Abstract A09: Cerebrospinal fluid (CSF) as a source of ctDNA for diagnosis and monitoring of pediatric patients with midline diffuse gliomas
Authors: Nobre Liana , Siddaway Robert , Johnson Monique , Ryall Scott , Zapotocky Michal , Bennet Julie , Tabori Uri , Hawkins Cynthia 
Published: June 2020

Abstract
Background: Midline diffuse gliomas are aggressive pediatric tumors, characterized molecularly by the presence of H3K27 mutation. These tumors are extremely malignant, with median overall survival of less than a year. Localized mainly in the pons or thalamic regions, they are not amenable to a gross total resection; however, a biopsy is needed for pathologic and molecular diagnosis. In this scenario, liquid biopsy is of utmost importance and has the potential to spare the risk of morbidity with surgical procedures, determine diagnosis and prognosis, as well as serve as a tool to identify potential targetable alterations. The objective of this pilot feasibility study is to explore the use of CSF ctDNA using ddPCR and next-generation sequencing tools to diagnose and monitor patients with midline diffuse gliomas.
Methods: We extracted CSF ct DNA collected from external ventricular drain in two patients diagnosed with midline diffuse glioma (one thalamic glioblastoma, one DIPG). ctDNA was extracted from 2 mL of CSF, quantification was done by quibit, and samples were processed first with ddPCR for detection of H3K27M point mutation followed by comprehensive profiling by whole-exome sequencing using Illumina HiSeq2500 sequencer. We assessed the feasibility of identifying diagnostic point mutations, as well as potential evolution of the tumor molecular landscape at disease progression.
Results: The first case, a thalamic GBM, had tissue available for molecular analysis; targeted RNA sequencing was performed on a clinical basis detecting H3K27M mutation (VAF=43%) and a PDGFRA N659K mutation (VAF=43%). ddPCR analysis of the CSF ctDNA identified the H3K27M mutation with VAF of 14%. Whole-exome sequencing (WES) confirmed the presence of H3K27M point mutation at VAF 11% and PDGFRA mutation at VAF 13%. This patient received radiation and chemotherapy but progressed rapidly and passed 7 months after the diagnosis. The second case, a DIPG diagnosed upon radiologicl and clinical criteria, was previously treated with conventional focal radiation and needed an EVD upon massive leptomeningeal progression with associated meningitis. CSF was then processed for ctDNA analysis. ddPCR confirmed the presence of H3K27M mutation with VAF of 48%. WES confirmed the presence of H3K27M mutation (VAF 14%) and additional TP53 R273C point mutation with VAF of 87%. This patient passed roughly 11 months post diagnosis.
Conclusion: We have demonstrated the feasibility of detection of molecular markers in CSF through ddPCR and NGS platform to not only to assist in diagnosis of pediatric midline diffuse gliomas, but also to monitor these patients for additional/new molecular events that may be targetable. Building on these results, we are currently implementing a targeted sequencing panel to establish the specificity of this method in a large pediatric brain tumor cohort.
Citation Format: Liana Nobre, Robert Siddaway, Monique Johnson, Scott Ryall, Michal Zapotocky, Julie Bennet, Uri Tabori, Cynthia Hawkins. Cerebrospinal fluid (CSF) as a source of ctDNA for diagnosis and monitoring of pediatric patients with midline diffuse gliomas [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 A09.