Prognostic Molecular Biomarker Assessment in Glioma Patients using CRISPR-Cas9-targeted Nanopore Sequencing
Analiz Rodriguez, David Ussery, JD Day, Thidathip Wongsurawat, Piroon Jejaroenpun, Intawat Nookaew
Introduction: Molecular classification of diffuse glioma enables more precise diagnosis, prognosis, and treatment decisions. Currently, combination of two molecular markers, isocitrate dehydrogenase 1 and 2 (IDH1/ IDH2) gene mutation information and O6-Methylguanine-DNA-methyltranferase (MGMT) methylation status, are the main prognostic biomarkers of newly diagnosed diffuse gliomas. Furthermore, an accurate interpretation of MGMT-promoter methylation status is essential to determining which patients benefit from temozolomide (TMZ) therapy.
Objective: The presence of an IDHmutation can be easily tested by PCR or next generation sequencing. However, there remains controversy with the identification of MGMT-promoter methylation status since there exists variable degrees of methylation and no clear consensus on cutoff values for “methylated” or “unmethylated” have been defined. To be best suited for routine clinical setting and research use, the optimal test should be reproducible, readily available, and timely. Therefore, we explored the feasibility of single-molecule nanopore third generation sequencing technology to comprehensively assess both mutation and methylation status simultaneously.
Methods: This technology allows methylation detection directly from the native DNA sequence without requiring bisulfite treatment which reduces processing time. To specifically study IDH1, IDH2,and MGMT-promoter loci, we combined the CRISPR-Cas9 system to cut desired DNA fragments in a non–amplification dependent fashion. In addition, a data analysis pipeline was developed to quantitatively detect methylation.
Results: We applied our approach on human DNA controls, glioma cell lines and 4 patient brain tumor samples and were enabled to assess correct mutation and methylation status of targeted loci within 1.5 days.
Conclusions: CRISPR-cas-9-targeted nanopore sequencing can potentially improve precision medicine and be applied to all cancer types.