Introduction

The recently published 2021 World Health Organization (WHO) classification, followed by the previous 2016 WHO classification, consistently emphasizes the role of molecular markers in the classification of adult-type diffuse gliomas, namely isocitrate dehydrogenase (IDH) mutation and 1p/19q codeletion status [1]. In the 2021 classification, there are three types of adult-type diffuse gliomas: oligodendroglioma, IDH-mutant, and 1p/19q-codeleted, astrocytoma, IDH-mutant, and glioblastoma, IDH-wildtype [1]. WHO grade 2 and 3 IDH-wildtype diffuse gliomas which lack testing of molecular markers sufficient for molecular glioblastoma are designated as IDH-wildtype diffuse gliomas, not otherwise specified (NOS) [2, 3]. This new classification emphasizes different prognosis within each type rather than comparing prognosis among different types within each WHO grade in the previous 2016 WHO classification.

Patient sex is recognized as a biologically relevant factor not only for cancer incidence but also for survival [4]. Sex differences have been consistently recognized by epidemiological studies in patients with gliomas [5, 6]. Majority of studies focused on incidence of glioma, reporting a higher incidence of glioma in males [7, 8, 9, –11]. However, less information on sex differences in survival of patients with glioma is available [6, 12]. While several large population-based studies have shown female survival advantage in glioblastoma [6, 13, 14, –16], these datasets lacked important molecular markers such as IDH mutation or O6-methylguanine-methyltransferase (MGMT) promoter methylation status. While both IDH mutation and MGMT promoter methylation status are important prognostic markers in glioblastoma [17, 18, 19, 20, –22], whether there is an independent effect of sex over well-acknowledged prognostic molecular markers is unknown. Moreover, survival difference according to sex among types of adult-type diffuse glioma other than glioblastoma is yet to be known; a recent study showed that non-glioblastoma patients revealed no sex difference in survival, but this study lacked type-specific analysis according to the molecular markers in the 2021 WHO classification [16].

Exploring sex differences in survival is important as this can be incorporated in implementing personalized treatment strategies and designing clinical trials. In addition, type-specific effects of sex in survival is also relevant as personalized treatment strategies can be tailored according to types of adult-type diffuse gliomas. Current overrepresentation of male in the clinical trials of adult-type diffuse gliomas may obscure key elements of sexual dimorphism in survival [23]. Hence, an integrated clinical and molecular analysis is warranted to elucidate type-specific sex differences in survival in adult-type diffuse gliomas.

Therefore, this study aimed to investigate whether type-specific sex differences in survival exist independently of clinical and molecular factors in adult-type diffuse gliomas according to the 2021 WHO classification.

Methods

Patient enrollment

Between January 2005 and October 2021, 1,458 patients with adult-type diffuse glioma from our institution were recruited. The inclusion criteria were as follows: (a) gliomas confirmed by histopathology, (b) known IDH mutation and 1p/19q codeletion status, and (c) age over 18 years. The exclusion criteria were as follows: (a) follow-up loss within 3 months excluding death (n = 92), (b) presence of H3 K27 alteration leading to the diagnosis of diffuse midline glioma, H3 K27-altered (n = 36), and (c) insufficient tissue for molecular diagnosis (n = 5). A total of 1325 patients were included in this study. Figure 1 shows the patient inclusion process.

Fig. 1
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Flowchart for patient selection

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Molecular classification

Diagnoses were made according to the WHO classification [24]. Immunohistochemical analysis was performed to detect IDH1 R132H mutation, and IDH1/2 status was confirmed by peptide nucleic acid-mediated clamping polymerase chain reaction in IDH1-negative patients on immunohistochemical analysis. Fluorescent in situ hybridization analysis was conducted to detect 1p/19q codeletion. The H3 K27 mutant protein was detected by immunohistochemistry analysis using polyclonal antibodies for the histone H3.3 tail. MGMT promoter methylation status was determined by methylation-specific polymerase chain reaction in all patients (n = 1325, 100%).

