Abstract B25: Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies
Authors: Breese Marcus R. , Shah Avanthi T. , Tanasa Bogdan , Lee Alex G. , Leung Stanley G. , Spillinger Aviv , Liu Heng-Yi , Hazard Florette K. , Sweet-Cordero Alejandro 
Published: February 2019

Abstract
Clinical use of gene-panel based tumor sequencing has expanded exponentially over the past few years. While in some cases this molecular testing identifies clinically actionable findings, these highly targeted approaches may miss unanticipated, clinically meaningful or novel alterations. In cancers with poorly understood etiologies, including many pediatric solid or high-risk tumors, an unbiased approach may prove more useful. We sought to explore the feasibility and utility of whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) in comparison to commercially available targeted gene-panel testing in pediatric oncology.
Herein we describe our experience with an initial cohort of 58 high-risk pediatric oncology patients (37 solid tumors, 11 brain tumors, and 10 leukemia/lymphomas). The majority of patients (n=40) had relapsed/refractory disease. An additional eighteen 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% survival. A total of 102 samples were obtained from these 58 patients, with 70 samples originating at the primary sites of disease and 32 samples from metastatic sites. Thirty-one samples were chemotherapy/radiation therapy naïve. A combination of WGS and RNA-seq were used to characterize available samples and compared to results from panel testing for that patient (performed as part of their clinical evaluation). Where possible, fresh frozen tissue (FFT) samples were obtained during clinically indicated surgical procedures. When FFT was unavailable, formalin-fixed, paraffin-embedded (FFPE) samples were used. When possible, multiple samples from an individual patient were collected (i.e., specimens obtained at biopsy, resection, relapse, and/or from metastatic sites). Germline DNA was isolated from peripheral blood, with the exception of leukemia patients where saliva was used. Somatic DNA samples were sequenced to an average depth of at least 60X and germline samples to at least 30X. Somatic RNA-seq was performed to a depth of at least 20 million paired-end reads for each sample. In-house as well as published tools and algorithms were used to analyze DNA samples for single-nucleotide variants (SNVs), structural rearrangements, and copy-number alterations. RNA samples were analyzed to identify known and novel gene fusions, to measure allele specific expression of SNVs, and to perform gene-expression outlier analysis.
Consistent with previous observations, the mutational burden across pediatric cancers was low. While common mutations were identified, there was a long tail of mutations that occurred at a low frequency. As anticipated, samples obtained post-chemotherapy had a higher mutational burden than treatment-naïve samples. TP53 was the most commonly mutated gene, but we also identified SNVs in other genes commonly mutated in cancer, such as ASXL1, NOTCH2, and RB1. Other novel recurring variants were discovered, further analysis of which is ongoing. Canonical gene fusions were detected in 8/8 patients as well as potentially novel fusions, confirmation of which is also ongoing. In nearly all patients, variants identified by gene panels were also identified through WGS/RNA-seq analysis; however, in 2 instances, variants reported by gene panel testing were reclassified as germline using our tumor/normal WGS analysis. These results indicate that integrated WGS and RNA-seq analysis is feasible in the clinical setting and can reliably identify variants reported on commercially available gene panel testing. However, this approach also resulted in additional clinically relevant findings and allows for novel discovery that will further advance our understanding of these rare and highly aggressive pediatric malignancies.
Citation Format: Marcus R. Breese, Avanthi T. Shah, Bogdan Tanasa, Alex G. Lee, Stanley G. Leung, Aviv Spillinger, Heng-Yi Liu, Florette K. Hazard, Alejandro Sweet-Cordero. Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B25.