Diffuse glioma samples
One hundred and twenty-four formalin-fixed paraffin-embedded (FFPE) tumor tissue samples were selected from the Central Nervous System Tumor Tissue Bank at Kagoshima University Hospital. The study was approved by the Institutional Review Board of Kagoshima University (approval number: 180104) and complied with the tenets of the Declaration of Helsinki. Informed consent was obtained from all patients. Resected tumors were fixed with phosphate-buffered 10% formalin within 24 h of sampling and routinely processed for paraffin embedding, followed by sectioning for hematoxylin and eosin staining. All tumors were originally classified according to the World Health Organization classification of 2016. The tumor series consisted of 10 grade III IDH-wildtype anaplastic astrocytomas (AAs) and 114 grade IV IDH-wildtype GBMs. All tissues were histologically evaluated by board-certified pathologists (M.K. and A.T.) to ensure an estimated tumor cell content of 30% or more. In all patients, when analyzing copy number variations, we sequenced leukocyte DNA for comparison against matched tumor DNA.
DNA extraction and quantification
For DNA preparation from FFPE samples, we used the Maxwell 16 FFPE Tissue LEV DNA Purification kit (Promega) according to the manufacturer's instructions. Thereafter, the concentration of DNA was measured using a Qubit 3.0 Fluorometer dsDNA BR Assay kit (Life Technologies), and DNA quality was monitored using the QIAseq DNA QuantiMIZE kit (QIAGEN). The extracted DNA was diluted to a concentration of 5–10 ng/μL as a template, and polymerase chain reaction (PCR) was performed using the QIAseq DNA QuantiMIZE kit.
Next-generation sequencing
NGS was performed using an amplicon-based glioma-tailored gene panel as described previously [15]. Amplicon sequences were aligned to the human reference genome GRCh37 (hg19) in the target region of the sequence. Data were analyzed using the QIAGEN Web Portal service (https://www.qiagen.com/).
Detection of EGFR variants
In all patients, to analyze rearrangement events as significant targets of intragenic copy number breakpoints, we sequenced leukocyte DNA for comparison against matched tumor DNA. The EGFR variants were detected by reduced read counts obtained for EGFR sequences in tumor DNA relative to those in matched leukocyte DNA. In addition, as a validation method, the EGFR variants were detected by determining the copy number (CN) of each exon in EGFR and identifying exon sites with -2SD below in CN compared with the average CN of all exons.
Complementary DNA analyses of clinical specimens
The total RNA from FFPE samples of clinical specimens was extracted using the Maxwell 16 LEV RNA FFPE Purification kit (Promega) and converted into cDNA using the ReverTra Ace qPCR RT Kit & Master Mix (TOYOBO Inc., Osaka, Japan). The cDNA fragments around the targeted site were amplified by PCR using the KOD One PCR Master Mix (TOYOBO Inc.) using the primers listed in Supplementary Table S1. The PCR products were analyzed by electrophoresis on 2% agarose gels. The PCR fragments were purified using the Exo-CIP Rapid PCR Cleanup kit (New England Biolabs Inc., Ipswich, MA) and analyzed by GENEWIZ Japan Corp.
Immunohistochemistry
Surgical specimens were fixed within 10 min of excision in 10% neutral buffered formaldehyde for 24 h, embedded in paraffin, cut into 3-μm-thick sections, and mounted on glass slides coated with poly-l-lysine. Subsequently, the sections were probed with 1:25 anti-EGFR (mouse monoclonal antibody, clone EGFR.113; Leica Biosystems) and 1:200 anti-EGFRvⅢ (rabbit monoclonal antibody, cat#64952; Cell Signaling Technology). The sections were then stained with diaminobenzidine tetrahydrochloride and hematoxylin.
TCGA and MSKCC data analyses
We retrieved the molecular characteristics of the GBM cohort from previous publications. After excluding H3F3A, IDH1/2, and BRAF V600E-mutant cases, we analyzed 343 cases from The Cancer Genome Atlas (TCGA) cohort and 456 cases from the Memorial Sloan Kettering Cancer Center (MSKCC) cohort [16]. All cases were conclusively diagnosed as IDH-wildtype GBM using the cBioPortal for Cancer Genomics (https://cbioportal.org).
Definition of actionable alterations
Actionable gene alterations were predicted to confer sensitivity to either an approved targeted agent or an experimental targeted agent currently under clinical trials. The JAX Clinical Knowledgebase (JAX-CKB) (https://ckb.jax.org/) was used to identify clinically actionable variants.
Data analysis
We used OncoPrinter (cbioportal.org/oncoprinter) and MutationMapper (cbioportal.org/mutation_mapper), which are tools in the cBioPortal for Cancer Genomics, to visualize and analyze our data [17,18]. Statistical analyses were performed using JMP Pro v13 software (SAS Institute, Cary, NC). Groups were compared using the χ2 test. Differences were considered significant at p < 0.05.