A genomic case study of desmoplastic small round cell tumor: comprehensive analysis reveals insights into potential therapeutic targets and development of a monitoring tool for a rare and aggressive disease

Abstract

Background: Genome-wide profiling of rare tumors is crucial for improvement of diagnosis, treatment, and, consequently, achieving better outcomes. Desmoplastic small round cell tumor (DSRCT) is a rare type of sarcoma arising from mesenchymal cells of abdominal peritoneum that usually develops in male adolescents and young adults. A specific translocation, t(11;22)(p13;q12), resulting in EWS and WT1 gene fusion is the only recurrent molecular hallmark and no other genetic factor has been associated to this aggressive tumor. Here, we present a comprehensive genomic profiling of one DSRCT affecting a 26-year-old male, who achieved an excellent outcome.

Methods: We investigated somatic and germline variants through whole-exome sequencing using a family based approach and, by array CGH, we explored the occurrence of genomic imbalances. Additionally, we performed mate-paired whole-genome sequencing for defining the specific breakpoint of the EWS-WT1 translocation, allowing us to develop a personalized tumor marker for monitoring the patient by liquid biopsy.

Results: We identified genetic variants leading to protein alterations including 12 somatic and 14 germline events (11 germline compound heterozygous mutations and 3 rare homozygous polymorphisms) affecting genes predominantly involved in mesenchymal cell differentiation pathways. Regarding copy number alterations (CNA) few events were detected, mainly restricted to gains in chromosomes 5 and 18 and losses at 11p, 13q, and 22q. The deletions at 11p and 22q indicated the presence of the classic translocation, t(11;22)(p13;q12). In addition, the mapping of the specific genomic breakpoint of the EWS-WT1 gene fusion allowed the design of a personalized biomarker for assessing circulating tumor DNA (ctDNA) in plasma during patient follow-up. This biomarker has been used in four post-treatment blood samples, 3 years after surgery, and no trace of EWS-WT1 gene fusion was detected, in accordance with imaging tests showing no evidence of disease and with the good general health status of the patient.

Conclusions: Overall, our findings revealed genes with potential to be associated with risk assessment and tumorigenesis of this rare type of sarcoma. Additionally, we established a liquid biopsy approach for monitoring patient follow-up based on genomic information that can be similarly adopted for patients diagnosed with a rare tumor.

Keywords: Desmoplastic small round cell tumor; EWS-WT1 gene fusion; Genomic profiling; Liquid biopsy; Personalized biomarker; Whole-exome sequencing.

Fig. 1

Network analysis by IPA. a Interaction network of genes harboring protein-affecting somatic mutations (network score = 45) is associated with the top disease and functions: cell death and survival, nervous system development and function, cellular compromise. b Interaction network of genes harboring rare polymorphisms detected in homozygosis in the patient (network score = 12) is associated with the top disease and functions: cell cycle, digestive system development and function, hair and skin development and function. c Interaction network of genes affected by compound heterozygous variants (network score = 25) is associated with the top disease and functions: cancer, organismal injury, abnormalities, and gastrointestinal disease. Continuous and dashed lines indicate direct and indirect interactions between molecules, respectively. Blue molecules represent the genes encountered in our analysis and blank molecules represent other genes automatically included by IPA. Molecules are displayed by various shapes depending on the functional class of the gene product, according to IPA Path designer shapes (Additional file 7)

Fig. 2

Array CGH profile showing the pattern of somatic copy number alterations detected in the DSRCT genome. a Copy number alterations detected by array CGH analysis using a 180-K platform with an effective resolution of ~70 Kb: aneuploidy of chromosomes 5 and 18 (gains, in blue), and partial losses of chromosome 13q, 11p, and 22q (in red). The green circle indicates the focal deletion of a segment of 1.3 Mb at 9p24.1. Arrows indicate chromosome 11 and chromosome 22 breakpoints, 11p13 and 22q12.2, respectively. b Circus plot shows the copy number alterations detected by array CGH and WES. Only the genomic regions affected by CNA events are represented. The numbers on each chromosome region are described in megabases. In blue, data from array CGH and in green data from WES. A great overlap of CNA detection can be observed using both approaches

Fig. 3

Use of the chromosomal translocation t(11;22)(p13;q12) as a personalized tool for patient monitoring along follow-up. a FISH analysis shows break apart probes for WT1 gene, indicating the occurrence of the fusion. b Mate-pair whole-genome sequencing detected paired reads mapping to EWS and WT1 genes. c PCR amplification followed by Sanger sequencing confirmed the breakpoint region involving intronic regions of EWS and WT1 genes. d Digital droplet PCR assays for detection of the somatic rearrangement EWS-WT1. Left panel—Screening of ctDNA from plasma samples collected serially along patient follow-up by ddPCR. No gene fusion was detected in ctDNA from the patient collected in four different time points after surgery, suggesting no relapse, recurrence, or progression of the disease. Presence of cell-free DNA is shown by detection of non-rearranged WT1 probes in the plasma samples from the patient and from control plasma sample. Middle panel—Serial dilutions of tumor DNA to check the sensibility of the approach in detecting the fusion event, starting from 60 ng of input following five dilution series of tenfold as indicated. Right panel—Detection of somatic rearrangement in different tumor DNA fractions, 1.0 and 0.1%