Paediatric cancer is the leading cause of death in children post infancy in the Western world. Solid tumours, including tumours of the central nervous system (CNS), neuroblastomas, and bone and soft tissue sarcomas, are amongst the poorest outcome cancers in children (5-year survival rates <50%). These account for >50% of cases and contribute to up to 60% of paediatric cancer deaths. Several clinical and genomic profiling studies in Europe, such as the UK Stratified Medicine Paediatrics (SMPaeds) programme, have shown that a substantial proportion of childhood malignancies contain actionable tumour alterations. This knowledge has guided the development and delivery of several successful clinical trials for targeted precision therapies. However, despite these important advances, the majority of children are still treated with multiple rounds of conventional chemotherapy, and approximately 40-60% of children with solid tumours relapse with chemo-resistant disease. At this stage, targeted therapies are the only chance of survival. Yet, only a small minority of patients (<20%) with refractory disease are currently enrolled in molecularly guided treatment regimens. This missed opportunity is the result of (1) delayed relapse detection due to the lack of feasible molecular tests to monitor disease progression; and (2) major sampling challenges associated with obtaining tissue material for genomic tumour profiling: in children, tissue biopsy requires general anaesthesia, is often not physically feasible, and commonly results in insufficient tissue for both pathology and molecular analysis.
Minimally invasive molecular profiling using liquid biopsy analyses could provide a powerful alternative to circumvent tumour sampling challenges. Liquid biopsy refers to the analysis of cell-free DNA (cfDNA) from body fluids, primarily blood, but also cerebrospinal fluid (CSF) or urine, for the detection of tumour-specific DNA aberrations. This is possible because in cancer patients cfDNA also contains small proportions of circulating tumour-derived DNA (ctDNA) released by cancer cells. Importantly, liquid biopsy analyses are highly amenable to serial sampling, thus allowing longitudinal monitoring of treatment response and disease evolution. Molecular profiling of cfDNA using DNA sequencing methods have already proven very promising for the detection of genomic aberrations in common adult malignancies as well as in some paediatric cancers. However, current liquid biopsy approaches relying on conventional sequencing are limited by (a) low sensitivity and specificity, in part owing to very low concentrations of ctDNA in the blood, (b) high upfront costs associated with current sequencing technologies; and (c) slow turnaround times. These challenges drastically limit large-scale implementation of ctDNA methods in most health-care settings across the EU. In contrast, newer long-read 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. In addition, real-time nanopore sequencing facilitates data analyses to be coupled to the sequencing process, which significantly reduces turnaround times from several weeks to hours. This is critical in clinical practice as long turnaround times delay therapeutic intervention. Importantly, nanopore sequencing is capable of reading individual, native DNA molecules without the need for preamplification steps. This allows simultaneous detection of alterations of the DNA sequence, including copy number aberrations (CNAs), single nucleotide variants (SNVs) and structural variants (SVs), as well as DNA modifications (i.e. DNA methylation) from the same assay. This multi-modality would be hugely beneficial for the genomic testing of paediatric cancers, and drastically increase testing sensitivity.
However, the utility of long-read sequencing for cfDNA analyses remains largely unexplored. Therefore, the overall aim of this project is to evaluate the clinical utility and feasibility of long-read sequencing for cfDNA analysis. Further, this project has the overarching aim of developing a novel multi-modal liquid biopsy test to enable highly accurate diagnosis, monitoring and early relapse detection of childhood cancers using long-read whole genome sequencing.
