Current and Investigational Drug Strategies for Glioblastoma

Clinical Oncology xxx (2014) 1e12 Contents lists available at ScienceDirect

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Overview

Current and Investigational Drug Strategies for Glioblastoma M. Ajaz *, S. Jefferies y, L. Brazil z, C. Watts x, A. Chalmers { * Surrey

Cancer Research Institute, University of Surrey, Guildford, UK Oncology Centre, Addenbrooke’s Hospital, Cambridge, UK z Guy’s, St Thomas’ and King’s College Hospitals, London, UK x Division of Neurosurgery, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK { Institute of Cancer Sciences, University of Glasgow, Glasgow, UK y

Received 25 March 2014; accepted 27 March 2014

Abstract Medical treatments for glioblastoma face several challenges. Lipophilic alkylators remain the mainstay of treatment, emphasising the primacy of good bloodebrain barrier penetration. Temozolomide has emerged as a major contributor to improved patient survival. The roles of procarbazine and vincristine in the procarbazine, lomustine and vincristine (PCV) schedule have attracted scrutiny and several lines of evidence now support the use of lomustine as effective single-agent therapy. Bevacizumab has had a convoluted development history, but clearly now has no major role in first-line treatment, and may even be detrimental to quality of life in this setting. In later disease, clinically meaningful benefits are achievable in some patients, but more impressively the combination of bevacizumab and lomustine shows early promise. Over the last decade, investigational strategies in glioblastoma have largely subscribed to the targeted kinase inhibitor paradigm and have mostly failed. Low prevalence dominant driver lesions such as the FGFR-TACC fusion may represent a niche role for this agent class. Immunological, metabolic and radiosensitising approaches are being pursued and offer more generalised efficacy. Finally, trial design is a crucial consideration. Progress in clinical glioblastoma research would be greatly facilitated by improved methodologies incorporating: (i) routine pharmacokinetic and pharmacodynamic assessments by preoperative dosing; and (ii) multi-stage, multi-arm protocols incorporating new therapy approaches and high-resolution biology in order to guide necessary improvements in science. Ó 2014 Published by Elsevier Ltd on behalf of The Royal College of Radiologists. Key words: Chemotherapy; clinical trials; glioblastoma multiforme; new agents

Statement of Search Strategies Used and Sources of Information This paper reflects expert opinion and current literature accessed by the authors; no formal search strategy has been defined.

Introduction Medical treatments for glioblastoma multiforme need to access and disrupt a particularly complex process. The classic cancer target, the malignant glial cell, is driven by a defined range of molecular lesions, largely shared with Author for correspondence: M. Ajaz, Surrey Cancer Research Institute, University of Surrey, Leggett Building, Daphne Jackson Road, Guildford GU2 7WG, UK. Tel: þ44-1483-688602. E-mail address: [email protected] (M. Ajaz).

other malignancies [1]. However, drug penetration is only one of several major challenges. The brain is uniquely a highly structured organ and malignant glial cells effectively reprise an embryonic development programme and disseminate early. Glioblastoma is therefore an organ- and tissue-level process as much as a classic cancer cell mass. The malignant cells themselves show hierarchical and evolutionary heterogeneity, affecting both the prevalence of and the response to simple drug targets. Multiple other cell types contribute to the process, including the brain’s resident macrophages, microglia. Immune and inflammatory reactions are largely ineffective or counterproductive. The most successful drug treatments for glioblastoma have been lipophilic alkylators, classic cytotoxic agents that are able to pass through the bloodebrain barrier (BBB) and deplete the cycling malignant cell population by DNA damage. The PCV schedule of procarbazine, lomustine and vincristine, established in the 1970s, remains a major component of clinical practice and as a clinical trial

0936-6555/$36.00 Ó 2014 Published by Elsevier Ltd on behalf of The Royal College of Radiologists. http://dx.doi.org/10.1016/j.clon.2014.03.012

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intervention. Temozolomide was not initially developed for glioblastoma, but attracted interest after observations in generic phase I trials. Results in recurrent disease were followed by the landmark demonstration of improved outcomes in combination with radiotherapy in 2005 [2], which persists as the single major advance in treatment of the last several decades. More recently, anti-angiogenic intervention using the anti-vascular endothelial growth factor (VEGF) antibody, bevacizumab, has gained attention. Because this agent class acts on the tumour vasculature, the need for parenchymal brain access is obviated. Outside the USA, widespread adoption of bevacizumab in recurrent disease has been hindered primarily by a lack of evidence from appropriately designed trials. Anecdotal experience continues to support its enthusiastic use by some, but the combination of the high cost and a lack of direct evidence presents difficulties. In the first-line setting, two large trials have recently reported showing that bevacizumab does not improve outcomes when added to first-line temozolomide and radiotherapy [3,4]. The last decade has seen a major expansion of investigational and approved new agents in oncology. Although typically commercially developed for other cancer indications first, multiple targeted agents have been repurposed for glioblastoma in early phase clinical trials, but with limited success. Immunotherapies have progressed to phase III trials, and a number of other investigational avenues are open. This overview will describe current treatments, the status of anti-angiogenic approaches, failures and future directions with new agents and trial design in glioblastoma.

