Nanopore sequencing of cell-free DNA for the sensitive detection, molecular profiling and monitoring of childhood cancers

Paediatric cancer is the leading cause of death in children post infancy in the Western world. The integration of high-throughput molecular profiling is now urgently needed to guide clinical decision-making and treatment stratification. However, access to adequate tumour material for genomic profiling remains highly challenging, particularly in children with refractory cancers, due to sampling difficulties and small sample volumes. The analysis of cell-free DNA (cfDNA) from liquid biopsies for the detection of circulating tumour DNA (ctDNA) could offer a powerful, minimally invasive alternative to tumour profiling, thus circumventing sampling limitations. However, current ctDNA approaches have limited specificity and sensitivity, and their clinical implementation is hindered by poor scalability and high costs associated with conventional sequencing technologies. In contrast, emerging nanopore sequencing platforms have faster turnaround times, are more affordable and significantly smaller than conventional sequencers, making them easy to deploy and implement in healthcare settings. However, their utility for cfDNA analyses remains largely unexplored. Here, using available data and liquid biopsy samples from ~500 children with cancer, I will develop computational tools for the detailed analysis of cfDNA nanopore sequencing data. By harnessing nanopores ability to sequence native DNA, thus allowing the detection of mutational DNA alterations and epigenetic modifications from the same assay, I will: (a) establish highly sensitive and specific approaches for the detection and molecular profiling of ctDNA, (b) develop methylation-based disease classifiers for accurate diagnosis and treatment stratification, and (c) evaluate the clinical utility of cfDNA nanopore sequencing to monitor treatment response and detect cancer relapse. The clinical implementation of these approaches could therefore significantly improve disease management and outcomes for children with cancer.