Capturing clinically actionable copy number alterations in Wilms tumor using nanopore sequencing

Copy number alterations (CNVs) involving chromosomes 1p, 1q, 16q, and 11p15 are key genomic markers used in the risk stratification of Wilms tumor (WT). These CNVs, when considered alongside disease stage and other clinical features, are associated with an increased risk of relapse. Accordingly, testing for these changes is recommended to guide treatment choices in children with favorable histology disease. Current methods for detecting segmental CNVs in WT, including single nucleotide polymorphism (SNP) arrays and short-read sequencing, require prolonged turnaround times, high cost, and do not capture loss of imprinting (LOI) at 11p15, a key adverse predictor for patients who would otherwise have very low risk disease. In this study, we assessed the feasibility of utilizing nanopore sequencing for CNV and 11p15 LOI analysis in WT.

We performed whole-genome sequencing (WGS) using the MinION platform on 15 patient-derived WT xenografts previously characterized by whole exome sequencing (WES) and multiplex ligation-dependent probe amplification (MLPA), applying adaptive sampling in a subset to enrich for clinically relevant regions. End-to-end sequencing analysis was completed within 4 days.

Despite low sequencing depth (average 6.7x, WGS; 8.5x, adaptive sampling sequencing), nanopore WGS detected 94% (16/17) of the CNVs that are clinically relevant in WT. No false-positive findings were observed. Adaptive sampling showed high concordance with WGS for CNV detection and allowed sample multiplexing. However, the sequencing depth obtained by nanopore WGS and adaptive sampling was insufficient for assessment of methylation status at the Imprinting Control Regions (ICR)1 and ICR2 at 11p15.

This study demonstrates the feasibility of using nanopore sequencing for the assessment of clinically relevant CNVs in WT and highlights the potential of this technology for the integrated evaluation of copy number variation and 11p15 methylation status with a much shorter turnaround time and lower cost than other conventional methods for CNV testing. Successful clinical integration will require higher throughput nanopore sequencing platforms, as indicated by the current findings.