A single assay can provide real-time, intraoperative methylome classification of central nervous system (CNS) tumours with complete diagnosis available within 24 hours, UK researchers report.
DNA methylation-based classification is now a fundamental criterion in the routine diagnosis of many CNS tumour sub-types, but traditionally this was a slow process due to technological limitations.
Nanopore sequencing has been shown to reliably classify the methylation status of CNS tumours, with much faster turnaround times than array-based assays, a research team led by the University of Nottingham and Nottingham University Hospitals NHS Trust wrote in the journal Neuro-Oncology.
However, full integrated diagnosis might require subsequent additional assays to detect pathognomonic somatic mutations and structural variants, which delayed time to final diagnosis.
Utilising nanopore sequencing technology
To overcome this, the team developed a software tool called ROBIN, which uses PromethION nanopore sequencing technology to provide real-time, intraoperative methylome classification and next-day comprehensive molecular profiling within a single assay.
The researchers tested ROBIN’s performance on 50 prospective intraoperative CNS tumour cases, finding it could achieve diagnostic turnaround time of two hours and generate robust tumour classifications within minutes of sequencing.
‘A key challenge for intraoperative tumour classification is balancing the competing needs of rapid sample processing and the requirement to generate DNA of adequate yield and read length for the subsequent detection of additional molecular features,’ the researchers wrote.
‘Previous implementations of intraoperative, ultra-fast classification do not generate libraries of sufficient read length and pore occupancy for detecting additional molecular features beyond methylation-based classification alone.’
In contrast, ROBIN was able to detect single nucleotide variants, copy number variants, and structural variants in real time, and was able to inform a complete integrated diagnosis within 24 hours.
‘Classifier performance demonstrated concordance with final integrated diagnosis in 90% of prospective cases,’ they wrote.
ROBIN operated through a web interface and ran on the local computer performing the sequencing, but its output could be viewed by a pathologist remotely, enabling a diagnosis to be made by a clinician in real time during sequencing and permitting geographically separate sequencing and clinical analysis.
Achieving comprehensive tumour classifications more quickly
Professor Matt Loose, a biologist from the School of Life Sciences at the University of Nottingham, developed a method to sequence specific parts of human DNA at higher depth using nanopore portable sequencing devices.
‘When we first were able to sequence an entire human genome in 2018, it took around five labs and six months to do, which obviously isn’t ideal when time is of the essence for a patient,’ he said.
‘This new method now allows us to choose the bits of DNA that we need to look at in order to answer specific questions, such as what type of tumour and how can it be treated.’
‘Combined with our later research where we were able to look at relevant parts of the human genome more quickly – then we now have a process where we can use ROBIN to create comprehensive classifications of tumours more quickly.’
Professor Loose said the new pathway was quicker, cheaper and more accurate than current methods, potentially costing £450 per person – or less – once scaled.
‘Our method can eliminate the need for four to five separate tests, reducing costs as a consequence as we are getting more information from the single test we do,’ he said.
‘Most importantly, it delivers results to the patients when they need them.’
‘Transformative for patients’
Dr Simon Newman, chief scientific officer at The Brain Tumour Charity, said the delivery of an accurate diagnosis within hours of surgery would be transformative for patients, ensuring rapid access to the optimal standard of care and removing weeks of uncertainty while waiting for diagnosis and prognosis.
‘The potential to combine so many separate tests into one and deliver at a localised level is a game changer for driving equity of access to rapid and accurate molecular diagnosis,’ he said.
‘The BRAIN MATRIX trial, funded by the Brain Tumour Charity, is now exploring how this technology can match patients to personalised clinical trials across the UK.’
