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For many pediatric cancer patients, commonly used gene-panel sequencing tests yield few actionable results, partly due to the complex genomic alterations present. We hypothesized that an unbiased approach, combining whole-genome (WGS) and RNA sequencing (RNAseq), could overcome this and lead to a more comprehensive understanding of these diseases. While prior studies have evaluated WGS and RNAseq in pediatric cancers, few focused primarily on metastatic or relapsed disease. We also placed special focus on longitudinal profiling of patients, including with additional deep sequencing, to capture tumor evolution at the primary and metastatic sites, and to quantify the utility of resampling. We assembled a cohort of 191 high-risk pediatric oncology patients, including solid tumors, CNS tumors, and leukemias/lymphomas. We have representation of patients with relapsed/refractory disease (68), metastatic disease at diagnosis (10), rare diagnoses (19), prior cancer history, and estimated overall survival Multiple sampling from the same patient identified drastic spatial and temporal differences in the genomes and transcriptomes of these tumors. Using the Jaccard index as a measure of concordance between samples shows dynamic changes between samples collected at different time points across multiple modalities (range 0-1, 1 is identical); SNVs ranged from 0.01-0.79, SVs 0.01-0.73, major CNAs 0.07-0.99, minor CNAs 0.38-0.99, up expression outliers 0.12-0.56, down expression outliers 0.04-0.54, and fusions 0-1. Potentially biologically significant differences in therapy-induced mutations by platinum agents were also observed, highlighting the impact of therapy on tumor evolution. Clonal architectures were extracted from deep resequencing and show extensive spatial, temporal, and metastatic heterogeneity in these rare and highly aggressive malignancies that is not captured by WGS alone. Identifying clinically relevant evolution remains a challenge in most patients, but our results suggest that resampling of pediatric tumors at relapse or metastasis will be important for the effectiveness of targeted therapies in the future. Citation Format: Henry J. Martell, Avanthi T. Shah, Alex G. Lee, Bogdan Tanasa, Stanley G. Leung, Aviv Spillinger, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, María V. Pons Ventura, Florette K. Hazard, Arun Rangaswami, Sheri L. Spunt, Norman J. Lacayo, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agrawal, Marcus R. Breese, Alejandro Sweet-Cordero. Longitudinal profiling of high-risk pediatric malignancies using a multiomics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1509.

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.

The use of sequencing-based assays for clinical management of pediatric cancer patients has become increasingly common. However, for many pediatric patients, gene panel based sequencing tests yield few actionable results. Given the complex genomic alterations present in many pediatric cancers, especially high-risk solid tumors, we hypothesized that an unbiased approach might reveal more actionable findings and lead to a more comprehensive understanding of these diseases. To accomplish this, we integrated whole-genome sequencing (WGS) with RNAseq in the analysis of a pediatric oncology cohort, with a focus on longitudinal cases to capture potential tumor evolution in metastatic or treated cases. Our cohort consists of 269 high-risk pediatric oncology patients, including patients with relapsed/refractory disease, metastatic disease at diagnosis, prior cancer history, a rare diagnosis, or an estimated overall survival Our results show that the integration of WGS and RNAseq can provide more and higher-quality actionable information than either modality alone, whilst also capturing the majority of actionable variants detected by panel sequencing. RNAseq identified not only druggable fusions and expression outliers, but also many rare and novel fusions. WGS provided fusion validation but highlighted the limitations of WGS alone in identifying fusions resulting from complex SVs. Conversely, WGS was adept at capturing genome-wide patterns of CNAs and loss of heterozygosity that are missed by gene-centric panels. Further RNAseq integration enabled prioritization of expressed SNVs as well as CNAs and SVs that significantly alter gene expression. We also used WGS to extract mutational signatures and tracked their evolution across longitudinal samples. We found potentially biologically significant differences in therapy-induced mutations caused by platinum and alkylating agents. Our unbiased approach has enabled further discovery that advances our understanding of these rare and highly aggressive malignancies. Citation Format: Henry J. Martell, Avanthi Tayi Shah, Alex G. Lee, Bogdan Tanasa, Stanley G. Leung, Aviv Spillinger, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, Maria V. Pons Ventura, Florette K. Hazard, Arun Rangaswami, Sheri L. Spunt, Norman J. Lacayo, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agrawal, Marcus R. Breese, E. Alejandro Sweet-Cordero. Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 54.

