EGFR is associated with WHO grade IV GBMs and is well-studied, yet our understanding of the mechanisms of amplification and its impact on tumor cell biology is incomplete. Originally, it was anticipated that higher levels of EGFR amplification in this cohort might correlate with poorer response to adjuvant therapy and thus have the worst outcomes. Yet the relationship between EGFR amplification and outcome was paradoxical, such that lower levels of amplification correlated with worse response to TMZ-containing adjuvant therapeutic regimens compared to GBMs with high amplification or none at all. Since amplification of EGFR and other receptor tyrosine kinases is seen in a variety of cancers, this phenomenon may apply to a broader range of neoplasms beyond the brain.
These results may help explain the incongruities seen in other studies evaluating EGFR
amplification as a glioma biomarker. Some have shown EGFR
amplification to be a negative prognostic marker in GBMs (14
), but others have not (3
); some have even suggested it could be a favorable marker, especially in older patients (2
). In our large cohort, pooling all EGFR
-amplified GBMs together and comparing them to non-amplified GBMs showed no difference in survival (). It was only when the EGFR
-amplified tumors were stratified according to degree of amplification that significant survival differences were unmasked (). Further unmasking was done via subgroup analyses according to postsurgical adjuvant therapy, which suggested that there was a survival difference only if TMZ was part of the regimen, and that low-to-moderately-amplified GBMs did not respond as well to TMZ (). Thus, it is possible that the discrepancies between previous cohorts may have been due to varying proportions of these subgroups of EGFR
-amplified GBMs as well as to treatment confounders. Since only 16 cases in the entire cohort were treated with other chemotherapies but without any TMZ, it was difficult to determine whether the adverse effects of low-to-moderate EGFR
amplification might be exclusive to TMZ or also be a feature of other chemotherapies. Still, low-to-moderate EGFR
amplification was an independent adverse prognostic factor in a model with TMZ versus no TMZ, as well as a model focusing only on TMZ-treated cases ().
The significantly better survival seen in GBMs with high EGFR
amplification compared to non-amplified tumors () may have been due to nonrandom enrichment of the latter group with GBMs not exposed to adjuvant treatment, with a concomitant reduction in the relative proportion of non-amplified cases treated with TMZ (). Why this happened is not clear, since 94% of all cases were obtained from 2005 and later when TMZ was standard-of-care in GBM treatment. When reasons for withholding TMZ were retrievable, the most common were adverse systemic reactions, poor overall patient health, voluntary patient/family decisions, and inability to cover treatment costs. None of these intuitively correlate with the presence or degree of EGFR
amplification. Therefore, our data as a whole suggest that high EGFR
amplification might not be a favorable marker per se, but rather that low-to-moderate amplification is an adverse marker, especially in TMZ-treated cases. Other well-known biomarkers of GBM outcome appeared to be evenly distributed between non-amplified, low-to-moderate, and high-amplified GBMs. Although advanced patient age is a powerful adverse prognostic marker (25
), there was no difference in patient age, gender, or surgery between non-amplified, low-to-moderate, and high-level EGFR
-amplified GBMs (). As expected, EGFR expression and frequency of 10q LOH were higher in the amplified versus non-amplified tumors, but there was no significant difference between the low-to-moderate and high amplifier subsets in any of the measured parameters. MGMT
promoter methylation showed no significant difference between amplification subtypes (), so while the number and followup interval of methylation-tested cases was not powerful enough for univariate and multivariate survival analyses, it is unlikely to be a confounding variable.
Although our understanding of gene amplification is rudimentary, some mechanistic insights have been discovered that allow for testable hypotheses. It was recently demonstrated that, of GBMs with EGFR
amplification, those with lower EGFR
copy numbers tended to feature interstitial amplification, wherein all the EGFR
genes were located exclusively within chromosome 7. In contrast, EGFR
in GBMs with higher copy numbers were mostly located in fragments of extrachromosomal DNA called double minutes (18
). Double minutes have been known to exist in a variety of cancers, arising through defects in DNA reproduction and faulty repair machinery (9
). Perhaps GBMs with high levels of EGFR
simply have more fragile genomes, thus responding better to DNA-damaging TMZ and offsetting the otherwise deleterious effects of EGFR
amplification. Indeed, other recent work has suggested that high chromosomal fragility can actually be a favorable prognostic marker in various carcinomas (5
) and even in other brain tumors like ependymomas (33
Since the size of the 7p12 amplicon varies, other nearby genes are often co-amplified with EGFR
and could impact tumor biology. LANCL2
, for example, is co-amplified and over-expressed in about 50% of EGFR
-amplified GBMs (8
encodes Lanthionine Synthetase C-like 2, which may promote sensitivity to chemotherapy (27
). About one-third of EGFR
-amplified GBMs also co-amplify ECOP
, encoding EGFR
-Coamplified and Overexpressed Protein, which upregulates the antiapoptotic activity of NF-κB (26
). The FISH probe used in this cohort for clinical testing covers the entire EGFR
gene and extends beyond its 3′ centromeric end, but does not reach LANCL2
(Supplemental Figure 2
). However, analysis of TCGA GBMs showed no difference in survival by co-amplification of LANCL2
(Supplemental Figure 3
) or by co-amplification of ECOP
= 0.61, not shown).
Studying additional retrospective cohorts will be of high interest given the nature of these findings. Analysis of TCGA GBMs weakly trended toward a similar paradoxic relationship between degree of amplification and outcome (Supplemental Figure 4
), though there are several possible reasons why a stronger relationship was not found. First, the TCGA cohort is 30% smaller than the current study. Second, the TCGA dataset is inherently biased toward cases in which large resections of highly viable tumors was possible. This cohort, in contrast, studied all cases regardless of sample size and degree of necrosis, as long as at least 60 scoreable tumor cells were in the specimen. Third, copy number in the TCGA was determined by SNP arrays and reported as log2 ratios (see Methods). While in principle this is analogous to FISH, in reality it proved very difficult to precisely extrapolate the FISH cutoffs to SNP array data. Still, the TCGA cohort at least suggests that the current cohort data is valid.
This study has key advantages of a very large cohort and prospective nature of the molecular data. Yet the survival analysis was retrospective, with all the attendant limitations thereof. Specifically, the correlation between low-to-moderate EGFRamplification and poorer TMZ response should be validated prospectively, and could readily be incorporated into any number of ongoing clinical trials. Nevertheless, this study provides the first large-scale evidence that the degree of EGFR amplification may impact the biological behavior of GBMs, though in a counterintuitive manner. This could account for conflicting results in prior outcome-based studies of EGFR amplification, and suggests that there might be more biologically relevant heterogeneity in EGFR-amplified tumors than has been previously assumed.