In this study, we collected higher number of samples than in our previous study . We found that the frequency of EGFR mutations was low but the frequency of EGFR mutations in the kinase domain was high in wildtype-GBMs. Moreover, we developed a method to detect EGFR transcriptional variants; we found a novel EGFR transcriptional variant and identified a case with a rare EGFR variant with longitudinal and temporal transformations of EGFRvⅢ.
EGFR amplification is detectable in approximately 40–50% of all GBMs [1–3]. The frequency of EGFR alterations in our cohort was lower than that in TCGA and MSKCC cohorts. However, a study from Japan reported that 25.5–33.1% of GBMs have EGFR amplifications [19, 20], which is consistent with our findings. Recent reports indicate that 14.4–26% GBMs harbor EGFR mutations [2, 21]; however, the EGFR mutation frequency in GBMs in our cohort (6.0%) was considerably lower than that in the previous study cohort. These results suggest that EGFR alterations may be less frequent in Japan than in other countries. Moreover, in our study, EGFRA289D/T/V was the most common missense mutation, and two cases harbored EGFRT790M missense mutations in the EGFR kinase domain. Previous studies have shown that approximately 4% EGFR mutations in GBM have mutations in the kinase domain [2, 21]; however, in our study, 27% EGFR mutations were in the kinase domain in GBMs, and this is higher than that reported previously.
Two cases (1 GBM and 1 AA) harbored EGFRT790M missense mutations in the kinase domain. EGFRT790M, which is commonly observed in lung cancer , is a very rare mutation in glioma, with only one case of GBM reported previously . Osimertinib is an oral, third-generation tyrosine kinase inhibitor (TKI) that irreversibly inhibits EGFR and was developed specifically to target the EGFRT790M-resistant mutation in EGFR-mutated non-small-cell lung cancer [22, 24, 25]. Makhin et al. reported a GBM case with two EGFR point mutations (C628F and A289V) that responded well to osimertinib. Thus, glioma cases with mutations in the EGFR kinase domain may benefit from EGFR TKIs . In our study, a high EGFR amplification was identified in 5/10 (50%) patients with IDH-wildtype AA, consistent with the findings of a previous study . In patients with IDH-wildtype AA, 33% EGFR mutations (2/6 mutation sites) had T790M and S768I mutations in the kinase domain. However, in IDH-wildtype AA, the distribution of mutations within the EGFR coding sequence has not been reported.
EGFRvⅢ is the most common EGFR splice variant. EGFRvⅢ activates multiple downstream signaling pathways and exhibits high tumorigenic potential [8, 11, 12]. Recent reports indicate that 50–60% EGFR-amplified GBMs harbor EGFRvⅢ variants [9, 10]. EGFRvⅢ positivity was detected in 2 of the 25 EGFR-amplified glioblastomas (8.0%) in our study, suggesting that EGFRvⅢ positivity may have been less frequent in our study than in previous studies. These discrepancies may be due to ethnic differences in patient cohorts or differences in analysis methods; we used NGS and not immunohistochemistry or RT-PCR. A comparative study found that RT-PCR was more sensitive and specific than immunostaining using two different EGFRvⅢ-specific antibodies . Another study showed that the sensitivity of NGS-based EGFRvⅢ detection is lower than that of immunohistochemistry or RT-PCR, reflecting that EGFRvⅢ may be restricted to small subclones of glioma cells, which may not lead to a detectable reduction in exon 2–7 gene dosage .
In this study, we identified one uncharacterized EGFR variant with deletions of exons 6–7 (Δe 6–7). Deletions of exons 2–14 (Δe 2–14) constitute a very rare variant in glioma, with only one case of GBM having been reported previously . In one case, the initial EGFRvⅢ (Δe 2–7) mutation transformed into a EGFR exon 2–14 deletion (Δe 2–14) at the time of recurrence. Recent reports indicate that 16–59% of GBMs that were initially EGFRvⅢ positive, lost EGFRvⅢ at recurrence [9, 10, 14]. Some reports suggest that the frequency of EGFRvⅢ loss at recurrence is altered by the treatment received , whereas others suggest that it is not dependent on the treatment received . In addition, it has been reported that the expression of EGFRvⅢ is a result of epigenetic regulation , but the mechanism by which EGFRvⅢ is lost at recurrence is unknown. Based on our findings, it is possible that the initial EGFRvⅢ mutation is transformed to other variants during recurrence. The functional significance of the novel EGFR variants needs to be analyzed in further studies.
In conclusion, we report the distribution of mutations within the EGFR coding sequence and two novel EGFR variants, one of which showed longitudinal and temporal transformation of EGFRvⅢ. In addition, we showed that the frequency of driver gene alterations in GBMs differs across cohorts. Thus, to implement personalized medicine, it is necessary to accurately assess the genetic profile of each cohort.