Despite encouraging results in clinical trials (1
), the benefit of bevacizumab in glioblastoma is typically transient, followed by tumor growth resumption, a phenomenon found in other cancers treated with anti-angiogenic therapy (5
) and associated with a poor prognosis (13
). Anti-angiogenic therapy resistance has been associated with increased tumor cell invasion (10
), although other mechanisms, including recruitment of bone marrow-derived cells to tumors (29
), have also been suggested.
In our radiographic analysis of 21 bevacizumab-resistant glioblastomas, over half, which we called enhancing bevacizumab-resistant glioblastomas (EBRGs), exhibited enhancing growth on MRI that has defined glioblastoma recurrence since the 1990 advent of the Macdonald response criteria (30
). The remaining cases, termed non-enhancing bevacizumab-resistant glioblastomas (NBRGs), were characterized by non-enhancing FLAIR-bright growth on MRI which has been associated with glioblastoma recurrence since revised treatment response criteria were put forth by the Response Assessment in Neuro-Oncology (RANO) group in 2010 (15
) to recognize changes in the presentation of recurrent glioblastoma since the advent of anti-angiogenic therapy. While the initial identification of non-enhancing recurrence after bevacizumab treatment led some to suggest that the phenomenon was common, subsequent studies (6
) and review of 74 glioblastomas progressing during anti-angiogenic therapy at our institution (unpublished observations) showed that 60–75% of glioblastomas progressing during anti-angiogenic therapy are enhancing local recurrences.
Other investigators have correlated glioblastoma PFS and OS after bevacizumab with the pre-treatment ratio of the FLAIR bright volume to the T1 gadolinium enhanced volume (31
). Our analysis expands upon these observations by finding 2 radiographic subtypes of glioblastomas progressing during bevacizumab treatment and showing distinct transcriptional phenotypes for each subtype, with the functional correlate of increased cellular invasiveness in one subtype. Invasion, proliferation, and hypoxia have not correlated well in prior glioblastoma studies (32
) and did not correlate well in our bevacizumab-naïve glioblastomas, so it is possible that the trend we observed of NBRGs becoming more hypoxic, more invasive, and equally proliferative relative to pre-treatment may occur specifically after resistance to anti-angiogenic therapy.
An unbiased gene selection approach in which differential gene expression measuring changes in expression after bevacizumab resistance compared to before was clustered on high variance probes revealed two clustering patterns, analysis of which confirmed one to be NBRGs and the other EBRGs. Immunohistochemistry showed that NBRGs, which exhibit FLAIR-bright non-enhancing tumor progression on MRI that has been associated with devascularized infiltration (6
), maintained the hypoxia and reduced vascularity reported after successful bevacizumab treatment (34
), and showed unchanged proliferation marker expression compared to before bevacizumab treatment. The unchanged tumor cell proliferation in NBRGs could reflect reliance on invasion, particularly perivascular invasion, to reduce angiogenesis dependence by allowing cellular migration away from devascularized areas into areas closer to blood vessels, rather than continuing the nodular enhancing exponential growth that is difficult to sustain during VEGF blockade. Conversely, EBRGs, which exhibit MRI enhancement potentially consistent with neovascularization, reacquired the reduced hypoxia and increased vascularity seen before bevacizumab treatment, which may have contributed to the observed increased cell proliferation compared to before bevacizumab treatment.
