Abstract 7179: Streamlined Glioma diagnosis from formalin-fixed paraffin-embedded (FFPE) tissue: One assay, a fraction of the cost and turnaround time
Authors: Mashiat Lamia Mimosa, Jared T. Simpson, Shreya Patel, Karel Boissinot, Mora Tiab, Ramzi Fattouh, Rola M. Saleeb
Journal: Cancer Research (2025)

Abstract We developed a single diagnostic assay to detect Glioma biomarkers including whole chromosomal and gene copy number variations (CNVs), and single nucleotide variations (SNVs). CNVs and SNVs are traditionally detected using different large/high-throughput platforms that can only exist in big genomic facilities. This leads to very high molecular costs and long turnaround times. The current complex diagnostic algorithm creates delays in patient management while increasing inequity in healthcare as smaller non-centralized labs are unable to conduct glioma molecular testing. To streamline glioma diagnosis and address these challenges, we aimed to use a third-generation sequencing platform called nanopore sequencing. It can simultaneously detect both CNVs and SNVs using small inexpensive tools, but is not yet compatible with formalin-fixed paraffin-embedded (FFPE) DNA. Therefore, we developed a PCR based assay to create clean nanopore compatible FFPE DNA copies to detect CNVs and SNVs. We established a single nucleotide polymorphism (SNP) microarray-based approach to detect the CNVs. Approximately 300 amplicons were included in the assay design including unique SNP-rich areas to detect the chromosomal heterozygosity. We included all glioma markers required by World Health Organization's (WHO) guidelines including chromosomal CNVs on chr 1, 7, 10, 19; gene CNVs on CDKN2A, CDKN2B, EGFR; and SNVs on IDH1, IDH2, TERT promoter, TP53, ATRX, H3-3A and H3C2. We optimized the testing conditions, and reduced cost and turn-around including streamlining the data analysis using a custom shell script. Lastly, we conducted a mini validation as well as a cost and turn-around time analysis using 40 Glioma FFPE samples. All our samples had a concordant molecular classification status with the reference results. The concordance, sensitivity and specificity (total accuracy) of the assay were all at 100%. A loss status of chr 1p, chr 19q and chr 10 were determined via a loss of heterozygosity (LOH), and gain status of chr 7 was observed as an allele gain 2:1 SNP pattern. Our total turnaround time (from DNA extraction to data analysis) is 3 business days for an entire batch of samples, which is at least 1/3rd the turnaround time of conventional methods. Our material cost is $150 CAD/sample (including DNA extraction, library preparation and sequencing) which is ∼10% of the existing assay costs. This is the first single streamlined diagnostic test to detect CNVs and SNVs in Gliomas using FFPE DNA. Our assay will increase healthcare equity by allowing smaller labs to adopt molecular testing due to its low assay/capital cost, shorter testing time, and streamlined workflow, helping to overcome many of the existing cancer diagnostic challenges. Citation Format: Mashiat Lamia Mimosa, Jared T. Simpson, Shreya Patel, Karel Boissinot, Mora Tiab, Ramzi Fattouh, Rola M. Saleeb. Streamlined Glioma diagnosis from formalin-fixed paraffin-embedded (FFPE) tissue: One assay, a fraction of the cost and turnaround time [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7179.