Targeted next-generation sequencing using the Illumina TruSight Tumor 170 panel was performed in 844 patients (63.7%) [25, 26]. Epidermal growth factor receptor (EGFR) amplification was considered when genes were with ≥ twofold-change relative to the average level. For telomerase reverse transcriptase promoter (TERTp), C228T and C250T mutations were evaluated using a pyrosequencing assay [27]. Combined gain of entire chromosome 7 and loss of entire chromosome 10 (chromosome + 7/− 10) was also analyzed. EGFR amplification, TERTp mutation, and + 7/− 10 chromosome copy number status were available in 565 (42.6%), 844 (63.7%), and 417 patients (31.5%), respectively. WHO grade 2 and 3 IDH-wildtype diffuse gliomas lacking testing of these molecular markers sufficient for molecular glioblastoma were assigned as IDH-wildtype diffuse gliomas, NOS [2, 3].

MRI protocol

Brain MRI scans, including T1-weighted image, T2-weighed image, pre-and postcontrast fluid-attenuated inversion recovery (FLAIR), and postcontrast 3D T1-weighted images were acquired with a 3T unit (Achieva or Ingenia; Philips Healthcare, Best, Netherlands).

Data collection

Data including age at initial diagnosis, sex, histological grade (grade 2, 3, or 4 based on histological features), molecular markers, treatment (such as radiation, temozolomide, or PCV [procarbazine, lomustine, and vincristine] therapy), date of death or last follow-up were collected. All patients underwent preoperative MRI examination for initial evaluation and postoperative MRI examination performed within 48 h of operation. The tumor location of geographic epicenter on the preoperative MRI was determined with the largest component of either contrast-enhancing or non-contrast-enhancing tumor. Lobar location included frontal lobe, temporal lobe, parietal lobe, occipital lobe, and insula, while nonlobar location included basal ganglia, thalamus, brainstem, cerebellum and corpus callosum [28, 29]. The extent of resection based on pre- and postoperative MRI (gross total resection, subtotal [tumor removal of ≥ 75% but < 100%], partial [tumor removal of < 75%] or biopsy) was determined by independent review of two neuroradiologists (M.K. and Y.W.P., with 8 and 11 years of experience, respectively). In the rare case of ambiguity, a senior neuroradiologist (S.S.A, with 18 years of experience) was consulted for the final decision.

Overall survival (OS) was defined as the time from the day of initial diagnosis of glioma until death or last follow-up.

Statistical analysis

The clinical and imaging characteristics of patients were compared according to types of adult-type diffuse gliomas (namely oligodendroglioma, IDH-mutant, and 1p/19q-codeleted, astrocytoma, IDH-mutant, glioblastoma, IDH-wildtype, and IDH-wildtype diffuse glioma, NOS), using the Chi-square for categorical variables and independent samples t-test or Mann–Whitney U test for continuous variables according to normality. The clinical and imaging characteristics were compared according to sex within each type of adult-type diffuse gliomas.

Cox regression analysis was performed to determine predictors of OS. Variables of interest in the univariable analysis were included in the multivariable models using backward elimination according to the likelihood ratio with a variable selection criterion of P < 0.05. The proportional hazards assumption was met in all models except for glioblastoma, IDH-wildtype, particularly for MGMT promoter methylation status (P = 0.047). However, as there was no significant interaction of MGMT promoter methylation status with time (P = 0.288), Cox analyses was pursued. As all glioblastoma, IDH-wildtype patients were included after 2005 and underwent Stupp protocol [5], treatment was not included in the Cox regression analysis in this type. Survival rates were determined using the unadjusted and adjusted Kaplan–Meier method, and curves were compared using the log-rank test. In glioblastoma, IDH-wildtype patients, an interaction between female sex and MGMT promoter methylation status was evaluated to assess whether the effect of female sex on survival was dependent of MGMT promoter methylation status. In addition, a likelihood ratio test was used in the context of Cox models to assess the contribution of sex in predicting survival beyond that provided by other predictors in the multivariable Cox model including MTMT promoter methylation status. The variance inflation factor was used to detect multicollinearity between variables; all variables included in the multivariable model showed a variance inflation factor < 10. Statistical analysis was performed using R statistical software (R version 4.0.2, R Core Team, Vienna, Austria). Statistical significance was set at P < 0.05. A biostatistician (with 15 years of experience) was consulted for statistical analysis.