Despite many clinical trials, outcome for osteosarcoma (OS) patients remains poor, especially for those with metastatic or relapsed/refractory disease. We leveraged network-based systems biology approaches to discover Master Regulator (MR) proteins representing pharmacologically accessible, mechanistic determinants of OS cell state, and to dissect tumor transcriptional heterogeneity. MRs were identified by interrogating an OS regulatory network—generated from 87 RNAseq profiles in the TARGET OS cohort—with gene expression signatures from 149 diagnostic OS samples, using the VIPER algorithm. RNAseq profiles were generated in four sarcoma cell lines following perturbation with ~400 oncology drugs, and used to identify drugs capable of inverting MR activity profiles with the OncoTreat algorithm. Unsupervised, protein activity-based clustering of the samples identified two clusters, characterized by a significant overall survival difference (76% vs 38%, log-rank p-val 0.0041). Patients in the high-risk cluster presented aberrant activity of proteins involved in cell cycle (FOXM1, CENPF, TOP2A) and epigenetic remodeling (EZH2, KDM1A), while those in the low-risk cluster had aberrant activity of proteins involved in immune response (VAV1, CD86, CEBPE), interferon gama signaling (IRF5, MNDA), and senescence control (CREG1, TFEC). We further identified patients with metastatic disease and particularly poor outcome ( Citation Format: Somnath Tagore, Jovana Pavisic, Aaron T. Griffin, Katherine Janeway, Andrew L. Kung, Filemon Dela Cruz, Alejandro Sweet-Cordero, Inge Behroozfard, Stanley Leung, Alex Lee, Darrell Yamashiro, Julia Glade Bender, Andrea Califano. A systems biology approach to defining tumor heterogeneity and prognostic and targetable master regulator protein signatures from bulk and single-cell RNAseq in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 488.

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.

Clinical use of gene-panel based sequencing has become increasingly common in the management of pediatric cancer patients. For many patients, gene-panel tests have identified clinically actionable findings. However, highly targeted approaches will miss unanticipated (but potentially clinically meaningful) or novel alterations. In diseases with unknown or complex etiologies, including many pediatric high-risk and solid tumors, an unbiased approach may yield more 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. Here we describe our experience with an expanded cohort of over 100 high-risk pediatric oncology patients, with a combination of solid tumors, brain tumors, and leukemia/lymphomas represented. The majority of patients were deemed high-risk due to relapsed/refractory disease. An additional group of patients were defined as high-risk at time of initial diagnosis due to metastatic disease, a rare tumor, prior history of another cancer type, an undifferentiated tumor, or less than 50% estimated overall survival. WGS (tumor/germline) and RNAseq were used to characterize available samples and compared to results from panel testing for each patient (performed as part of their clinical evaluation). When possible, multiple samples from an individual patient were collected (i.e. specimens obtained at biopsy, resection, relapse, and/or from metastatic sites). Somatic DNA samples were sequenced to an average depth of at least 60X and germline samples to at least 30X. RNAseq was performed to a depth of at least 20 million paired-end reads for each sample. WGS samples were analyzed for single nucleotide variants (SNVs), structural rearrangements (SV), and copy-number alterations (CNA). RNAseq samples were analyzed to identify known and novel gene-fusions, to measure allele specific expression of SNVs, and to 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 was 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. However, the use of WGS/RNAseq resulted 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: Marcus R. Breese, Avanthi T. Shah, Alex G. Lee, Bogdan Tanasa, Stanley G. Leung, Aviv Spillinger, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, Florette K. Hazard, Arun Rangaswami, Sheri L. Spunt, Norman J. Lacayo, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agrawal, E. Alejandro Sweet-Cordero. Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3665.