We sought to analyse the androgen receptor (AR) in glioblastoma (GBM) due to the location of the AR gene on chromosome X, often reported with shorter survival and higher prevalence of GBM among males. Copy number (CN) and mRNA expression of AR were tested with droplet digital PCR in 91 fresh-frozen GBM samples and 170 formalin-fixed, paraffinembedded samples collected at Link€ oping University Hospital. The freshfrozen cohort was also subjected to pyrosequencing methylation analysis of 17 CpG sites in the AR promoter. Additionally, the gene expression of AR was analysed in the fresh-frozen cohort and The Cancer Genome Atlas (TCGA) cohort of isocitrate dehydrogenase wild-type primary GBM (135 females and 219 males). The association of AR expression and overall survival (OS) was tested with Kaplan-Meier log rank analysis after dichotomisation by maximally selected rank statistics. We found that AR CN alterations were more common in female GBM. AR gene expression correlated with methylation levels of different CpG sites in males and females but there was no difference in expression between sexes. Survival analysis of TCGA cohort revealed the opposite effect of AR overexpression on OS of males and females, with high AR expression correlating with shorter OS in females and longer OS in males. Additional gene set enrichment analysis showed that AR expression correlated with DNA repair response, especially in the male group. In summary, we found that high AR gene expression in GBM exhibits sex-dependent effects on patient survival, which, for males, is linked to DNA repair response.

BACKGROUND The mRNA and protein expression of androgen receptor (AR) are upregulated in glioblastoma (GBM) and studies show that use of AR antagonists leads to apoptosis of GBM cells in vitro and reduction of tumor volume in a mouse model. With increased prevalence of GBM among males, the role and genetic alterations of AR are worth investigating, especially taking into account the location of AR on chromosome X and possible sex differences associated with it. MATERIAL AND METHODS Copy number (CN) and mRNA expression of AR were tested with droplet digital PCR in 106 fresh frozen GBM samples (34 females and 72 males) collected at Linköping University Hospital. This cohort was also subjected to AR promoter methylation analysis, where 17 CpG sites were the target of pyrosequencing. Methylation levels were then correlated with mRNA gene expression, independently for each sex, using Pearson correlation coefficient. Gene expression of AR was also analyzed in The Cancer Genome Atlas cohort of primary IDH wild type GBM (135 females and 219 males) and association of AR expression and overall survival (OS) was tested with Kaplan-Meier log rank analysis after dichotomization by maximally selected rank statistics. RESULTS DNA amplifications, as well as deletions of the AR were detected, with a higher frequency of alterations found in females (26.4% vs. 8.3%). AR gene expression correlated with methylation levels of two CpG sites in females (chrX:67543271, chrX:67543762) and three different CpG sites in males (chrX:67543889, chrX:67543895, chrX:67543899). There was no difference in the AR expression between males and females in neither of the cohorts, but significantly higher AR expression was found in the classical subtype of GBM in TCGA. Survival analysis of the TCGA cohort revealed the opposite effect of AR expression on OS of males and females, with high AR expression correlating with shorter OS in females (13.6 vs.15.7 months, p=0.035) and longer OS in males (16.6 vs. 12.2 months, p=0.04). Additional gene set enrichment analysis of these samples showed that high AR expression was strongly correlated with DNA repair but only in the male group. CONCLUSION Our results show that high AR mRNA expression in GBM exhibits different effects on patients’ survival depending on their sex, despite common occurrence of high AR expression and CN changes in males and females. The reasons for potential protective influence of AR in males remain unclear and require further investigations.