Patient demographic and tumor characteristics
We identified 121 adult GBM cases with available molecular and immunohistochemistry data between 2016 and 2018 (Additional file 2). We excluded histologic GBMs containing IDH R132 and H3F3A mutations from statistical analyses (n = 11 and n = 1, respectively), for the reasons noted above [15].
Within this cohort (n = 109), the average age of patients was 60 years (range 18–84 years). Genetic alterations in the TERT gene were detected in 93 tumors; 92 were TERTp mutant (84.4%), and an additional case had a TERT-SUB fusion. The remaining 16 patients (14.7%) had TERTp-wt GBM (Fig. 1 and Additional file 2). The average age of patients with TERTp-wt GBMs was 53.2 years, which was significantly younger than the average age of their counterparts with TERTp mutant GBMs (60.7 years, p = 0.0096), and significantly older than the average age of patients with IDH mutant GBMs (38.6 years, p = 0.0041).
Across the cohort of IDH-wt GBM, the male to female ratio was 1.66. TERTp-wt GBMs did manifest a numerically higher proportion of male patients (13/16, 87.5%), compared with 55/93 male patients (59%) with TERTp mutant GBMs, but this difference was not statically significant (p = 0.103).
We examined the location of the primary tumor presentation. In the TERTp-wt group, the primary tumors were mainly found in a supratentorial (13) and thalamic/midline location (1), but also in a cerebellar site (3 cases). In contrast, in the TERTp mutant group, the tumors were exclusively located supratentorially (91) or thalamic/midline (2), with none found in the cerebellum. Consequently, a significant correlation between TERTp-wt status and cerebellar location (p = 0.0027) was observed. Of note, one of the cerebellar GBMs occurred in a patient with a NF1 germline mutation (Neurofibromatosis type 1).
The median time of follow-up in surviving patients was 189 days for the TERTp-wt group and 246 days for the TERTp mutant group. Due to the short follow-up time, survival analyses may be underpowered to detect differences. Nonetheless, no detectable difference in survival was observed between the two groups (p = 0.74).
Genetic and epigenetic correlation
We examined genetic and epigenetic correlations between TERTp-wt versus mutant tumors. Four TERTp mutant cases were found to harbor a hotspot BRAF V600E mutation, which is characteristic of epithelioid GBM [14]. NF1 mutations were more commonly seen in TERTp-wt GBMs (6/16, 37.5%), in comparison with 18/93 (19%) in the TERTp mutant GBM cohort, however, this was not a statistically significant difference (p = 0.11). Also, we did not observe a significant difference in MGMT promoter methylation status in the TERTp-wt group vs. the mutant group (7/14 vs. 36/90, p = 0.56).
Activating alterations in the PI3K pathway (mainly PIK3CA or PIK3R1) were detected in 25 out of 109 cases in the cohort (23%) (Additional file 3). Interestingly, we observed a strong correlation between TERTp-wt status and mutations targeting the PI3K pathway: 9/16 (56%) of TERTp-wt GBMs contained a PI3K pathway alteration, while only 16/93 (17%) of mutant GBMs harbored these alterations (p = 0.0018) (Fig. 1). Furthermore, we detected an inverse correlation between PIK3CA/PIK3R1 and EGFR alterations. Only 2/25 cases (8%) with a PI3K pathway alteration had an EGFR mutation or EGFRvIII, whereas 38/82 of PI3K wild-type GBM had an EGFR alteration (46.3%, p = 0.0003).
Moreover, as expected, ATRX mutations were detected by sequencing in 6/16 (37.5%) TERTp-wt GBMs, while only 6/93 (6.5%) of TERTp mutant GBMs had an ATRX mutation. Consequently, this manifested as a significant correlation between TERTp-wt status and ATRX mutation (p = 0.0022). Of note, our workflow for assigning mutation was highly sensitive, leading to potential false positive assignments of ATRX candidate alterations that may not functionally inactivate the protein product. The further assessment of ATRX loss-of-expression using immunohistochemistry revealed a similarly significant result: 4/13 (31%) of TERTp-wt GBMs had ATRX loss vs. 0/80 mutant GBMs (p = 0.0002) (Fig. 2).
Finally, we noted that 8/16 (50%) of TERTp-wt GBMs harbored mutations in the BAF complex gene family (SMARCA4, SMARCB1, ATRX, and ARID1A), compared with only 8/93 of TERTp mutant GBMs (p = 0.0002). Given the role of ATRX in telomere maintenance, mutations in either group (ATRX vs SWI/SNF) may be unrelated. Nevertheless, we found that this association remained significant when excluding ATRX (3/16 (18.8%) of TERTp-wt GBMs harboring mutations compared with only 2/93 of TERTp mutant GBMs, p = 0.022). When combined with our analyses above, we detected a significant difference in co-occurrence between mutations in the BAF complex and PI3K pathway genes by comparing the TERTp-wt (n = 5/16) and TERTp mutant groups (n = 1/93, p = 0.0002) (Fig. 3).