Targeted Analysis of Cell-free Circulating Tumor DNA is Suitable for Early Relapse and Actionable Target Detection in Patients with Neuroblastoma

Abstract

Purpose: Treating refractory or relapsed neuroblastoma remains challenging. Monitoring body fluids for tumor-derived molecular information indicating minimal residual disease supports more frequent diagnostic surveillance and may have the power to detect resistant subclones before they give rise to relapses. If actionable targets are identified from liquid biopsies, targeted treatment options can be considered earlier. Experimental Design: Droplet digital PCR assays assessing MYCN and ALK copy numbers and allelic frequencies of ALK p.F1174L and ALK p.R1275Q mutations were applied to longitudinally collected liquid biopsies and matched tumor tissue samples from 31 patients with high-risk neuroblastoma. Total cell-free DNA (cfDNA) levels and marker detection were compared with data from routine clinical diagnostics. Results: Total cfDNA concentrations in blood plasma from patients with high-risk neuroblastoma were higher than in healthy controls and consistently correlated with neuron-specific enolase levels and lactate dehydrogenase activity but not with 123I-meta-iodobenzylguanidine scores at relapse diagnosis. Targeted cfDNA diagnostics proved superior for early relapse detection to all current diagnostics in 2 patients. Marker analysis in cfDNA indicated intratumor heterogeneity for cell clones harboring MYCN amplifications and druggable ALK alterations that were not detectable in matched tumor tissue samples in 17 patients from our cohort. Proof of concept is provided for molecular target detection in cerebrospinal fluid from patients with isolated central nervous system relapses. Conclusions: Tumor-specific alterations can be identified and monitored during disease course in liquid biopsies from pediatric patients with high-risk neuroblastoma. This approach to cfDNA surveillance warrants further prospective validation and exploitation for diagnostic purposes and to guide therapeutic decisions.

Combined low-pass whole genome and targeted sequencing in liquid biopsies for pediatric solid tumors

“…Establishing the clinical applicability of plasma-based liquid biopsies in pediatric cancer patients versus adult patients has been challenging due to the limited sample volumes from infants and young children, small cohort sizes for individual tumor types, as well as the varied nature of the genomic alterations that characterize pediatric solid tumors. Nevertheless, several studies have described the feasibility of NGS-based approaches in pediatric solid tumors 19 , 21 – 24 , 30 , 31 , 35 – 37 . For example, to detect neuroblastoma-specific markers for LB biobanking strategies in 84 infants, Lodrini et al showed that as little as 1–2 ml of blood plasma, CSF, or urine had sufficient cfDNA for disease monitoring and ultimately clinical implementation of LB assays 23 .…”

“…The feasibility of employing NGS and droplet digital PCR-based assays utilizing cfDNA derived from CSF, plasma, or the aqueous humor of the eye for pediatric central nervous system (CNS) tumors, solid tumors, or retinoblastoma, respectively, has recently been described 10 , 19 – 27 . In the diagnostic setting, ultra-low-pass WGS (ULP-WGS) analysis of plasma ctDNA was effectively used to distinguish malignant peripheral nerve sheath tumors (MPNST) from the benign lesion, plexiform neurofibroma (PN), in patients with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome 28 .…”

Cell-Free RNA from Plasma in Patients with Neuroblastoma: Exploring the Technical and Clinical Potential

“…The presence of neuroblastoma-specific mRNA in the cellular compartment of blood and bone marrow, such as PHOX2B , TH and CHRNA3 , has been shown to correlate with outcome, enabling response monitoring in patients with high-risk disease [ 6 , 7 , 8 , 9 ]. Additionally, several targets in cell-free DNA (cfDNA) from plasma have been described to track therapy response, disappearing as tumor burden decreases and re-appearing as the disease relapses [ 10 , 11 , 12 , 13 ]. However, the presence of tumor-specific mRNA is often attributed to circulating tumor cells, which are not always present in every stage of the disease.…”

Extracellular Vesicles: A New Source of Biomarkers in Pediatric Solid Tumors? A Systematic Review

“…The presence of the methylated tumor suppressor gene RASSF1A can be detected in plasma for several types of pediatric solid tumors, and can be used to monitor therapy response ( 25 , 26 ). For neuroblastoma, tumor-specific aberrations in the MYCN and ALK genes (mutations and copy number alterations) can be monitored during the course of the disease ( 27 , 28 ). Copy number profiling can be performed on cell-free DNA to detect a tumor-derived signal, and this can be combined with the copy number profile from the primary tumor, offering a more comprehensive overview of the genetic landscape of the tumor and its metastatic lesions ( 29 ).…”