Research ArticleCancer

Sex differences in GBM revealed by analysis of patient imaging, transcriptome, and survival data

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Science Translational Medicine  02 Jan 2019:
Vol. 11, Issue 473, eaao5253
DOI: 10.1126/scitranslmed.aao5253
  • Fig. 1 Sex differences in MRI-based metrics of therapeutic responses and their correlation with survival.

    (A) Tumor growth velocities calculated from serial T1 gadolinium (Gd)–enhanced MR images exhibit progressive decline for female (n = 23) but not for male (n = 40) patients with GBM. (B) Velocity of tumor growth (low velocity, green line; high velocity, purple line) over the first TMZ imaging interval (1 to 3, 28-day cycles of TMZ) stratifies female survival (P = 0.00817, log rank) but not male survival (P = 0.263). (C) Histograms of pretreatment D and rho values in all available MRI cases (independent 53 and 318 GBM case series) for male (n = 227) and female (n = 144) patients. (D) Pretreatment D significantly stratifies survival among females (n = 144; P = 0.0071, log rank) and not among males (n = 227, P = 0.61). (E) High pretreatment rho is associated with worse survival outcomes for both females (n = 144; P = 0.032, log rank) and males (n = 227, P = 0.0037).

  • Fig. 2 Heat maps of joint and sex-specific expression components of TCGA GBM transcriptome data revealed by JIVE.

    The heat maps visualize each expression component. Each row represents a gene, and each column a patient sample. For each patient, there are two color codes presented above the heat map. These identify their assignment to sex-specific clusters and to TCGA molecular subtypes (gray indicates unassigned samples). Samples were ordered by sex-specific clusters. The original female (A) and male (B) expression data were decomposed into the shared expression component common to both sexes (“Joint”) and the expression component individual to each sex (“Female specific” and “Male specific”) and residuals as indicated. The female-relevant heat maps (A) show 283 signature genes that define the five female-specific clusters, and the male-relevant heat maps (B) show 293 signature genes that define the five male-specific clusters. (C) The Venn diagram of male and female signature genes indicates that 116 genes are in common.

  • Fig. 3 Sex-specific survival effects of IDH mutation.

    (A) OS benefit of fc3 and mc5 is demonstrated in the combined TCGA, GSE13041, GSE16011, and REMBRANDT datasets. See table S3 for P values and hazard ratios (HRs). (B) OS for IDH1 WT cases indicates that both fc3 and mc5 exert effects on survival in the absence of IDH1 mutation. (C) OS in IDH1 mutant cases indicates that male-specific clusters are still associated with an effect on survival. The numbers of female IDH1 mutant cases not assigned to fc3 are n = 3, 2, 3, and 6 in fc1, fc2, fc4, and fc5, respectively, using TCGA and GSE16011 samples in combination (see table S7). (D) IDH1 mutation confers a similar survival benefit in males and females with GBM. (E) The survival benefit of fc3 is independent of IDH1 status. In contrast, IDH1 status exerts a significant effect on survival in mc5 cases. P = 4.3 × 10−4 for the comparison between mc5 cases with and without IDH1 mutation. Overall log rank test P value is shown comparing across all the groups presented in each panel (table S8 shows the P values and HRs for all pairwise comparisons).

  • Fig. 4 DFS of sex-specific clusters in TCGA GBM dataset and OS of sex-specific clusters in three independent datasets combined.

    (A) DFS in TCGA-derived female clusters (fc1–5). (B) DFS in TCGA-derived male clusters (mc1–5). (C) DFS in TCGA-derived female clusters (fc1–5) in which IDH1 mutant cases are plotted as an independent cluster. (D) DFS in TCGA-derived male clusters (mc1–5) in which IDH1 mutant cases are plotted as an independent cluster. Independent samples combining the GSE13041, GSE16011, and REMBRANDT datasets were assigned to sex-specific clusters, and the superiority of OS of fc3 (E) and mc5 (F) was validated in the independent samples. Overall log rank test P value is shown comparing across all the groups presented in each panel (see tables S3 and S4 for the P values and HRs for all pairwise comparisons).