Conventional Cytotoxic Chemotherapy Lomustine (Figure 1A) is a nitrosurea that is rapidly hydroxylated on first passage through the liver to alkylating metabolites with a half-life of 16e48 h and with good BBB penetration. Clinical dosing is 100e130 mg/m2 on day 1 of a treatment cycle that is 6 weeks in length due to prolonged myelosuppression. Since a 1976 report [5], lomustine has typically been partnered in clinical use by procarbazine and vincristine, giving the PCV schedule. Until recently, this combination has been largely immutable due to accumulated clinical precedent and continued usage in major trials, predominantly in low-grade glioma [6,7]. However, the superiority of PCV over single-agent nitrosurea rests on limited evidence [8e10] and lomustine has shown unexpectedly high single-agent activity as a control arm in modern trials, with 6 month progression-free survival rates of 19% [11] and 25% [12]. The role of vincristine is being increasingly questioned. Disrupting microtubule dynamics is a rational strategy in glioblastoma, not only to target mitosis, but also as a way to attenuate glioma cell migration [13]. However, it is doubtful whether the central nervous system (CNS) pharmacokinetics of vincristine allow these effects to be realised: it is poorly suited to BBB penetration on account of high molecular mass, polar surface area and efflux pump liability. A Korean phase II trial is appraising the effect of vincristine omission from PCV [14] and the NOA-05 trial established precedent for the procarbazineelomustine doublet in gliomatosis cerebri [15].

Fig 1. The structures of (A) lomustine; (B) temozolomide; and (C) vascular endothelial growth factor-A (VEGF-A) [63], the antigenic ligand of bevacizumab. Please cite this article in press as: Ajaz M, et al., Current and Investigational Drug Strategies for Glioblastoma, Clinical Oncology (2014), http:// dx.doi.org/10.1016/j.clon.2014.03.012

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Procarbazine is a methylhydrazine derivative with multiple mechanisms of action, including direct alkylation and inhibition of DNA, RNA and protein synthesis [16]. Low molecular weight, logP (partition coefficient, a measure of lipophilicity) and polar surface area predict good BBB penetration. There are limited data on single-agent efficacy. In a phase II comparison with temozolomide it was associated with a 6 month progression-free survival of only 8% [17]. In combination with thalidomide, no responses were seen in recurrent glioblastoma [18]. The addition of procarbazine to nimustine and radiotherapy increased toxicity without increasing overall survival [19]. Therefore, procarbazine may also be poorly contributory to the PCV schedule, lending further indirect support for the use of single agent lomustine. In the pre-temozolomide era, the addition of chemotherapy to primary radiotherapy for glioblastoma was assessed in two meta-analyses [20,21], including a large Medical Research Council trial [22]. Although the Medical Research Council trial was itself negative, the meta-analyses suggested modest improvements in survival, particularly when drugs were given concurrently as well as adjuvantly. The addition of temozolomide to radiotherapy for glioblastoma is now standard practice on the basis of a doubling of 2 year survival and the emergence of a long tail of patients who remain progression-free for some years [23]. In this schedule, temozolomide is given at 75 mg/m2 throughout 6 weeks of radiotherapy and at 150e200 mg/m2 every 4 weeks thereafter for 6 months. Temozolomide (Figure 1B) undergoes spontaneous degradation at physiological pH to 3-methyl-(triazen-1-yl) imidazole-4-carboxamide (MTIC), whose major toxic DNA lesion is methylation at the O6 position of guanine. Repair of this damage by O6-methylguanine methyltransferase (MGMT) is associated with resistance to temozolomide, and methylation of the MGMT promoter, which inactivates transcription of the gene, predicts drug sensitivity [24], possibly only in classical subtype glioblastoma [25]. Pharmacological down-regulation of MGMT activity has not proved clinically successful. In order to optimise the deployment of this clear leader among glioblastoma drugs, alternative dosing schedules have been explored. A phase II trial compared dose-dense and metronomic dosing in the post-chemoradiation adjuvant phase (followed by 13-cisretinoic acid until tumour progression) [26]. The phase III trial RTOG 0525 compared dose-dense temozolomide with standard adjuvant dosing. Neither showed a clear advantage to the dose-dense schedule. There is some suggestion that adjuvant temozolomide is more important than the low-dose concomitant treatment [27]. In North America, there has been widespread adoption of extended adjuvant phase temozolomide from 6 months to 12 months, 24 months or until progression. Retrospective reports have suggested better outcomes with extended adjuvant temozolomide [28e30], but a gap in the evidence remains. In relapsed disease, a three-way comparison of temozolomide on a 5 day or 21 day schedule and PCV found no survival difference between temozolomide and PCV, and that the standard 5 day schedule was associated with better

