Subventricular zones: new key targets for glioblastoma treatment

Glioblastoma is the most common primary brain tumor among adults, with poor outcome following chemoradiotherapy, despite deeper insights into molecular biology and significant advances in therapeutics [1]. Like other cancers, glioblastomas are characterized by a high intratumoral heterogeneity in a wide range of phenotypic and functional features [2‐4]. The stochastic model has traditionally been accepted as the basis of tumor heterogeneity, wherein a small population of genetically unstable clonal cells randomly acquires the appropriate somatic mutations necessary to confer extensive proliferative capabilities [5]. However, recent evidence suggests that according to the alternative cancer stem cell model [6], glioblastomas are rather organized hierarchically as they contain a subpopulation of cancer cells with stem cell characteristics, including self-renewal capacity and multilineage potency, as well as tumor-initiation/proliferation and migration ability [7‐11].

The origin of brain tumor stem cells is still controversial, but neural stem cells are highly likely to be candidate as both populations share many similarities, including molecular pathways (Notch receptor activation [12, 13], inactivation of PTEN tumor suppression gene [14]) and markers of gene expression (like Nestin, CD133 or Sox [15]). A growing body of evidence has also suggested that any glioma cell could acquire a stem cell phenotype in response to hypoxia, or even radiation, contributing to GBM radioresistance [16‐18]. Stem cells of various tissues exist within protective niches composed of a number of differentiated cell types providing direct cell contacts and secreting factors that maintain stem cells primarily in a quiescent state [19]. It is then hypothesized that tumor stem cells might arise from normal stem cells that have acquired mutations which enable them to escape from niche control or after deregulation of extrinsic factors within the niche, leading to uncontrolled proliferation of stem cells and tumorigenesis [20‐22]. The subventricular zone (SVZ) is the largest niche of neurogenesis in the adult mammalian brain [23, 24], so that an emerging hypothesis is that brain tumor stem cells could derive from the SVZ [11]. It should be noted that the subgranular zone abutting the hippocampal dentate gyrus is a secondary nich in adult mammalian brains, but where neurogenesis only occurs in foci closely associated with blood vessels [25].

Many data have already provided interesting relationships between SVZ involvement in glioblastoma and outcome or pattern of relapse, strongly suggesting that tumors contacting SVZ are associated with multifocal presentation at diagnosis, multifocal or distant progression [26‐29] and above all decreased survival [27, 29‐31]. This stresses the need to better understand the role of the SVZ as a potential source of glioblastoma through initiation and promotion, a potential source of relapses through repopulation of tumor, and as a target for glioblastoma treatment.

Glioblastoma stem cells are known to be inherently resistant to chemotherapy and radiotherapy [32, 33]. One strategy to overcome glioblastoma stem cells radioresistance could consist in increasing the radiation dose to the SVZ. The impact of incidental radiation dose to the SVZ during the course of a standard chemoradiotherapy on the outcome of patient has already been assessed, but mostly in inhomogeneous series and with somewhat contradictory results [34‐41]. These results are all the more confusing that such irradiation to neural stem cells is source of radiation-induced neurotoxicity [42], and is in contradiction with hippocampal sparing strategies [43].

Therefore, the objective of this retrospective study was to identify SVZ-related prognostic factors of survival and patterns of recurrence, with regard to both surgical and radiotherapeutic management, to better integrate SVZ in the global therapeutic strategy of glioblastoma patients, with as few neurotoxicity as possible.

Owing to complex relationships between brain tumor stem cells and neural stem cells, the optimal management of neurogenic regions of patients with glioblastoma still remains controversial. Herein, we focused on the survival rates and the recurrence patterns of glioblastoma with respect to SVZ-related factors.

In spite of inherent biases due to its retrospective design, this study has several strengths. Indeed, it included a uniform cohort of consecutively-treated glioblastoma patients with the same first-line therapy (surgical resection and adjuvant temozolomide-based chemoradiation with uniform dose/fractionation scheme), and uniform follow-up using most updated criteria, of more than 6 months after surgery, to avoid any pseudo-progression. Volume delineation, plan evaluation and approval were ensured by a single experienced radiation oncologist (ECJM). MGMT methylation status was available for almost the whole population, as well as all classical glioblastoma prognostic factors. Our salvage therapies were not homogenous (of the 38 patients who benefited a salvage treatment, 17 received a re-resection, most of them received in situ carmustine wafers (CW), and the 21 remaining patients received bevacizumab as second line, temozolomide or fotemustine); however this cofounding factor could influence OS but not TTP. Finally, our multivariate analysis aimed to control any other confounding factors. It should there be mentioned that as compared to intensity modulated radiotherapy which uses inverse planning with predefined constraints to structures, 3DCRT uses a number of fields and a beam arrangement defined by the operator, which can introduce some confounding factors in the assessment of dose to SVZ too difficult to take into account and therefore not included in our multivariable model.

