Integrated diagnosis of adult‐type glioma according to 2021 World Health Organization classification: Analysis of 184 cases using a custom‐made next‐generation sequencing panel

To the Editor, Central nervous system (CNS) tumors are classified based on an integrated histological and molecular analyses approach according to 2016 World Health Organization (WHO) classification, updated version of the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT‐NOW), and the new 2021 WHO classification. According to the 2021 WHO classification, IDH‐wildtype diffuse gliomas corresponding to grade II or III diffuse gliomas in the previous classification would be diagnosed as GBM if these tumors harbor more than one of the following molecular features: TERT promoter (TERTp) mutation, EGFR amplification, and concurrent gain of whole chromosome 7 and loss of whole chromosome 10 (ch+7/−10) (molecular GBM). IDH‐mutant diffuse astrocytomas corresponding to grade II or III diffuse IDH‐mutant astrocytomas in 2016 WHO classification would be diagnosed as IDH‐mutant grade 4 astrocytomas if homozygous deletion (HD) of CDKN2A/B is detected. A substantial part of the 2021 WHO classification is extracting molecular glioblastoma (GBM) from IDH‐wild type glioma, and molecularly defining IDH‐mutant astrocytoma grading. Previously, we developed a small glioma‐tailored 48‐gene custom panel to detect 1p/19q codeletions and driver gene mutations such as IDH1/2, TP53, ATRX, TERTp, and CDKN2A/B. Here, we analyzed 184 cases of adult‐type diffuse glioma in an ordinary pathology laboratory equipped with next‐generation sequencing (NGS) and related apparatus at a single institution. Using the NGS panel mentioned above, we successfully detected gene alterations necessary for integrated diagnosis according to the newest classification of adult‐type gliomas. Study specimens were collected from cases registered in the CNS Tumor Tissue Bank, Kagoshima University Hospital from 2016 to 2021. Biopsied or resected tumors were routinely processed for formalin‐fixed and paraffin‐embedded (FFPE) tissue preparation, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC). All tumors were first classified according to the 2016 WHO classification and then the 2021 WHO classification using HE‐ stained sections, IHC for IDH1 (R132H) and H3 K27M, and NGS analysis. DNA extraction from the FFPE tissues, quantification, library preparation, and NGS analyses using a small glioma‐tailored 48‐gene custom NGS panel were performed as previously reported. The study was approved by the Ethics Committees for Clinical and Epidemiologic Research at Kagoshima University (approval no: 180104) and complied with the Helsinki Declaration. Written informed consent was obtained from each participant and from a parent for participants under 20 years of age. Among 245 CNS tumors registered in the CNS Tumor Tissue Bank at Kagoshima University Hospital, 184 were adult‐type diffuse gliomas, which were subjected to NGS analysis, except three cases histologically diagnosed as glioblastoma, IDH‐wildtype, oligodendroglioma, and diffuse astrocytoma, IDH‐mutant with low extracted DNA quality. Among them, 116 were grade IV glioblastomas (115 IDH‐wildtype and 1 IDH‐mutant), comprising the majority of the grade IV diffuse gliomas. Other grade IV diffuse gliomas included gliosarcoma (six cases) and diffuse midline glioma, H3 K27m‐mutant (three cases). Two IDH‐wildtype glioblastoma cases were reclassified as diffuse midline glioma, H3 K27m‐mutant, after NGS analysis and subsequent immunohistochemical analysis for positive H3 K27M expression in both cases. Sixteen cases were IDH‐wildtype diffuse gliomas (seven grade II, nine grade III). Other 16 cases were IDH‐mutant diffuse astrocytomas (10 grade II, six grade III). Twenty‐seven cases were diagnosed as IDH‐mutant oligodendroglial tumors, with 1p/19q codeletion (12 grade II, five grade III).