Circulating Cell-Free DNA Assessment in Biofluids from Children with Neuroblastoma Demonstrates Feasibility and Potential for Minimally Invasive Molecular Diagnostics

Abstract

Liquid biopsy strategies in pediatric patients are challenging due to low body weight. This study investigated cfDNA size distribution and concentration in blood, bone marrow, cerebrospinal fluid, and urine from 84 patients with neuroblastoma classified as low (n = 28), intermediate (n = 6), or high risk (n = 50) to provide key data for liquid biopsy biobanking strategies. The average volume of blood and bone marrow plasma provided ranged between 1 and 2 mL. Analysis of 637 DNA electropherograms obtained by Agilent TapeStation measurement revealed five different major profiles and characteristic DNA size distribution patterns for each of the biofluids. The proportion of samples containing primarily cfDNA was, at 85.5%, the highest for blood plasma. The median cfDNA concentration amounted to 6.28 ng/mL (blood plasma), 58.2 ng/mL (bone marrow plasma), 0.08 ng/mL (cerebrospinal fluid), and 0.49 ng/mL (urine) in samples. Meta-analysis of the dataset demonstrated that multiple cfDNA-based assays employing the same biofluid sample optimally require sampling volumes of 1 mL for blood and bone marrow plasma, 2 mL for cerebrospinal fluid, and as large as possible for urine samples. A favorable response to treatment was associated with a rapid decrease in blood-based cfDNA concentration in patients with high-risk neuroblastoma. Blood-based cfDNA concentration was not sufficient as a single parameter to indicate high-risk disease recurrence. We provide proof of concept that monitoring neuroblastoma-specific markers in very small blood volumes from infants is feasible.

MYCN Impact on High-Risk Neuroblastoma: From Diagnosis and Prognosis to Targeted Treatment

“…In particular, CTCs can provide comprehensive tumor profiling involving RNA, protein and/or metabolic information, while cfDNA only contains a genomic statement [ 45 , 46 ]. Of course, this method is more expensive and slower than cfDNA use [ 47 ], but it can be extensively considered as complementary in HR-NB evaluation, as suggested in other cancer studies [ 48 , 49 ].…”

Combined low-pass whole genome and targeted sequencing in liquid biopsies for 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 . However, whether this was a disease-specific finding, and whether cfDNA yield is correlated with location, histology, or tumor burden has yet to be determined.…”

Circulating tumor cells in neuroblastoma: Current status and future perspectives

“…One proof‐of‐concept study reported the feasibility of monitoring NB‐specific genomic alterations from ctDNA in minimal volumes of biofluids from infants (recommendations: 1 ml of blood and BM plasma, 2 ml of cerebrospinal fluid). 88 However, the clinical application of ctDNA analysis also has its inevitable drawbacks. First, ctDNA is thought to originate from lytic, apoptotic or necrotic tumor cells.…”

“…Moreover, it also requires smaller sampling volumes of biofluids, which facilitates longitudinal sampling during the disease course. One proof‐of‐concept study reported the feasibility of monitoring NB‐specific genomic alterations from ctDNA in minimal volumes of biofluids from infants (recommendations: 1 ml of blood and BM plasma, 2 ml of cerebrospinal fluid) 88 . However, the clinical application of ctDNA analysis also has its inevitable drawbacks.…”