  • Fig. 5 Analysis of genes and pathways that mediate better survival.

    (A) In the combined dataset, the survival of females assigned to fc3 (median survival, 1172 days) was compared with the survival of males assigned to mc5 (median survival, 620 days). (B to D) Genes that distinguished fc3 and mc5 from other female and male clusters, respectively, were compared (see table S2). Pathways in all analyses were prioritized by the combination of the numbers of genes from the pathway involved and the corrected P value for the relevance of the pathway. (B) Calcium/calmodulin signaling was the most significantly involved shared pathway between fc3 and mc5 (adjusted P < 0.001). (C) The integrin signaling pathway was the most significant female-specific pathway (adjusted P < 0.001; table S9). Genes that were up- and down-regulated in fc3 compared with the other female clusters are in red and blue boxes, respectively. (D) Cell cycle regulation was the most significant male-specific pathway (adjusted P < 0.001; table S9). Genes that were up- and down-regulated in mc5 compared with the other male clusters are in red and blue boxes, respectively. See table S2 for the complete gene lists and statistics for each analysis.

  • Fig. 6 mc5-defining genes and OS in the merged TCGA, GSE16011, and GSE13041 datasets.

    (A) Density plots for sex-specific expression of male (in blue) and female (in red) GBM specimens of three mc5-defining genes (BIRC5, KIF20A, and CCNB2). The overlay in male and female plots indicates near-identical expression in the populations. (B) Expression of each gene by sex and sex-specific clusters is presented as boxplots. (C) High and low expression groups for each gene were defined relative to the level of expression that distinguished mc5 from the other male clusters (see the “Overall sex-specific survival effects” section in the Supplementary Materials). The survival effects of differences in expression were determined for males and females. Each gene exerted a greater effect on survival in males compared with females. P values from the Cox regression model are labeled in red for comparisons between survival curves of female patients with GBM with low versus high expression of each and labeled in blue for the same survival analysis of male patients with GBM. The P value labeled in green refers to the interaction of sex and expression of a gene in the Cox regression models. Parallel analyses of the fc3-defining genes and the other mc5-defining genes are presented in figs. S9 and S10, respectively.

  • Fig. 7 Expression of cluster-defining genes and response to common chemotherapeutics in vitro.

    (A) Absolute IC50 values for TMZ, etoposide, CCNU, and VCR for five male and four female patient-derived GBM cell lines were calculated from six-point dose-response curves for each cell line. Boxplots of IC50 across cell lines by sex are presented (horizontal bar indicates median). Median male and female IC50 values were not significantly different based on two-sample t test. Spearman correlation coefficients of IC50 values for each drug with expression of mc5.17 genes (B), fc3.9 genes (C), or random gene sets are shown in male and female cell lines. For mc5.17 and fc3.9 genes, box plots represent the distribution of the 17 or 9 cluster-defining genes, respectively, and for random gene sets, the box plots represent the distribution of the Olkin-averaged Spearman correlation coefficient (54) of 17 or 9 randomly selected genes per random gene set for 1000 random gene sets. *P < 0.01 compared with random gene sets for each sex. (D) Quantification of the percentage of phospho-histone H3 (pHH3)–positive nuclei in male GBM cells implanted in male (black bars) or female (white bars) nude mice. Tumor-bearing mice were treated with vehicle [dimethyl sulfoxide (DMSO)], TMZ (21 mg/kg per day × 5 doses), or etoposide (20 mg/kg every other day × 3 doses), and pHH3 positivity was determined in a blinded fashion.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/473/eaao5253/DC1

    Detailed Materials and Methods

    Fig. S1. Sample timeline for MRI analysis of patient data.

    Fig. S2. Relation between RANO criteria, first tumor growth velocity, and survival.

    Fig. S3. Validation of sex differences in correlations between presurgical D and rho and survival.

    Fig. S4. Geometric illustration of JIVE decomposition.

    Fig. S5. Joint structure of male and female GBM samples relative to TCGA subtype signatures.

    Fig. S6. Expression variation in TCGA data explained by the JIVE components.

