrows-Wheeler Aligner algorithm. Somatic mutations and copy number alterations (CNAs) were identified using MuTect2 and EXCAVATOR2, respectively. Biological functions associated with altered genes were investigated through Ingenuity Pathway Analysis (IPA) software.

Results: A total of 137 unique somatic mutations were identified: 133 mutated genes were case-specific, and 2 were mutated in two cases but in different positions. Among the 135 mutated genes, 27% were related to two biological categories: DNA damage-response (DDR) network that was also identified through IPA and mesenchymal-epithelial reverse transition (MErT)/epithelial-mesenchymal transition (EMT) already demonstrated to be relevant in DSRCT. The mutated genes in the DDR network were involved in various steps of transcription and particularly affected pre-mRNA. Half of these genes encoded RNA-binding proteins or DNA/RNA-binding proteins, which were recently recognized as a new class of DDR players. CNAs in genes/gene families, involved in MErT/EMT and DDR, were recurrent across patients and mostly segregated in the MErT/EMT category. In addition, recurrent gains of regions in chromosome 1 involving many MErT/EMT gene families and loss of one arm or the entire chromosome 6 affecting relevant immune-regulatory genes were recorded.

Conclusions: The emerging picture is an extreme inter-tumor heterogeneity, characterized by the concurrent deregulation of the DDR and MErT/EMT dynamic and plastic programs that could favour genomic instability and explain the refractory DSRCT profile.

Keywords: Chromosome imbalance; Copy number alterations; DNA damage response; Desmoplastic small round cell tumor; Genomic stability; Immune response; Mesenchymal–epithelial reverse transition/epithelial–mesenchymal transition; Somatic mutations; Whole-exome sequencing.

Fig. 1

Mutation spectrum of desmoplastic small round cell tumor (DSRCT). a Color-coded heatmap reporting the number of observed somatic mutations identified in each chromosome for each case. The total number of mutations for single case and their chromosome locations are reported in the vertical and horizontal bar plots, respectively. The cases are classified into three groups (see coloured top horizontal bar). b Distribution of somatic variant types for each case. MErT, mesenchymal–epithelial reverse transition; hybrid, hybrid/partial epithelial–mesenchymal transition; EMT, epithelial–mesenchymal transition

Fig. 2

Inter-patient heterogeneity of mutated genes in DSRCTs. Heatmap, coloured according to the variant type, representing the 135 mutated genes across six cases of DSRCT. The cases are classified into three groups (see coloured top horizontal bar). MErT, mesenchymal–epithelial reverse transition; hybrid, hybrid/partial epithelial–mesenchymal transition; EMT, epithelial–mesenchymal transition. For the entire name of the genes, reported as gene ID, see Additional file 2: Table S1

Fig. 3

Comparison between the DSRCT mutational profile and mutational signatures from Catalogue Of Somatic Mutations In Cancer (COSMIC). a Observed mutational profile of the aggregated list of the 137 somatic mutations categorized according to the 96 possible tri-nucleotide variations. The fraction of mutations found in each trinucleotide context is displayed. b Reconstruction of the observed DSRCT profile according to the 30 COSMIC mutational signatures; known COSMIC signatures which gave the major contribution (weights) are reported at the top of the panel. c Differences between the observed and the reconstructed mutational profiles; the mean error is reported at the top of the panel

