Abstract A25: Cell-free DNA for noninvasive molecular profiling and response monitoring in pediatric cancers
Authors: Kothari Prachi , Yang Julie , Stewart Caitlin , Gedvilaite Erika , Stephens Dennis , Berger Michael F. , Ortiz Michael V. , Shukla Neerav , Slotkin Emily , Modak Shakeel , Tsui Dana W. Y. 
Published: June 2020

Abstract
Background: Cell-free DNA (cfDNA) is a promising tool to noninvasively profile the cancer genome and track response to treatment. Its utility in adult cancers has been widely demonstrated; however, evidence in pediatric cancers is relatively limited. To address that, we performed targeted sequencing to screen for actionable mutations in plasma, and evaluated the utility of shallow whole-genome sequencing (sWGS) as (i) a tool to estimate cfDNA tumor fractions to guide data interpretation in plasma, and (ii) a mutation-agnostic approach to monitor treatment responses and at the same time screen for actionable somatic copy number alterations from cfDNA, in pediatric cancer patients with neuroblastoma (NB), osteosarcoma (OS), and Wilms’ tumor (WT).
Methods: In the first part of the study, we analyzed cfDNA by a ~400 gene targeted sequencing assay, MSK-IMPACT, and found mutations in 62% (31/50) of patients. In parallel, we performed sWGS (~0.1x) in the same cfDNA samples to estimate the fraction of tumor-derived DNA (cfDNA tumor fraction), which is established based on global copy number alterations and distinct tumor-derived cfDNA size profiles as features, that we learned in an independent adult cancer dataset using logistic regression model. Then we further evaluated the utility of sWGS-estimated tumor fractions to track responses in 28 patients (18 OS, 7 WT, and 3 NB) for whom longitudinal plasma samples were available.
Results: Plasma samples with an estimated high tumor fraction tend to show better agreement between tumor and plasma. Among the three cancer types, MSK-IMPACT targeted sequencing analysis of cfDNA from relapsed neuroblastoma revealed actionable mutations, including alterations in ALK, NRAS, and TSC1. Wilms’ tumor cfDNA revealed prognostic biomarkers, such as somatic mutations in TP53 and 1q gains, which are associated with worse prognosis and require more aggressive therapy. The sWGS analysis also revealed somatic focal copy number amplifications that are potentially actionable such as CDK4 and MDM2 from the OS patients. When analyzing longitudinal plasma samples, we found that sWGS-derived cfDNA tumor fractions closely tracked the dynamics of disease response to treatment and surgical resection. In two OS patients where no mutations were detected by targeted sequencing but cfDNA tumor fraction was high, we performed exome sequencing on the cfDNA and found BRCA signature, which is potentially actionable in OS.
Conclusions: Our results show that plasma profiling reveals actionable alterations across different types of pediatric cancers. Gene-panel targeted sequencing can reveal important somatic mutations, and low-pass sWGS allows longitudinal monitoring of tumor responses and detects clinically relevant copy number alterations. It also helps to identify cfDNA samples that have sufficient tumor-derived DNA for more comprehensive molecular profiling such as exome sequencing to screen for mutational signature that has clinical implication.
Citation Format: Prachi Kothari, Julie Yang, Caitlin Stewart, Erika Gedvilaite, Dennis Stephens, Michael F. Berger, Michael V. Ortiz, Neerav Shukla, Emily Slotkin, Shakeel Modak, Dana W. Y. Tsui. Cell-free DNA for noninvasive molecular profiling and response monitoring in pediatric cancers [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A25.