Study design
The analyses of this retrospective study included two steps. The detail of this study design and flowchart of patient selection are provided below and are summarized in Fig. 1. Two cohorts were collected as described below: the Japanese and TCGA cohorts. Briefly, Step1 aimed to investigate the somatic landscape including CNAs in the primary IDH wild-type GBM and compared the somatic landscapes of the two cohorts with each other. In Step2, we investigated the clinical impact of CNA profiles in primary IDH wild-type GBM cases treated with chemoradiotherapy with TMZ after initial surgery using the Japanese and TCGA cohorts [26]. The Japanese cohorts collected for Step1 and 2 analyses were Cohort K1 and K2, respectively. Similarly, TCGA cohorts subjected to each step were Cohort T1 and T2, respectively.
A Japanese GBM cohort of Kansai molecular diagnosis network for CNS tumors (KNBTG)
Japanese cohort was collected from the cases registered in Kansai Molecular Diagnosis Network for CNS tumors (KNBTG): a consortium where neuro-oncologists, neurosurgeons, pediatricians, pathologists, and basic scientists are conducting cooperative researches for malignant brain tumor [25]. The researchers are affiliated to university hospitals, regional medical centers, and research institutes mainly in the Kansai area of Western Japan. This network routinely collects glioma cases registered from the participating institutions. Tumor samples and clinical information of patients treated at the affiliated institutions are collected and registered in this data bank after obtaining written informed consent. The detailed clinical information including preoperative Karnofsky Performance status (KPS), extent of resection, and adjuvant therapy are routinely obtained from each institution. Since this is a population-based study in Japan, subjects are exclusively composed of East Asians and under the universal health care insurance system of Japan.
The initial screening criteria from enrolled gliomas in KNBTG was as follows: local diagnosis of primary GBM and availability of genomic DNA for molecular analysis of the naïve specimens. Primary GBM was clinically regarded as the GBM arising de novo, with no known lower-grade precursor lesion. The inclusion criteria for Cohort K1 was as follows: central review of a histopathological diagnosis of IDH-wild-type GBM based on the 2016 CNS WHO, molecular data available for analysis, and absence of IDH1/2 and H3F3A mutation, and 1p/19q codeletion. Out of 234 primary GBM initially collected from seven institutions participating in this study, 212 cases were enrolled as Cohort K1.
To analyze the prognostic impact of CNAs, cases in which patients underwent radiotherapy (RT) of 50–65 Gy and concurrent TMZ were further selected from Cohort K1. Those treated with either RT alone, TMZ alone, or short-course RT plus TMZ were excluded from any survival analysis. Cases ineligible for treatment criteria and lacking in KPS score were excluded. In addition, those participating in clinical studies for testing experimental treatment add-on to RT plus TMZ (n = 8) were excluded. Finally, 140 cases were available for Step2 analysis as Cohort K2.
Central pathology review of KNBTG cohort
All molecular-pathological diagnosis of IDH-wild-type GBM eligible for this investigation had reached an agreement with an experienced neuropathologist (Y.K.) and was according to the 2016 WHO classification for central nervous system tumors.
Molecular analysis of KNBTG cohort
All tissue specimens were obtained at the time of the initial treatment before chemoradiation between December 2006 and November 2017. Tumor genomic DNA was extracted with NucleoSpin Tissue kit (Macherey-Nagel, Inc., Bethlehem, PA) or DNeasy Blood & Tissue Kit (Qiagen, Tokyo, Japan), according to the manufacturer’s protocol. The same batch of DNA samples was used for each technique as follows.
Mutational status of IDH1/2, TERT promoter, H3F3A, HIST1H3B, and TP53 were determined using the Sanger technique. The details of Sanger sequencing have been previously reported [1] and is additionally provided in the supplementary information (SI). The methylation status of the MGMT promoter was assessed using quantitative methylation specific PCR (qMSP) following the bisulfite modification of tumor genomic DNA. The details of the qMSP protocol for testing MGMT methylation are described in SI.
