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.

Keywords: ALK mutation; ALK-inhibitor; MYCN amplification; cancer; detection of therapeutic targets; liquid biopsy; minimal residual disease; precision medicine; real-time monitoring of therapeutic efficacy.

Figure 1

Overview of liquid biosamples from patients with neuroblastoma. (A) Line chart summarizing patient age at diagnosis and patient age at individual sample time point for the sequentially collected biofluids. (B) Bar chart indicating the percentage of biofluids according to sample volume. (C) Dot chart demonstrating blood plasma volumes provided from patients ≤ and >18 months of age. ** p < 0.01. (D) Multiple pie charts visualizing the distribution of biosamples collected in this study according to treatment stratification of patients with neuroblastic tumors [38].

Figure 2

Analytical specification of cell-free DNA in biofluids. (A) DNA extracted from biofluids was analyzed using the cell-free DNA ScreenTape assay (Agilent) and the Agilent 4200 TapeStation System. Shown are five electropherogram profiles of cfDNA samples that represent differing sample quality dependent on the amount of high molecular weight material detected and the identification of the typical peak at approximately 170 bp representing the mononucleosome. Vertical arrows indicate peaks characteristic for the cfDNA fraction and/or the high molecular weight genomic DNA. The predominant DNA fraction is highlighted with a black arrow, whereas the smaller DNA fraction is marked with a white arrow. The broad peak characteristic in some electropherograms is indicated with a solid black horizontal line. (B) Multiple pie charts demonstrating the distribution of electropherogram profiles characterized by (i) no prominent peak, (ii) primarily cfDNA, (iii) similar amounts of cfDNA and high molecular weight genomic DNA, (iv) primarily high molecular weight genomic DNA and (v) a dominant broad peak among all blood plasma, bone marrow plasma, cerebrospinal fluid and urine samples collected from patients with neuroblastoma, pediatric controls and healthy individuals.

Figure 3

Summary of total cell-free DNA concentrations in biofluids from patients with neuroblastoma and controls. Shown are cfDNA concentrations in blood plasma, bone marrow plasma, cerebrospinal fluid and urine from patients with neuroblastoma and, where applicable, from pediatric controls or healthy individuals. Each symbol depicts an individual measurement. Data are categorized into five different subgroups according to their specific DNA electropherogram. n.s., not significant; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 4

Comparison of cell-free DNA availability with minimum and optimum input requirements for cfDNA analysis. (A) Schematic diagram summarizing the sample requirements of selected sequencing and PCR technologies [16,41,42,43]. (B) Percentage of samples fulfilling the DNA input thresholds as indicated. CNA, copy number alteration; SNV, single nucleotide variation.

Figure 5

Longitudinal monitoring of cfDNA concentration in blood plasma from patients with high-risk neuroblastoma demonstrates highly variable baseline cfDNA concentrations and rapid cfDNA clearance following favorable treatment response. Total blood-based cfDNA concentrations from patients with a high cfDNA baseline concentration (>50 ng/mL plasma) are summarized in (A) while those from patients with a low baseline cfDNA concentration (≤50 ng/mL) are shown in (B). Retrospective cfDNA quantification in sequentially collected blood samples from patients with relapsed high-risk neuroblastoma is shown in (C). Total cfDNA concentrations were quantified using the Agilent 4200 TapeStation System. Dashed lines indicate the median cfDNA concentration in the pediatric control cohort. The treatment modules according to the German NB2004 Trial (EudraCT 20661) and the consecutive 2017 Guidelines for Diagnosis and Treatment of Patients with Neuroblastic Tumors are indicated above the plots. HDCT, high-dose chemotherapy; ↯, radiation; S, surgery.

Figure 6

Overview of molecular ctDNA profiling results in 3 selected infants <18 months with neuroblastoma. Shown is the timeline from diagnosis through therapeutic interventions including drug therapies and surgery. Selected images reflect the tumor burden at initial diagnosis for patients B8 (left panel), B67 (middle panel) and B93 (right panel). Time points of bone marrow assessment by (immuno)cytology and droplet digital PCR (ddPCR) are summarized below the timelines. Sample volumes and cfDNA content available for ddPCR are also indicated. N4, chemotherapy regimen according to the NB2016 registry: doxorubicin, vincristine, cyclophosphamide; VAC, chemotherapy regimen according to the Cooperative Soft Tissue Sarcoma Study Group of the German Society of Pediatric Hematology and Oncology (GPOH): vincristine, actinomycin-D, cyclophosphamide. B, biopsy; BM, bone marrow; BW, body weight; R, resection; DD, disease detectable; NDD, no disease detectable.

Figure 7

Graphical abstract demonstrating the workflow of liquid biopsy-based cfDNA diagnostics in patients with neuroblastoma.