These differences in tumor cells and the tumor microenvironment between EBRGs and NBRGs were reflected in the genes found to be differentially transcribed between EBRGs versus NBRGs. Specifically, the transcriptional data showed upregulation in NBRGs of integrin α5β1 and two of its ligands, fibronectin and laminin. Increased tumor cell expression of α5β1 could bind upregulated fibronectin or laminin in the vascular basement membrane, possibly promoting perivascular invasion as a mechanism of anti-angiogenic therapy resistance. Conversely, EBRGs exhibited increased expression of aquaporin 4, which promotes vascular proliferation (35
), possibly allowing EBRGs to reacquire their pre-treatment vessel densities and obtain the blood supply needed for the increased cellular proliferation we found in EBRGs, as evidenced by increased Ki-67 staining and increased MAPK4 and MAPK10 expression. We confirmed a functional impact of these molecular differences identified in the microarray analysis by demonstrating NBRG cells to be more invasive in matrigel-coated Boyden chambers than EBRG cells. The hypothesis that the transcriptional reprogramming we identified is specific to bevacizumab resistance rather than merely reflecting differences between non-enhancing versus enhancing glioblastoma is supported by 3 lines of evidence. First, array data on glioblastomas and their paired bevacizumab-naïve recurrences did not cluster towards either an EBRG or NBRG pattern using the gene set we compiled by microarray analysis. Second, confirmatory RT-PCR showed no alteration in bevacizumab-naïve recurrent glioblastomas relative to their paired initial tumors of transcripts altered in NBRGs or EBRGs. Third, genes we identified to be differentially expressed between EBRGs versus NBRGs did not overlap with previously reported genes differentially expressed between enhancing versus non-enhancing glioblastoma, such as the 79 genes differentially expressed between incompletely versus completely enhancing glioblastomas (36
) or the 643 genes differentially expressed between the enhancing periphery and the central necrotic core of glioblastoma (37
It has been suggested that EMT, a phenomenon recognized in non-CNS malignancies and associated with a worsened prognosis, metastases, and chemotherapy resistance (21
), can occur after anti-angiogenic therapy (38
). Some have proposed EMT in glioblastoma (39
), but it remains unclear what the equivalent of a non-migratory epithelial state or a mesenchymal state with metastatic potential is in glioblastoma. While the glioblastoma subtypes of Phillips et al. (22
) and the TCGA subtypes (28
) included a mesenchymal subtype, it is unclear whether these mesenchymal subtypes embody EMT features, and the unchanging nature of TCGA subtypes implies that they do not detect EMT. Regardless, our findings that NBRGs had (i) examples that converted to the Phillips et al. (22
) mesenchymal subtype after bevacizumab resistance; (ii) increased frequency of N-cadherin expression, a feature of EMT in non-CNS tumors (21
); (iii) increased expression of TWIST1, an EMT-regulating transcription factor (40
); and (iv) mesenchymal morphology with structures resembling pseudopodia (41
) suggest that further work to determine what constitutes EMT in glioblastoma and under what conditions anti-angiogenic therapy promotes EMT is warranted.
While immunostaining primary tumor cells from BRGs revealed differences in N-cadherin expression between NBRGs and EBRGs, N-cadherin gene expression was unaltered by adjusted p-value in microarray analysis. This discrepancy between microarray data and immunostaining suggests possible post-transcriptional or translational regulation in addition to the transcriptional differences we identified, a possible subject for future work.
There are limitations to any study utilizing infrequently available clinical specimens. The first limitation, present in over half the cases analyzed, is that bevacizumab is usually combined with other treatments. However, there was no differential tendency for combination treatment in EBRGs versus NBRGs. Furthermore, the large number of genes changes in common between tumors, regardless of other treatments received, along with evidence supporting these genes as potential mediators of anti-angiogenic therapy resistance (28
), suggests that additional treatments did not confound our analysis. A second limitation is small sample size, reflecting the few cases undergoing surgery after progression during bevacizumab treatment for which pre-treatment tissue was available. While it is possible that an expanded sample size could uncover other resistance patterns with mediators distinct from those listed here, our sample size was large enough to uncover statistically significant clustering of tumors resistant to anti-angiogenic therapy by differentially expressed genes and provides a platform to guide further efforts to define and disrupt resistance to anti-angiogenic therapy. A third limitation is that our study selected BRGs amenable to surgical resection. While one might hypothesize that selecting surgically resectable cases would bias our study towards enhancing local recurrences, we found 57% of resected recurrences to be EBRGs, less than the 74% of overall progressions on bevacizumab treatment that were enhancing local during this time period (unpublished observations). Regardless, by studying cases of bevacizumab resistance leading to surgery, a necessity to study this tissue, our findings may not be applicable to unresectable bevacizumab-resistant tumors. A fourth limitation is that, while a comparison of NBRGs to EBRGs revealed several genes with adjusted p values below 0.05, no differentially expressed oncologically pertinent transcripts in NBRGs or EBRGs relative to their paired pre-bevacizumab-treated tumors had significant adjusted p-values, despite having significant raw p-values, and some exhibited low upregulation (). However, these oncologically pertinent transcripts had prior evidence (28
) supporting their potential involvement in anti-angiogenic therapy resistance, reducing concerns about needing to adjust for multiple testing (49
) and suggesting that their raw p-values being statistically significant is sufficient to render them appropriate for further investigation, while the fold changes we detected by real time RT-PCR were higher than those detected by microarray and, have been suggested by some to better reflect changes in individual transcript levels (49
Thus, just as prolonged treatment with temozolomide, the current standard of care for newly diagnosed glioblastoma, can create a “hypermutator” phenotype associated with recurrence (50
), VEGF-targeted treatments like bevacizumab may cause “hyperinvasive” (NBRG) or “hyperproliferative” (EBRG) phenotypes associated with glioblastoma recurrence. These findings provide important biologic insight into how tumors counteract anti-angiogenic treatments, responses that have unfortunately limited the efficacy of anti-angiogenic therapies used in patients to date.