To assess CNAs in GBM, we performed Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA KIT P105 (version D2) and P088 (version C2), in accordance with the manufacturer’s protocol (MRC Holland, Amsterdam, Netherland) [15]. The P105 kit is designed to detect CNAs typical in gliomas, and includes probes against PDGFRA, EGFR, CDKN2A, PTEN, TP53, CDK4, MDM2, and NFKBIA genes. P088 kit was designed to assess mainly 1p/19q codeletion, and we used this kit only for the IDH-mutated-GBM to exclude oligodendroglial tumors. Based on previous publications, the CNA category was classified by the following thresholds: homozygous deletion (x ≤ 0.4), hemizygous deletion (0.4 < x ≤ 0.7), gain (1.3 ≤ x < 2.0), amplification (x ≥ 2.0) [16]. Any other details of the MLPA protocol and its data analysis to assess CNAs are described in SI.
The cancer genome atlas (TCGA)
An independent and extensive molecular data and clinical information including survival data and treatment of GBM in TCGA (n = 577) were collected from cBioPortal for Cancer Genomics (https://cbioportal.org) [6, 11] and the supplemental data of the previous publication by TCGA [3, 5]. Based on the racial distribution available from cBioPortal (n = 550), whites accounted for a much higher percentage (88.5%, n = 487) than blacks (9.1%, n = 50) and Asians (2.4%, n = 13).
Out of 577 cases initially collected, 359 cases were enrolled as Cohort T1, who were conclusively diagnosed with primary IDH-wild-type GBM. Recurrent, secondary, or IDH-mutant-GBMs along with those that lacked any such information were differentially excluded from this step. For the purpose of survival analysis in Step1, only one case was additionally excluded due to a lack of censoring data.
From Cohort T1, 152 cases receiving TMZ chemoradiation as initial treatment were selected for external validation (Cohort T2) [3]. Those initially treated with either RT alone, TMZ alone, or alkylating chemotherapy other than TMZ, along with those where any such information was unavailable, were excluded. Thus, the inclusion criteria of the cohort from TCGA were similar to that for the Japanese cases in each step.
Molecular and clinical data acquisition from a larger dataset in TCGA
All data regarding CNA in each patient from TCGA were downloaded from the online resource provided in cBioPortal for Cancer Genomics on July 15th, 2018. To identify CNA profiles, we employed putative copy number calls generated by the GISTIC or RAE algorithms in the portal, such as “-2”, “-1”, “0”, “1”, and “2” [6, 11]. These acquired calls were converted to foregoing categories as follows: − 2 to homozygous deletion, − 1 to hemizygous deletion, 0 to neutral, 1 to gain, and 2 to amplification. The mutation status in TP53 was also downloaded from the portal. Further molecular data about MGMT methylation and various clinical information were available from the supplemental data of the previous publication by TCGA [3, 5]. IDH mutational status could be obtained from both data sources and obtainable results were fully cross-validated with each other. There were few data regarding TERT promoter mutation in any of the data sources.
Statistical analysis
All statistical analyses were performed using a JMP Pro version 13 software (SAS Institute, Cary, NC, USA). The difference was considered significant if the p-value was < 0.05.
Patients were subdivided into two groups on the basis of age (≥ 65 or < 65 years), preoperative KPS (≤ 70 or 80–100%), and extent of resection (< 90% or 90–100%) for the purposes of statistical analysis.
Pair associations of molecular variables were evaluated using a two-tailed Fisher’s exact test and computing the log odds ratio (LOR). Concurrent with significant correlation (p < 0.05, Fisher’s exact), LOR below − 2.0 and above 2.0 were respectively defined as association toward mutual exclusivity and co-occurrence. LOR from − 2.0 to − 1.5 and 1.5 to 2.0 were defined to have a tendency for these associations.
For survival analysis, overall survival (OS) was defined as the interval between the initial operative day and the date of either death or the last follow-up date on which the patient was known to be alive, with a cut-off date of 28 February 2018 for the KNBTG cohort. Patients who were still alive at the last follow-up were considered as a censored event. The survival data were analyzed using the log-rank test and Cox regression analyses. In Cox analysis, the hazard ratio (HR) and p-value were computed. Variables with a significant p-value in univariate analysis were subsequently used to build a multivariate Cox model. Stepwise procedure was used for constructing multivariate Cox regression model for survival. After excluding non-significant factor one by one in the multivariate analysis, the remaining variables (p < 0.05) were considered to be independent predictors of survival. Extent of resection and TERT promoter mutation status were not considered in Cox regression model for the TCGA cohort because of a lack of information for all or the majority of patients in this dataset.
