A tailored molecular profiling programme for children with cancer to identify clinically actionable genetic alterations

Abstract

Background: For children with cancer, the clinical integration of precision medicine to enable predictive biomarker-based therapeutic stratification is urgently needed.

Methods: We have developed a hybrid-capture next-generation sequencing (NGS) panel, specifically designed to detect genetic alterations in paediatric solid tumours, which gives reliable results from as little as 50 ng of DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. In this study, we offered an NGS panel, with clinical reporting via a molecular tumour board for children with solid tumours. Furthermore, for a cohort of 12 patients, we used a circulating tumour DNA (ctDNA)-specific panel to sequence ctDNA from matched plasma samples and compared plasma and tumour findings.

Results: A total of 255 samples were submitted from 223 patients for the NGS panel. Using FFPE tissue, 82% of all submitted samples passed quality control for clinical reporting. At least one genetic alteration was detected in 70% of sequenced samples. The overall detection rate of clinically actionable alterations, defined by modified OncoKB criteria, for all sequenced samples was 51%. A total of 8 patients were sequenced at different stages of treatment. In 6 of these, there were differences in the genetic alterations detected between time points. Sequencing of matched ctDNA in a cohort of extracranial paediatric solid tumours also identified a high detection rate of somatic alterations in plasma.

Conclusion: We demonstrate that tailored clinical molecular profiling of both tumour DNA and plasma-derived ctDNA is feasible for children with solid tumours. Furthermore, we show that a targeted NGS panel-based approach can identify actionable genetic alterations in a high proportion of patients.

Keywords: Circulating tumour DNA; Clinical targeted sequencing; Paediatric oncology; Personalised medicine.

Fig. 1

Study overview. After obtaining informed consent, tumour and blood samples were collected. DNA was extracted, and sequence libraries were prepared using the capture-based paediatric solid tumour panel. After sequencing, samples underwent an in-house data analysis pipeline that detects mutations, structural variants and copy number changes. Genomic alterations were manually reviewed by two independent scientists and then discussed in a molecular tumour board before a clinical report was issued. FFPE, formalin-fixed paraffin-embedded.

Fig. 2

Tumour samples submitted for sequencing. Summary of sample flow and the total number of samples successfully sequenced (A). Distribution of tumour types among reported cases (B). DSRCT, desmoplastic small round cell tumour; CNS, central nervous system.

Fig. 3

Overview of sequencing results. Oncoprint represents somatic mutations and gains, amplification and deletions detected in genes that are covered by the targeted panel. Samples are grouped in columns with genes displayed along rows. Samples are arranged according to the tumour type and genes sorted by frequency. Panel version, sample type, molecular annotations and diagnosis are provided as bars according to the included key (A). Bar plot of most recurrent altered genes, sorted by frequency and colour coded according to the tumour type (B). FFPE, formalin-fixed paraffin-embedded; DSRCT, desmoplastic small round cell tumour; CNS, central nervous system; FF, fresh frozen.

Fig. 4

Clinical actionability. Somatic alterations were defined according to OncoKB levels of evidence. Actionability tiers are described in the key. Distribution of actionability tiers for the entire sequenced cohort (A). Distribution of actionability tiers across common tumours, colour coded according to the tumour type (B). DSRCT, desmoplastic small round cell tumour; CNS, central nervous system.

Fig. 5

Comparison of results from paired samples, sequenced at different time points. Venn diagrams compare the genetic findings in eight patients. Shared alterations are illustrated by the intersection of the two ovals. Alterations detected at only the 1st time point are represented in the pink oval, and alterations identified at the 2nd time point only are represented in the green oval. The size of the oval represents the number of variants identified in each patient.

Fig. S1

Validation metrics for version 2 of the paediatric panel. Comparison of quality control metrics in ten patient samples run in version 1 and version 2 of the paediatric panel: number of reads (A), percentage of unique on-target reads (B), percentage of mapped unique on-target reads (C) and percentage of duplicates (D). Correlation of allele frequencies obtained by NGS and ddPCR for cancer variants in horizon cell blends (76 SNPs and 28 indels) (E). Correlation of allele frequencies obtained by version 1 and version 2 for cancer variants in FFPE samples (60 SNPs and 2 indels) (F). NGS, next-generation sequencing; indels, insertions and deletions; VAF, variant allele frequency; FFPE, formalin-fixed paraffin-embedded; SNP, single nucleotide polymorphism; ddPCR, droplet digital polymerase chain reaction.

Fig. S2

Illustration of structural variants detected by the panel. A snapshot of IGV showing spanning reads covering the structural variant region is shown on the top, and a cartoon illustration of the structural variant is displayed in the bottom. FGFR1 tandem duplication from exon 9 to exon 18 detected in a patient with glioma (A); fusion between exon 2 of c11orf95 and exon 2 of RELA detected in a patient with ependymoma (B); fusion between exon 5 of SQSTM1 and exon 20 of ALK detected in a patient with an inflammatory myofibroblastic tumour (C). IGV, integrative genomics viewer.