Fig. 4

Pathways and genes mutated in DSRCT. a Network representing the six biological pathways containing protein-encoding mutated genes from our list significantly enriched according to Ingenuity Pathway Analysis (IPA^®, Qiagen; Bioinformatics, Redwood City, CA, USA; http://www.qiagen.com/ingenuity). b Heatmap reporting the 26 mutated genes across the six cases by the three DNA damage response (DDR) network subsets: core, RNA-binding proteins (RBPs), and RNA machinery (either directly or indirectly related). c Heatmap representing the distribution of the 10 mutated genes belonging to the MErT/EMT process across the six cases. The cases are classified into three groups (see coloured vertical bars on the left). Genes with a deleterious mutation are highlighted in bold. Genes belonging to both DDR network and MErT/EMT are marked by an asterisk. Genes are coloured according to the type of mutation. MErT, mesenchymal–epithelial reverse transition; hybrid, hybrid/partial epithelial–mesenchymal transition; EMT, epithelial–mesenchymal transition. Gene names: ADAD1, adenosine deaminase domain containing 1; RRM2, ribonucleotide reductase regulatory subunit M2; USP9X, ubiquitin specific peptidase 9 X-linked; WWC1, WW and C2 domain containing 1, WWC1; ATR, ATR serine/threonine kinase; CW22, CWC22 spliceosome associated protein homolog; ARID1A, AT-rich interaction domain 1A; U2AF1, U2 small nuclear RNA auxiliary factor; NUP214, xx; ZNF254, zinc finger 254; ZNF219, zinc finger 219; TP53, Tumor protein p53; DBR1, debranching RNA lariats 1; PIF1, PIF1 5′-to-3′ DNA helicase; ZNF600, zinc finger 600; ZNF708, zinc finger 708; RBM45, xx; ZNF568, zinc finger 568; ZNF676, zinc finger 676; BCLAF1, BCL2 associated transcription factor 1; EIF4G1, eukaryotic translation initiation factor 4 gamma 1; HTATSF1, HIV-1 Tat specific factor 1; ZNF208, zinc finger 208; PCF11, xx; ZNF225, zinc finger 225; DSG2, xx; TAGLN, transgelin; TBL1XR1, transducing beta like 1 X-linked receptor 1; CARF, calcium responsive transcription factor; ACTL8, actin like 8; TYRO3, TYRO3 protein tyrosine kinase; GRM7, glutamate metabotropic receptor 7; NUS1, NUS1, dehydrodolichyl diphosphate synthase subunit

Fig. 5

Copy number alteration (CNA) landscape of DSRCTs. a Genome-wide frequency of CNAs according to EXCAVATOR2 in the six cases of DSRCT; copy number gains and losses are reported in red and blue, respectively. b Gains and losses across the six cases for DDR network (left part) and MErT/EMT (right part). CNA events are colored according to EXCAVATOR2 copy number call. On the top are the genes belonging to these CNAs and their corresponding cytoband. Numbers on the box represent the number of genes involved. The cases are classified into three groups (see coloured vertical bars on the left). MErT, mesenchymal–epithelial reverse transition; hybrid, hybrid/partial epithelial–mesenchymal transition; EMT, epithelial–mesenchymal transition. Gene names: ATXN2, ataxin 2; TAF7, TATA box-binding protein-associated factor; BRWD1, bromodomain and WD repeat domain containing 1; HMGN1, high mobility group nucleosome-binding domain 1; PCDH, protocadherin; KRTAP, keratin-associated protein; LCE, late cornified envelope; SPRR, small proline-rich protein; CRCT1, cysteine-rich C-terminal 1; ETS2, ETS proto-oncogene 2; FOXQ1, forkhead box Q1; FOXF2, forkhead box F2

Fig. 6

Chromosome 1 gains in DSRCTs. a Plot representing genomic location of amplified segments of chromosome 1 in the six DSRCT cases. Recurrent gains were identified in four cases (DSRCT2, 3, 5, and 7). The cases are classified into three groups (see coloured vertical bars on the left). b Network showing genes mapping to chromosome 1 and associated to the functional categories by Ingenuity Pathway Analysis (IPA^®, Qiagen; Bioinformatics, Redwood City, CA, USA) and significantly enriched in the list of 410 genes commonly amplified in the four cases. The most enriched categories were “Cell movement” and “Cell migration” (both P < 0.001). See also Additional file 2: Table S6 for gene overlapping between the two pathways. For the entire name of the genes, reported as gene ID, see Additional file 2: Table S5

Fig. 7

Chromosome 6 losses in DSRCTs. a Plot representing genomic location of deleted segments of chromosome 6 in the six DSRCT cases. Loss of the entire chromosome 6 is present in DSRCT3 and 7, but restricted to the long arm in DSRCT5 (see also Additional file 1: Figure S3). The cases are classified into three groups (see coloured vertical bars on the left). b Network, identified by Ingenuity Pathway Analysis (IPA^®, Qiagen; Bioinformatics, Redwood City, CA, USA; http://www.qiagen.com/ingenuity): 21 genes present on the deleted chromosome 6q and belong to the pathway “formation of nucleosome” (total 22 genes) are members of the histone H1 (HIST1H) family