Our results suggest first that initial contact to SVZ is a strong prognostic factor for TTP in multivariate analysis. This result is in accordance with several other similar reports [27, 29‐31], and could be explained because glioblastoma arising in SVZ might have a higher percentage of more potent cells, making them more invasive and infiltrative. This hypothesis is reinforced by an established correlation between SVZ involvement and invasive tumor phenotype with distant or multifocal progression [26‐29].

From a biological point of view, such an invasive phenotype is supported by experiments showing that SVZ neuronal progenitor cells have a higher migratory potential compared to non-SVZ ones [49].

Contrary to several studies [30, 44, 50], we failed to correlate the surgical status to survival. This could be explained either by an underpowered sample size, or because initial contact to SVZ, even in case of GTR or NTR, would lead to a microscopic residual disease due to the aggressive phenotype of tumor cells from SVZ.

Interestingly, 83.3% of our patients with distant relapse presented a recurrence within SVZ, and all but one had a GTR, highlighting the role of neurogenic regions on initiation, promotion but also repopulation of tumors. Brain stem tumor cells in neurogenic regions seem to constitute a source for a subsequent relapse.

Overall, survival data and recurrence patterns from both literature and our study suggest that a “prophylactic” irradiation to SVZ could be necessary to perform a therapeutic intensification among contacting tumor patients to encompass therapeutic resistance, but also to eradicate potential “niches” of relapse among other glioblastoma patients. Several studies have established a correlation between a dose level to SVZ and survival outcomes [34‐40]. The first ever study from Evers et al. (UCLA, USA) found that mean dose >43 Gy to bSVZ was an independent factor for PFS (HR = 0.73, 95%CI = [0.57; 0.95]), but not for OS on multivariate analysis [34]. These results were not confirmed in the series from VUMC (The Netherlands) which could not find an association between this dose to SVZ and PFS [35]. This may be due to the more aggressive population of exclusively grade 4 patients in the VUMC study (compared to a mix grade 3/grade 4 glioma population in the UCLA study), which might require a higher dose to bSVZ. Given these heterogeneous results, Lee et al. updated and pooled these data restricted to only glioblastoma patients, but they failed to find a correlation between bSVZ dose and PFS as in Evers’s study. However, they found that increasing mean dose to iSVZ (>59.4 Gy) was an independent factor for PFS on multivariate analysis (HR = 0.45, 95%CI = [0.25; 0.82]) [36], similarly to Gupta et al., who found it was an independent factor for OS (HR = 0.87, 95%CI = [0.77; 0.98]) [37]. This benefit of increased iSVZ dose on PFS and/or OS was confirmed by Chen et al. with a 40 Gy cut-off, but only in the GTR group, hypothesizing that STR patients preferentially relapsed from residual tumor mass outgrowth and thus highlighting the role of such irradiation to eradicate stem cells “niches” (HR for PFS of 0.385, 95%CI = [0.165–0.901]) and HR for OS of 0.385, 95%CI = [0.165–0.895]) [38], and more recently by Adeberg et al. with the same cut-off but only in univariate analysis (median PFS : 8.5 versus 5.2 months; p = 0.013) [40]. Interestingly, contradictory results came from a recent study by Elicin et al. which found a worse PFS among patients with high iSVZ dose (>62.25 Gy) in both subgroups of good performance status and SVZ without tumoral contact. A worse PFS was also found among patients with dose to cSVZ >59.2 Gy (7.1 vs 10.4 months), but it was not confirmed in multivariate analysis [39]. Gupta et al. also found a worse PFS and OS with higher cSVZ doses, but patients in higher dose group had actually larger tumors, more often crossing the midline and with less GTR so that it was not confirmed in multivariate analysis [37]. On the contrary, Adeberg et al. found a better PFS among patients receiving a mean dose to cSVZ ≥30 Gy (HR in multivariate analysis of 0.45, 95%CI = [0.20–0.98]) [40]. A summary table of all previous results and our main results is presented (Table 6).

However, several limits should be noted in these retrospective studies, which could explain some of these discordances. Population was not always homogenous : grade 3 gliomas were included in Evers’ study [34] and patients could receive other drugs than temozolomide in two studies [34, 36]. Methylation status, which is a strong predictive factor of outcome [51], was missing or insufficiently reported except in two studies [37, 40]. Variability in the radiotherapy target volume delineation and dose-prescription between studies (one vs. two dose-level prescription depending on European or American guidelines) but also within a series [34, 36, 40], and variability of the SVZ delineation between studies (with [37] vs. without temporal horn [34, 36, 38, 39]) and within a series (3–5 mm thick), should also be mentioned. Above all, only mean dose to SVZ was considered in all studies, and thresholds were defined using only median mean doses or 75^th percentile.

