A novel Cas9-targeted long-read assay for simultaneous detection of IDH1/2 mutations and clinically relevant MGMT methylation in fresh biopsies of diffuse glioma

Thidathip Wongsurawat,David W Ussery,Shuk-Mei Ho,Analiz Rodriguez,Yuet-Kin Leung,Annick De Loose,John D Day,Piroon Jenjaroenpun,Intawat Nookaew,Duah Alkam

doi:10.1186/s40478-020-00963-0

Thidathip Wongsurawat, David W Ussery + Show 8 more

Open Access

https://doi.org/10.1186/s40478-020-00963-0

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Abstract

Molecular biomarkers provide both diagnostic and prognostic results for patients with diffuse glioma, the most common primary brain tumor in adults. Here, we used a long-read nanopore-based sequencing technique to simultaneously assess IDH mutation status and MGMT methylation level in 4 human cell lines and 8 fresh human brain tumor biopsies. Currently, these biomarkers are assayed separately, and results can take days to weeks. We demonstrated the use of nanopore Cas9-targeted sequencing (nCATS) to identify IDH1 and IDH2 mutations within 36 h and compared this approach against currently used clinical methods. nCATS was also able to simultaneously provide high-resolution evaluation of MGMT methylation levels not only at the promoter region, as with currently used methods, but also at CpGs across the proximal promoter region, the entirety of exon 1, and a portion of intron 1. We compared the methylation levels of all CpGs to MGMT expression in all cell lines and tumors and observed a positive correlation between intron 1 methylation and MGMT expression. Finally, we identified single nucleotide variants in 3 target loci. This pilot study demonstrates the feasibility of using nCATS as a clinical tool for cancer precision medicine.

Highlights

Diffuse gliomas (DG) comprise 80% of primary malignant central nervous system tumors in adults and traditionally were diagnosed with pathological criteria to define histological type and malignancy grade [1,2,3]
We explore the use of nanopore Cas9-targeted sequencing to accomplish these goals
NCATS allowed for simultaneous evaluation of isocitrate dehydrogenase 1 and 2 (IDH1/2) mutational status and methylguanine-DNA methyltransferase (MGMT) methylation level in a streamlined workflow, resulting in biomarker assessment within 36 h (Fig. 1c)

Summary

Introduction

Diffuse gliomas (DG) comprise 80% of primary malignant central nervous system tumors in adults and traditionally were diagnosed with pathological criteria to define histological type (e.g., astrocytoma, oligodendroglioma, or oligoastrocytoma) and malignancy grade (e.g., grades I-IV) [1,2,3]. In 2016, the World Health Organization (WHO) diagnostic guidelines incorporated molecular markers into the classification of DGs [4, 5]. Many of these diagnostic biomarkers serve as prognostic indicators, and the neuro-oncology community has supported this integration of molecular markers into clinical practice [6]. The use of novel sequencing techniques that can assess multiple biomarkers simultaneously is an attractive option to overcome current clinical practice limitations. In this pilot study, we explore the use of nanopore Cas9-targeted sequencing (nCATS) to accomplish these goals

Methods

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Conclusion