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Most circulating tumor DNA (ctDNA) assays are designed to detect recurrent mutations. Pediatric sarcomas share few recurrent mutations but rather are characterized by translocations and copy-number changes. We applied Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) for detection of translocations found in the most common pediatric sarcomas. We also applied ichorCNA to the combined off-target reads from our hybrid capture to simultaneously detect copy-number alterations (CNA). We analyzed 64 prospectively collected plasma samples from 17 patients with pediatric sarcoma. Translocations were detected in the pretreatment plasma of 13 patients and were confirmed by tumor sequencing in 12 patients. Two of these patients had evidence of complex chromosomal rearrangements in their ctDNA. We also detected copy-number changes in the pretreatment plasma of 7 patients. We found that ctDNA levels correlated with metastatic status and clinical response. Furthermore, we detected rising ctDNA levels before relapse was clinically apparent, demonstrating the high sensitivity of our assay. This assay can be utilized for simultaneous detection of translocations and CNAs in the plasma of patients with pediatric sarcoma. While we describe our experience in pediatric sarcomas, this approach can be applied to other tumors that are driven by structural variants.

Overlapping myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are clonal hematopoietic disorders with features of myelodysplasia and myeloproliferation. The only well-characterized MDS/MPN in children is juvenile myelomonocytic leukemia, an aggressive disorder of infants and toddlers. The biochemical hallmark of this disease is hyperactivation of the Ras/MAPK signaling pathway caused by mutations in Ras pathway genes in more than 90% of patients. Translocations involving receptor tyrosine kinases have been identified in rare cases. Here, we report a 2-year-old patient who presented with MDS/MPN driven by a cytogenetically cryptic NUP98-NSD1 fusion, a translocation thought to exclusively occur in patients with acute myeloid leukemia.

Targeted gene panel sequencing has become increasingly common in the management of pediatric cancer patients. For some patients, these cancer gene panel tests have identified clinically actionable findings, but for many pediatric patients, no actionable alterations are identified. This is in part due to the low mutational burden of pediatric malignancies; thus, an unbiased approach may shed light on potentially actionable findings. To accomplish this, we examined the feasibility and utility of whole-genome sequencing (WGS) and RNA sequencing (RNAseq) in the management of high-risk pediatric oncology patients. We describe our experience with a cohort of over 100 high-risk pediatric oncology patients, with a combination of solid tumors, brain tumors, and hematologic malignancies. The majority of patients were deemed high-risk due to relapsed/refractory disease. A second group of patients was defined as high-risk at time of initial diagnosis due to the presence of metastatic disease, an estimated overall survival of less than 50%, a rare tumor, an undifferentiated tumor, or prior history of another malignancy. When possible, multiple samples from an individual patient were collected (i.e., specimens at biopsy, resection, relapse, and/or from metastatic sites) to allow for evaluation of inter- and intratumoral heterogeneity. Close to 200 tumor samples were available for analysis using WGS and/or RNAseq analysis. Somatic DNA samples were sequenced to an average depth of 60X and germline samples to 30X. WGS samples were analyzed for SNVs, structural rearrangements (SVs), copy-number alterations (CNAs), and mutational signatures. RNAseq was performed to a depth of at least 20 million paired-end reads for each sample. These samples were analyzed to identify known and novel gene-fusions, measure allele specific expression of SNVs, and perform gene-expression outlier analysis. Expression of variants (SNV/SV) identified using WGS were confirmed using RNAseq. For gene expression outliers detected using RNAseq, the WGS data were used to predict possible mechanisms for the aberrant expression (such as CNA, gene fusions, or promoter hijacking). This analysis suggests that WGS and RNAseq analysis is feasible in a clinical setting and can reliably identify variants reported on gene panel tests. Furthermore, the use of WGS/RNAseq results in additional clinically informative findings while also enabling novel research to further advance our understanding of these rare and highly aggressive pediatric malignancies. Citation Format: Avanthi T. Shah, Marcus R. Breese, Alex G. Lee, Henry J. Martell, Bogdan Tanasa, Stanley G. Leung, Aviv Spillingeer, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, Florette K. Hazard, Soo-Jin Cho, Arun Rangaswami, Norman J. Lacayo, Sheri L. Spunt, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agarwaal, Alejandro Sweet-Cordero. Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B20.

Gliomatosis peritonei is a rare pathologic finding that is associated with ovarian teratomas and malignant mixed germ cell tumors. The occurrence of gliomatosis as a mature glial implant can impart an improved prognosis to patients with immature ovarian teratoma, making prompt and accurate diagnosis important. We describe a case of recurrent immature teratoma in a 10-yr-old female patient, in which comparative analysis of the RNA sequencing gene expression data from the patient's tumor was used effectively to aid in the diagnosis of gliomatosis peritonei.