Results

Patient characteristics

This study included 1325 patients with adult-type diffuse glioma (mean age ± standard deviation [SD], 54.0 ± 15.0 years) consisting of 569 females (42.9%) and 756 males (57.1%) with a median follow-up period of 19.3 months (interquartile range [IQR], 11.0–40.5). There were 183 patients (13.8%) with oligodendroglioma, IDH-mutant, and 1p/19q-codeleted, 211 patients (15.9%) with astrocytoma, IDH-mutant, 800 patients (60.4%) with glioblastoma, IDH-wildtype and 131 patients (9.9%) with IDH-wildtype diffuse glioma, NOS. The median OS was 29.9 months (IQR, 14.2–164.4), and 701 patients (52.9%) expired. Amongst all types of adult-type diffuse gliomas (oligodendroglioma, IDH mutant, 1p/19q-codeleted, astrocytoma, IDH mutant, glioblastoma, IDH-wildtype and IDH-wildtype diffuse glioma, NOS), there were significant differences in the age (P < 0.001), sex (P = 0.038), histological grade (P < 0.001), molecular markers (P < 0.001 for MGMT promoter methylation, EGFR amplification, TERTp mutation, and chromosome + 7/− 10), tumor location (P < 0.001 for frontal and nonlobar location, and P = 0.008 for infratentorial location), treatment (P < 0.001 for gross total resection, radiation therapy, temozolomide, and PCV therapy), and death (P < 0.001). Patients with glioblastoma, IDH-wildtype had the shortest median OS (17.3 months; IQR 10.6–33.0) followed by IDH-wildtype diffuse glioma, NOS (33.0 months; IQR 16.8–59.4), and both astrocytoma, IDH-mutant and oligodendroglioma, IDH mutant, and 1p/19q-codeleted did not reach median OS within the study period (P < 0.001). The clinical characteristics of the study patients are presented in Table 1. The Kaplan–Meier curves of different types of adult-type diffuse glioma is presented in Supplementary Fig. 1.

Table 1 Characteristics of adult-type diffuse glioma patients
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Difference of patient characteristics according to sex in each type

The clinical characteristics according to sex in each type of adult-type diffuse gliomas are presented in Supplementary Table 1. In glioblastoma, IDH-wildtype, female sex showed significantly higher prevalence of MGMT promoter methylation (40.2% vs 32.0%, P = 0.017) and nonlobar location (27.9% vs 21.3%, P = 0.030) than male sex. No significant differences were observed in other characteristics in glioblastoma, IDH-wildtype. In astrocytoma, IDH-mutant, female sex showed a significantly higher prevalence of MGMT promoter methylation (82.8% vs 70.2%, P = 0.037). In other types of adult-type diffuse gliomas, there were no significant differences in characteristics according to sex.

Overall survival in patients with glioblastoma, IDH-wildtype according to sex

In univariable analysis, female sex was significantly associated with better OS (hazard ratio [HR] = 0.81, 95% confidence interval [CI] = 0.68–0.96; P = 0.014). Age, histological grade 4, MGMT promoter methylation status, nonlobar tumor location, and gross total resection were also identified to be significant predictors of OS. In multivariable analysis, female sex remained as an independent predictor of OS (HR 0.91, 95% CI 0.83–0.99; P = 0.031), along with age, histological grade 4, MGMT promoter methylation status, and gross total resection. The interaction between female sex and MGMT promoter methylation status was not significant (P-interaction = 0.194) indicating that female sex impacts survival independently of MGMT methylation status. In addition, the likelihood ratio test showed additional effect of sex in model fitness (P = 0.015). Results of univariable and multivariable analyses for determining the predictor of OS in patients with glioblastoma, IDH-wildtype are summarized in Table 2. The unadjusted and adjusted Kaplan–Meier curves showed a significant difference in OS by sex (log-rank test, P = 0.009 and P = 0.031 respectively) (Fig. 2). The median OS for females were 19.2 months (IQR, 12.3–35.0) and 16.2 months (IQR, 10.5–30.6) for males.