    Fig. S7. Identification of the sex-specific clusters by consensus clustering for female and male GBMs.

    Fig. S8. Sex-specific cluster survival benefits in merged TCGA, GSE16011, and GSE13041 datasets.

    Fig. S9. fc3-defining genes and survival.

    Fig. S10. mc5-defining genes and survival.

    Fig. S11. TMZ, etoposide, VCR, and CCNU dose-response curves for five male and four female primary GBM cell lines.

    Fig. S12. Western blot analysis of MGMT expression in female and male patient-derived GBM cell lines.

    Fig. S13. Distribution of the coefficients of variation in the TCGA data.

    Fig. S14. Analysis flowchart.

    Table S1. Sex and TCGA subtype assignments in the four datasets.

    Table S2. Gene expression signatures for sex-specific clusters.

    Table S3. Median survival time, P values, and HRs associated with survival differences in sex-specific clusters.

    Table S4. Survival by IDH1 status—Female IDH1 WT.

    Table S5. Survival by IDH1 status—Male IDH1 WT.

    Table S6. Survival by IDH1 status—Male IDH1 mutant.

    Table S7. Survival by IDH1 status—Female IDH1 mutant.

    Table S8. Survival analysis of sex—IDH1 interaction.

    Table S9. Pathway analysis of fc3 and mc5.

    Table S10. Statistical analysis of the interaction between sex and chemotherapy treatment in vivo.

    Table S11. Patient-specific proliferation, invasion, hypoxia, necrosis, angiogenesis (PINHA) values.

    Table S12. Parameters for calculation of tumor growth velocities.

  • The PDF file includes:

    • Detailed Materials and Methods
    • Fig. S1. Sample timeline for MRI analysis of patient data.
    • Fig. S2. Relation between RANO criteria, first tumor growth velocity, and survival.
    • Fig. S3. Validation of sex differences in correlations between presurgical D and rho and survival.
    • Fig. S4. Geometric illustration of JIVE decomposition.
    • Fig. S5. Joint structure of male and female GBM samples relative to TCGA subtype signatures.
    • Fig. S6. Expression variation in TCGA data explained by the JIVE components.
    • Fig. S7. Identification of the sex-specific clusters by consensus clustering for female and male GBMs.
    • Fig. S8. Sex-specific cluster survival benefits in merged TCGA, GSE16011, and GSE13041 datasets.
    • Fig. S9. fc3-defining genes and survival.
    • Fig. S10. mc5-defining genes and survival.
    • Fig. S11. TMZ, etoposide, VCR, and CCNU dose-response curves for five male and four female primary GBM cell lines.
    • Fig. S12. Western blot analysis of MGMT expression in female and male patient-derived GBM cell lines.
    • Fig. S13. Distribution of the coefficients of variation in the TCGA data.
    • Fig. S14. Analysis flowchart.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Sex and TCGA subtype assignments in the four datasets.
    • Table S2 (Microsoft Excel format). Gene expression signatures for sex-specific clusters.
    • Table S3 (Microsoft Excel format). Median survival time, P values, and HRs associated with survival differences in sex-specific clusters.
    • Table S4 (Microsoft Excel format). Survival by IDH1 status—Female IDH1 WT.
    • Table S5 (Microsoft Excel format). Survival by IDH1 status—Male IDH1 WT.
    • Table S6 (Microsoft Excel format). Survival by IDH1 status—Male IDH1 mutant.
    • Table S7 (Microsoft Excel format). Survival by IDH1 status—Female IDH1 mutant.
    • Table S8 (Microsoft Excel format). Survival analysis of sex—IDH1 interaction.
    • Table S9 (Microsoft Excel format). Pathway analysis of fc3 and mc5.
    • Table S10 (Microsoft Excel format). Statistical analysis of the interaction between sex and chemotherapy treatment in vivo.
    • Table S11 (Microsoft Excel format). Patient-specific proliferation, invasion, hypoxia, necrosis, angiogenesis (PINHA) values.
    • Table S12 (Microsoft Excel format). Parameters for calculation of tumor growth velocities.

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