NCM 2021: Nanopore-based copy number variation approach for adult glioma classification — WHO 2021
The next speaker in the panel, Thidathip Wongsurawat introduced how she is researching the potential of nanopore sequencing to perform copy number variation-based classification of adult glioma, in line with WHO 2021 criteria. Thidathip explained that glioma is a type of brain tumour that develops from glial cells; it has high mortality and is most common in those aged 45–65. In 2007, the WHO glioma classification criteria were based entirely on phenotypic data; in 2016, ‘for the first time’, the WHO incorporated genotypic data alongside phenotypic data in glioma classification; and finally, in the updated 2021 criteria, the guidelines included an extended list of genetic markers. Thidathip highlighted a new marker, CDKN2A/B deletion, which, she had been informed, currently has no test for its identification; this deficiency isn’t just found within Thailand, it is a worldwide problem.
Many labs currently perform FISH for tumour classification. Problems with this method include that the pathologists need time to interpret FISH results, and that there is no standard method for CDKN2A/B deletion detection using FISH. Thidathip’s research goal is to develop an approach for identifying CDKN2A/B deletion that is simple to perform, has a quick turnaround time, and, ideally, can produce more information than that provided by FISH; for example, detection of both whole-arm or partial loss of the chromosome. Thidathip hypothesised that nanopore technology could have the potential to achieve these goals, and so to investigate this, Thidathip is comparing results from such a nanopore-based approach against parallel results obtained from currently used testing methods, such as FISH, EPIC arrays, and short-read whole-genome sequencing.
For this research, Thidathip was supplied with 19 samples which were known to have IDH mutations (mutations in IDH1/2 are included in the current typical testing algorithm for adult glioma). Thidathip adapted the ‘SMURF-seq’ protocol (Rishvanth K. Prabakar et al. Genome Biol. 2019), using the RAD004 or RBK004 rapid-chemistry-based kits for library preparation, and the MinION Mk1B and Mk1C devices for sequencing, followed by CNV calling. She shared results showing ‘the power’ of this adapted technique, with detection of 1p deletion, 19q deletion, and CDKN2A/B deletion. Furthermore, ‘even eight minutes after nanopore sequencing, we got the answer’ with this method. This would save both time and money compared to currently used methods (e.g. FISH). There was also high correlation between the CNV results using Thidathip’s method and those from short-read sequencing.
After analysis of all 19 samples, the nanopore-based method showed 100% concordance with current testing methods, and at a lower cost. And ‘the important thing is, because we use MinION… almost any lab worldwide’ can access it. Thidathip also noted some ‘good news’: that they got the same results using the Flongle device. Lastly, she presented a hypothetical working day incorporating this CNV workflow, explaining how, if you started at 9 am, the CNV profile would be reported by 1 pm, even including a 1-hour lunch break!
View Thidathip’s peer-reviewed publication on the simultaneous detection of IDH1/2 mutations and MGMT methylation in glioma research biopsy specimens, using Cas9 targeted sequencing: https://doi.org/10.1186/s40478-020-00963-0