Instead, our approach consisted of exploring dose distributions to SVZ during radiotherapy through a more complete analysis of the SVZ dose-volume histogram, in order to better understand the role of low/mid doses to SVZ in addition to high dose. This innovative approach had never been assessed before. We also tried to find an optimal cut-off for relevant dose-volume parameters, through a statistical method. It is there worth mentioning that due to the exploratory nature of the analysis, no adjustment for multiple testing was used. This led us to identify a V20Gy >84% to bSVZ (without temporal horn) as an independent prognostic factor for better TTP. The multivariate analysis included contact to SVZ and radiation dose to SVZ as co-variables and could then take into account the fact that patients with contacting tumors inherently received a higher dose to SVZ. Interestingly, in univariate analysis, a mean dose to bSVZ >40Gy was also a factor for better TTP, in the same way as Evers [34]. Last, it should be noted that a poorer TTP and OS has been found among patients with mean dose to iSVZ (with temporal horn) >43 Gy; however this did not reach statistical significance, and additionnally this was probably due to a larger proportion of contacting tumors in the iSVZ high dose group (80%) compared to the low dose group (30%).

Another feature of our approach was to explore two SVZ delineation methods (based on a standard and reproducible 5-mm lateral margin of the lateral ventricle [47]) : with (TH+) or without (TH-) temporal horn. The aim was to assess an hippocampal sparing method (TH-), as most neurogenic regions with potential brain tumor stem cells stand lateral to the body of lateral ventricles and biologically equivalent doses in 2-Gy fractions to 40% of the bilateral hippocampi greater than 7.3 Gy was shown to be associated with long-term neuro-cognitive impairment [52]. Hippocampal avoidance during whole-brain radiotherapy for brain metastases was associated with preservation of memory and quality of life in a phase II trial [43]. Hippocampal avoidance is not assessed in glioblastoma trials because optimal PTV coverage is the main objective. But when it comes to performing “prophylactic” irradiation to SVZ, neurocognitive preservation should be envisaged, as we confirmed that SVZ irradiation is compatible with hippocampal sparing through TH- delineation method. Chen et al. argued that iSVZ dose ≥40 Gy with TH+ method did not correlate with worsened patient KPS score at the end of radiation treatment, however neurocognitive performances are not precisely assessed in KPS [38]. We thus believe that delineation for glioblastoma patients should then include bSVZ delineation without temporal horn, as well as hippocampi delineation, with use of intensity modulated radiotherapy planning to ensure a sufficient coverage to bSVZ while ensuring an hippocampal avoidance according to previous constraints.

Regarding patterns of recurrence, larger proportion of patients with initial SVZ contacting tumor recurred within SVZ compared to patients with non-contacting tumors. This seems to be due to the in-field pattern of recurrence typically seen in glioblastoma following radiotherapy as most relapses occur within 2 cm beyond the contrast enhancement, and almost exclusively in the edema area [53‐55]. Additionally, patients with V20Gy >84% turned out to relapse away from SVZ contrary to those with V20Gy ≤84%, who mainly relapsed within SVZ. Radiobiological considerations could provide some interesting explanations to this pattern, as there is strong evidence that sublethal irradiation promotes glioblastoma cells migration, via an increase in the αvβ3 integrin expression [56]. Therefore, hypothesizing that SVZ is a potential source of recurrences through repopulation of tumor by remaining brain tumor stem cells following chemoradiation [11], the level of bSVZ irradiation could explane the location of recurrence within vs without SVZ.

Overall, these data could support two hypotheses. The simplest one would be that patients who received a V20Gy >84% to bSVZ really benefited from such a prophylactic irradiation through a brain tumor stem cells eradication. An additional and/or alternative one would be that some of patients who received a higher bSVZ irradiation experienced an “artificial” increase in TTP via a more durable transition through a migratory phenotype, before engaging towards a proliferative one. Then a strategy to encompass such a mechanism of radioresistance would be to associate prophylactic irradiation of bSVZ with intensity modulated radiotherapy (and hippocampal sparing) ensuring a V20Gy >84%, and inhibitors of migration which could target integrin-mediated signalling pathway. This strategy has never been assessed but could provide promising results.

Meanwhile, some more robust data should soon come from three ongoing or recently concluded clinical trials regarding the impact of SVZ irradiation in GBM (NCT01478854 exploring a deliberate sparing of neurogenesis niches, contrary to NCT02039778 and NCT02177578, exploring a deliberate SVZ irradiation).

To conclude, our data from a homogeneous patient cohort suggest that contact to SVZ could be an independent poor prognostic factor for TTP, as well as bSVZ insufficient dose coverage such as a V20Gy ≤84%. We also provide data suggesting that the pattern of recurrence is influenced by contact to SVZ and by radiation dose to bSVZ. These results highlight the prominent role of the SVZ in the prognostic of glioblastoma patients through mechanisms of therapeutic resistance, and therefore raise the question of their role in the optimal management of glioblastoma. Further prospective clinical trials assessing prophylactic irradiation of bSVZ by intensity modulated irradiation, with or without migration-inhibitors, would be critical in determining whether such strategy could improve devastating outcomes of glioblastoma patients.