Table 2 Univariable and multivariable cox regression analysis to determine predictors of overall survival in patients with glioblastoma, IDH-wildtype
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Fig. 2
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(a) Unadjusted and (b) adjusted Kaplan–Meier curves according to sex in glioblastoma, IDH-wildtype patients

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Overall survival in patients with astrocytoma, IDH-mutant according to sex

In univariable analysis, there was no statistical difference in OS according to sex (HR 0.92; 95% CI 0.49–1.70; P = 0.781). In univariable analysis, nonlobar tumor location, gross total resection, and temozolomide therapy were identified to be significant predictors of OS. In multivariable analysis, gross total resection and temozolomide therapy were identified to be significant predictors of OS. Results of univariable and multivariable analysis for determining predictors of overall survival in patients with astrocytoma, IDH mutant are summarized in Supplementary Table 2. The Kaplan–Meier curves did not show a significant difference in OS according to sex (log-rank test, P = 0.781) in astrocytoma, IDH-mutant (Supplementary Fig. 2).

Overall survival in patients with oligodendroglioma, IDH-mutant, and 1p/19q-codeleted according to sex

In univariable analysis, there was no statistical difference in OS according to sex (HR 0.68, 95% CI 0.24–1.82; P = 0.442). In univariable analysis, age and histological grade 3 were identified to be significant predictors of OS. In multivariable analysis, age and histological grade 3 continued to be significant predictors of OS. Results of univariable and multivariable analysis for determining predictors of overall survival in patients with oligodendroglioma, IDH mutant, and 1p/19q-codeleted are summarized in Supplementary Table 3. The Kaplan–Meier curves did not show a significant difference in OS according to sex (log-rank test, P = 0.439) in oligodendroglioma, IDH mutant, and 1p/19q-codeleted (Supplementary Fig. 2).

Overall survival in patients with IDH-wildtype diffuse glioma, NOS according to sex

In univariable analysis, there was no statistical difference in OS according to sex (HR 1.03, 95% CI 0.67–1.58; P = 0.904). In univariable analysis, age, histological grade 3, and gross total resection were identified to be significant predictors of OS. In multivariable analysis, age, histological grade 3, and gross total resection continued to be significant predictors of OS. Results of univariable and multivariable analysis for determining predictors of OS in patients with IDH-wildtype diffuse glioma, NOS are summarized in Supplementary Table 4. The Kaplan–Meier curves did not show a significant difference in OS according to sex (log-rank test, P = 0.904) in IDH-wildtype diffuse glioma, NOS (Supplementary Fig. 2).

Discussion

Sex as a prognostic factor in adult-type diffuse gliomas may deserve more attention and systematic investigation in the molecular era. In this study, we undertook an integrated clinical and molecular analysis to investigate whether type-specific sex differences in survival exist independently of clinical and molecular factors in adult-type diffuse gliomas according to the 2021 WHO classification. In glioblastoma, IDH-wildtype, female sex remained as an independent prognostic factor even after adjusting for MGMT promoter methylation status, suggesting that significant sexual dimorphism in survival exists. Sex disparity in survival was not observed in other types of adult-type diffuse gliomas, indicating that there are type-specific sex differences in survival in adult-type diffuse gliomas. Therefore, sex may be considered as a relevant factor in designing clinical trials and planning treatment strategies to avoid over- or under-treatment of glioblastoma, IDH-wildtype, but not in other types of adult-type diffuse gliomas.

