METHYLATION BASED LIQUID BIOPSY IN HIGH-RISK NEUROBLASTOMA

 

Gal-Mark, Nurit, Division of Pediatric Hematology-Oncology, Schneider Children's Medical Center of Israel, Felsenstein Medical Research Center, Tel Aviv University;
Grunwald, Assaf, School of Chemistry, Sackler Faculty of Exact Sciences, Tel Aviv University; Gahramanov, Valid, Schneider Children's Medical Center, Tel Aviv University;
Birger, Yehudit, Division of Pediatric Hematology and Oncology, Schneider Children's Medical Center, Department of Human Molecular Genetics & Biochemistry, Sackler Faculty of Medicine, Felsenstein Medical Research Center, Tel Aviv University;
Izraeli, Shai, Division of Pediatric Hematology and Oncology, Schneider Children's Medical Center, Department of Human Molecular Genetics & Biochemistry, Sackler Faculty of Medicine, Felsenstein Medical Research Center ;
Berko, Esther, Division of Pediatric Hematology and Oncology, Schneider Children's Medical Center, Faculty of Medicine, Tel Aviv University​

 

Objectives
Pediatric solid tumors harbor a paucity of tractable oncogenic mutations, characterized instead by chromosomal alterations, limiting applications of targeted therapy and studies of tumor evolution.  Neuroblastoma (NBL) is a common pediatric cancer of the peripheral nervous system, where patients with high-risk neuroblastoma (HR-NBL) have dismal outcomes despite toxic and morbid multimodal therapy.  While liquid biopsies are utilized to track disease evolution and identify genetic resistance in the subset of patients with ALK-driven HR-NBL, their utility is limited for the majority of NBL and pediatric solid tumor patients whose tumors lack oncogenic driver mutations. Here, we sought to develop a mutation-agnostic liquid biopsy approach, utilizing whole genome and epigenome based sequencing methodology.

 

Methods
We performed Oxford Nanopore Technology (ONT) sequencing of diagnostic HR-NBL tumor tissues, obtaining genome-wide mutational, copy number, and CpG methylation profiles.  We utilized the published tissue specific CpG methylation atlas to identify differentially methylated regions (DMRs) with CpG methylation profiles specific to NBL. We incorporated these regions into the methylation atlas to perform cell-type specific deconvolution of cell free DNA (cfDNA).

 

Results
Diagnostic tumor ONT sequencing identified 152 DMRs specific to HR-NBL, in pathways linked to neuronal development, transcriptional regulation, and signaling.  After sequencing cfDNA samples from patients with NBL, healthy controls, and patients with other malignancies, we selected the 25 best regions as NBL biomarkers, and validated performance in an independent cfDNA cohort. Utilizing the combined methylation atlas with NBL biomarkers, we performed cell-type deconvolution, sensitively and accurately detecting NBL in the cfDNA.  Serial cfDNA tracking with our NBL score correlated with disease burden and response to therapy. 

 

Conclusions & References 
ONT sequencing identifies HR-NBL specific DMRs, which can be utilized to non-invasively monitor disease in cfDNA.  Further efforts to identify disease molecular-subtype specific DMRs and their evolution during disease response and resistance are ongoing.