Minimally invasive classification of paediatric solid tumours using reduced representation bisulphite sequencing of cell-free DNA: a proof-of-principle study

Abstract

In the clinical management of paediatric solid tumours, histological examination of tumour tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumour is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumour samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of paediatric tumours using cell-free reduced representation bisulphite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumour type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in paediatric oncology in a cost-effective and minimally invasive manner.

Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome

“…In order to evaluate changes in cell contributions to cfDNA content, we performed non-negative least squares (NNLS) on the cfDNA samples in combination with a previously published cfDNA reference atlas (generated on Illumina arrays) [3]. In order to compare cf-RRBS with Illumina HM450K or EPIC, we grouped individual CpGs into regions as described before [2]. We observed similar proportions of erythrocyte progenitors as described by Moss et al .…”

“…Unmethylated lambda phage DNA (0.005 ng, or 0.5 µL of a 0.01 ng/µL solution) was added to the eluate after the SpeedVac step. [2] Libraries prepared using the cf-RRBS protocol were cleaned by magnetic bead selection (AMPure XT beads -NEB) and eluted in 0.1× TE buffer. The libraries were visualized with the Fragment Analyser (Agilent) and quantified using the Kapa library quantification kit for Illumina platforms (Kapa Biosystems).…”

“…In order to evaluate tube stability across time points, we determined 6 metrics per blood collection tube: [1] percentage of reads mapping within MspI regions, [2] evolution of relative DNA concentration (as assessed by the ratio of reads mapping to the human genome versus reads mapping to the lambda genome), [3] genome-wide CpG methylation percentage, [4] Total number of CpGs covered with at least 15 reads, [5] reproducibility of individual cytosine in CpG context calls by calculating the area-left-of-the-curve [22] (ALC) and [6] the evolution of the immune cell content.…”

“…The analysis of cell-free tumour-derived DNA (ctDNA) isolated from liquid biopsies, including blood plasma, has emerged as a complementary assay for tumour tissue genomic profiling [1,2]. The methylation pattern of cell-free DNA is currently under investigation as a biomarker for the non-invasive diagnosis of both benign [3] and malignant [4] conditions.…”

High throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by Methylated DNA sequencing (MeD-seq) of LpnPI digested fragments

“…The fact that most blood samples only yield small amounts of fragmented cfDNA combined with the low recovery of DNA after bisulfite conversion (22-66%) (11), renders conventional genome-wide DNA methylation analyses like whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) on cfDNA technically challenging. However, a recent proof-of-principle study in paediatric solid tumours demonstrates the feasibility of an adapted RRBS protocol for cfDNA analyses (12); (13). Similarly, Shen et al developed a method for high throughput sequencing of immune-precipitated methylated cfDNA (cfMeDIP-seq), which uses antibody-based enrichment of methylated DNA sequences and is therefore not dependent on bisulfite treatment (14).…”