Previous studies that had reported female survival advantage in patients with glioblastoma were performed prior to the 2021 WHO classification and included patients with both IDH-wildtype and IDH-mutant glioblastomas.[6, 13, 14, –16] The previous so-called IDH-mutant glioblastoma is now classified as astrocytoma, IDH-mutant, grade 4, which is a completely different type from glioblastoma, IDH-wildtype in the new classification, albeit representing a small proportion of previous glioblastoma. Also, the effect of MGMT promoter methylation status, which is a crucial prognostic marker in glioblastoma, was not taken into consideration in the previous studies proposing sex disparity in survival [30, 31].

In this study, we were able to demonstrate that female survival advantage in glioblastoma, IDH-wildtype was independent of MGMT promotor methylation status. There was a higher proportion of MGMT methylation status in females in glioblastoma, IDH wildtype which was in accordance with the results of previous studies in glioblastoma [31, 32], but sex remained as a significant prognostic factor with no interaction between female sex and MGMT promoter methylation status. Our study showed overall slightly lower level of MGMT promoter methylation as only IDH-wildtypes were included while astrocytoma, IDH-mutant, grade 4 were included as glioblastomas in the previous studies [3, 31, 32]. Interestingly, MGMT promoter methylation does not seem to occur uniformly in sex-bound fashion in all cancer types. A meta-analysis of the role of MGMT promoter methylation in non-small cell lung cancer showed no correlation with sex [33]. This suggests that the higher proportion of MGMT promoter methylation and better response to alkylating treatment seen in females in glioblastoma, IDH-wildtype is a tumor-specific phenomenon.

While female survival advantage in cancer has been attributed to a variety of environmental, genetic, immunologic, and hormonal factors [34, 35], sex differences in survival was only observed in glioblastoma, IDH-wildtype, and not in other types of adult-type diffuse gliomas. IDH-wildtype and IDH-mutant gliomas are thought to be distinct diseases with different pathogenesis and genetic profiles. IDH mutation with or without 1p/19q codeletion occurs in the early stage of gliomagenesis of IDH-mutant gliomas, and are thought to be the driver mutation [36]. On the other hand, IDH-wildtype gliomas undergo completely different driver events with a cascade of core signaling pathways [37]. It may be postulated that sex modulates specific tumorigenic pathways that bear prognostic implication in glioblastoma, IDH-wildtype. However, to fully uncover hidden elements of pathophysiology in the type-specific sex effects in survival, an integrated multilevel and transdisciplinary research approach, involving molecular cell biology, preclinical and clinical studies, needs to be undertaken [38, 39].

In our study, IDH-wildtype diffuse gliomas, NOS, did not show female survival advantage, which may be explained by the heterogeneous nature of this group. IDH-wildtype, NOS encompasses WHO grade 2 and 3 astrocytic tumors without the results of the relevant molecular markers in the 2021 WHO classification. Previous studies reported the incidence of molecular GBM with a wide range of 44.0–81.6% within IDH-wildtype grade 2 or 3 patients [40, 41, 42, 43], while the remainders in this group may be classified to diverse types such as diffuse astrocytoma, MYB- or MYBL1-altered, diffuse low-grade glioma, MAPK pathway-altered, or diffuse hemispheric glioma, H3 G34-mutant [3]. This heterogeneity probably accounts for female sex not exerting prognostic effect in IDH-wildtype diffuse gliomas, NOS.

There were several limitations in this study. First, this was a single-center, retrospective study with patients enrolled over a long period. There were many changes in diagnoses and treatment strategies of glioma patients other than glioblastoma, IDH-wildtype. Multicenter analysis is warranted to generalize the findings of our study. Second, Karnofsky performance status was not included in the analysis. However, clinical significance of Karnofsky performance status is limited due to its retrospective and subjective nature [44].

In conclusion, female survival advantage was seen in glioblastoma, IDH-wildtype but not in other types of adult-type diffuse gliomas, suggesting type-specific sex effects solely in glioblastoma, IDH-wildtype. This may have implications for designing personalized treatment strategies in glioblastoma, IDH-wildtype but not necessarily in other types of adult-type diffuse gliomas.