Liquid biopsy detection of genomic alterations in pediatric brain tumors from cell-free DNA in peripheral blood, CSF, and urine

Abstract

Background: The ability to identify genetic alterations in cancers is essential for precision medicine; however, surgical approaches to obtain brain tumor tissue are invasive. Profiling circulating tumor DNA (ctDNA) in liquid biopsies has emerged as a promising approach to avoid invasive procedures. Here, we systematically evaluated the feasibility of profiling pediatric brain tumors using ctDNA obtained from plasma, cerebrospinal fluid (CSF), and urine.

Methods: We prospectively collected 564 specimens (257 blood, 240 urine, and 67 CSF samples) from 258 patients across all histopathologies. We performed ultra-low-pass whole-genome sequencing (ULP-WGS) to assess copy number variations and estimate tumor fraction and developed a pediatric CNS tumor hybrid capture panel for deep sequencing of specific mutations and fusions.

Results: ULP-WGS detected copy number alterations in 9/46 (20%) CSF, 3/230 (1.3%) plasma, and 0/153 urine samples. Sequencing detected alterations in 3/10 (30%) CSF, 2/74 (2.7%) plasma, and 0/2 urine samples. The only positive results were in high-grade tumors. However, most samples had insufficient somatic mutations (median 1, range 0-39) discoverable by the sequencing panel to provide sufficient power to detect tumor fractions of greater than 0.1%.

Conclusions: Children with brain tumors harbor very low levels of ctDNA in blood, CSF, and urine, with CSF having the most DNA detectable. Molecular profiling is feasible in a small subset of high-grade tumors. The level of clonal aberrations per genome is low in most of the tumors, posing a challenge for detection using whole-genome or even targeted sequencing methods. Substantial challenges therefore remain to genetically characterize pediatric brain tumors from liquid biopsies.

Keywords: ULP-WGS; circulating tumor DNA; hybrid capture sequencing; liquid biopsy; pediatric brain tumors.

Fig. 1

cfDNA sample collection. (A) Workflow. (B) Histopathological diagnoses of the tumors included in the cohort. (C) Types of liquid biopsies collected for the 251 patients. Abbreviation: cfDNA, cell-free DNA.

Fig. 2

ULP-WGS for copy number detection in cfDNA. (A) Analysis method to compare copy number profiles of cfDNA samples to those of their matched tumors. (B) Copy number profiles in the tumors of 132 patients with matched cfDNA. Gains are in red and losses are in blue. (C) Pearson correlation coefficients from the copy number comparison. The dotted line indicates the threshold for a statistically significant correlation. Samples with detected ctDNA are highlighted in red. (D) Copy number ratios from a single tumor (horizontal axis) against the copy number ratios from cfDNA from paired CSF (top) and plasma (bottom). r represents the Pearson correlation coefficient. (E) Genome-wide copy number profiles of DNA obtained from the tumor, CSF, and plasma from panel D. Gains are in red and losses are in blue. Abbreviations: cfDNA, cell-free DNA; CSF, cerebrospinal fluid; ctDNA, circulating tumor DNA; ULP-WGS, ultra-low-pass whole-genome sequencing.

Fig. 3

Hybrid capture sequencing for ctDNA detection—raw-reads analysis. (A) Number of callable mutations and rearrangements detected per primary tumor. (B) Occasional matches in raw reads reflect false-positives. Mutant allelic fraction in raw reads at four glioma hotspot mutation positions (BRAF V600E, IDH1 R132H, H3F3A K27M, HIST1H3B K27M) in cfDNA samples from 22 patients diagnosed with gliomas. Abbreviations: cfDNA, cell-free DNA; ctDNA, circulating tumor DNA.

Figure 4.

Hybrid capture sequencing for ctDNA detection—duplex-reads analysis. (A) Total number of duplex reads per cfDNA sample. Samples with detected ctDNA are indicated in red. (B) ctDNA detected in a plasma cfDNA sample from a patient diagnosed with an HGG harboring mutations in PTEN, FGFR4, and SMARCA4. Comparison of the mutant allelic fractions between duplex (top panels) and raw reads (bottom panels) at each mutation locus indicates the level of background noise when using raw reads. Abbreviations: cfDNA, cell-free DNA; ctDNA, circulating tumor DNA; HGG, high-grade glioma.

Fig. 5

Probability of detection ctDNA (vertical axis) against the number of cfDNA molecules sequenced (horizontal axis). Only DNA molecules that cover locations found to be mutant in the paired tumor are considered. Detection probabilities are shown for cfDNA tumor fractions of 1%, 0.1%, and 0.01%. Vertical lines represent actual numbers of cfDNA molecules sequenced across samples in this study. Samples for which ctDNA was detected are indicated by red lines; and negative samples are in gray. The observed mutant allelic fractions for the positive cfDNA samples are indicated at the top. Abbreviations: cfDNA, cell-free DNA; ctDNA, circulating tumor DNA.