1. What are the key questions and challenges in studying Desmoplastic small round cell tumor (DSRCT)?
Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive pediatric cancer that presents in the abdominal or pelvic region. It is caused by a translocation between the Ewing sarcoma breakpoint region 1 (EWSR1) gene on chromosome 22 and the Wilms tumor 1 (WT1) gene on chromosome 11, leading to the EWSR1-WT1 fusion oncogene. Despite chemotherapy facilitating tumor shrinkage, recurrence is frequent, and the survival rate is only 15-25%. DSRCT is also known for its high level of metastasis, with 90% of patients having intraperitoneal metastases and 25-43% having extraperitoneal metastases. The rarity of DSRCT, estimated at fewer than 50 cases per year in the United States, has limited sample procurement for scientific discovery. The cell of origin and the genomic breakpoints leading to the EWSR1-WT1 fusion gene remain unknown. Recent studies have utilized RNA-sequencing and exome sequencing to identify adjoining exons and genomic breakpoints, but the specific breakpoints in DSRCT are still uninvestigated. Additionally, there is a lack of research comparing primary and recurrent/metastatic DSRCTs. Understanding the mechanisms DSRCT employs to evade chemotherapy and the immune system is crucial for developing effective treatments. In this study, a novel pair of primary and recurrent DSRCTs derived from the same patient five years apart is analyzed to investigate these outstanding questions and establish the first-in-kind map of DSRCT genomic breakpoints, suggesting the role of microhomology-mediated end-joining (MMEJ) in the formation of the EWSR1-WT1 fusion gene. RNA sequencing analysis also identifies pathways likely to play a role in chemotherapy evasion, tumor recurrence, and metastasis, including increases in homologous recombination, alteration in RNA splicing, and reduction in cytokine secretion. The study also analyzes the role of the EWSR1-WT1 signature in recurrent/metastatic DSRCTs and the potential susceptibility of these tumors to fusion gene targeting treatments.
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2. How is genomic DNA isolated from DSRCT specimens?
Genomic DNA is isolated from frozen primary DSRCT specimens using DNazol or a standard extraction buffer. The process involves proteinase K digestion, phenol/chloroform extraction, and ethanol precipitation. In some cases, DNA is further purified using miniprep DNA columns before PCR amplification. This method ensures the isolation of high-quality genomic DNA for subsequent analysis and identification of genomic breakpoints.
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3. How is RNA sequencing library constructed?
RNA sequencing libraries are constructed from 500 ng of total RNA using the Illumina TruSeq Stranded Total RNA kit with ribo zero. The process follows the manufacturer's instructions. The fragment size of RNA-seq libraries is verified using the Agilent 2100 Bioanalyzer, and the concentrations are determined using the Qubit instrument. The libraries are then loaded onto the Illumina NovaSeq 6000 for 75 bp paired-end read sequencing. Fastq files are generated using bcl2fastq software. Gene counts are generated by pseudoalignment to the human ENSEMBL transcriptome version 109 using kallisto. Only genes with TPM >0.5 for both samples are included for further analysis.
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4. What primers were used for PCR in DSRCT identification?
PCR primers were designed in exon 7 of EWSR1 and exon 10 of WT1 to account for all potential breakpoint locations. This design was used to amplify the EWSR1-WT1 fusion gene in DSRCT samples, confirming the tumor identity as DSRCT. The primers helped detect the presence of the fusion gene in tumor samples and cell lines, validating the DSRCT diagnosis. The PCR amplicons for the primary and recurrent/metastatic tumor samples were identical and the same size as the BER-DSRCT amplicon, suggesting the fusion in these tumors adjoined exon 7 of EWSR1 to exon 8 of WT1. Sanger sequencing of the PCR products further validated the precise joining of EWSR1 exon 7 to WT1 exon 8 in RNA transcripts from both the primary and metastatic tumor samples.
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