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survival and quality of life outcomes than the 21 day schedule [31]. Although PCV has traditionally been used at first relapse in glioblastoma, there is a case for rechallenge with temozolomide, at least in previous responders [32], with lomustine-based therapy reserved for conclusively temozolomide-resistant disease. Lastly, temozolomide may be a reasonable alternative to radiotherapy as first-line treatment for glioblastoma in elderly patients if they have MGMT promoter methylation [33e35]. It may also be used as first-line treatment for gliomatosis cerebri to defer the toxicity of wide-field irradiation [36]. Several other cytotoxic chemotherapy approaches are of interest. Fotemustine is an alkylator that has been popular in France and Italy [37]. Bendamustine has shown major success in lymphoma and penetrates the BBB, making its lack of efficacy in glioblastoma a disappointment [38]; additional dose escalation may represent a residual investigative approach. VAL-083 is a first-in-class bifunctional N7 DNA alkylating agent that crosses the BBB and accumulates in brain tissue. In a phase I/II trial in recurrent glioblastoma, disease stabilisation or regression was reported in 2/8 patients in the first three dose cohorts [39]. Cyclophosphamide crosses the BBB and has anti-tumour activity in glioblastoma [40], as well as representing an immunotherapeutic strategy [41,42]. Paclitaxel poliglumex (Opaxio) is an agent that links paclitaxel to a biodegradable polyglutamate polymer. When bound to the polymer, the paclitaxel is rendered inactive, potentially reducing normal tissue exposure to high levels of active chemotherapy and its associated toxicities. In a recent study, treatment with OPAXIO added to standard temozolomide and radiotherapy resulted in a median progression-free survival of 13.5 months in the subset of patients with glioblastoma (n ¼ 17). The median overall survival has not yet been reached, with 50% of patients having at least 22 months of follow-up since completing therapy [43]. A phase II study is underway assessing efficacy in MGMT unmethylated patients. GRN1005 is paclitaxel conjugated to angiopep-2, the ligand for low-density lipoprotein receptor-related protein expressed at the BBB. A phase I trial showed target tissue concentration, one complete response and two partial responses [44], but commercial development is currently suspended. Verubulin is a microtubule-destabilising agent with high brain penetration, which (in combination with carboplatin) achieved responses in 2/19 relapsed glioblastoma cases and stabilisation in a further five [45]. Vinblastine and vinorelbine have been used in the paediatric setting [46e48], and with the potential for chronic oral dosing, vinorelbine is an intriguing trial candidate for glioblastoma. Oral etoposide [49] and irinotecan [50,51] have been used as monotherapy and in combination, with little impact in individual studies. A recent meta-analysis confirmed inferior outcomes with irinotecan, but notably a survival advantage in patient cohorts treated with etoposide [52]. Platinum compounds are effective in vitro [53], as well as in the paediatric setting [54], but drug delivery challenges prevail in adult glioblastoma [55,56]. Attempts to deliver

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hydrophilic platinum illustrate the spectrum of investigational approaches for BBB circumvention, including intraarterial administration [57], liposomal [58] and micelle [59] carriage, focused ultrasound [60] and convectionenhanced delivery [61,62].

Anti-angiogenic Approaches Bevacizumab is a humanised monoclonal antibody to VEGF-A (Figure 1C) [63]. VEGF-A sequestration prevents signalling at the VEGF receptors VEGFR-1 and -2. There have been several drivers behind the pursuit of this approach in glioblastoma. First, glioblastoma is a highly vascular tumour and disruption of the tumour blood supply is a paradigmatic anti-cancer strategy [64]. Second, the vascular normalisation hypothesis [65] proposes that mid-way in therapy between aberrant tumour blood flow and full vascular ablation, there is a window of relatively normal vessel function that facilitates drug delivery. (In practice there is no evidence that this is achievable in a concerted, exploitable manner in humans.) Third, VEGF signalling increases vascular permeability, so that blockade reduces peritumoural oedema, achieving at least a potent steroid-sparing effect [66]. Fourth, malignant glial cells are themselves activated by VEGF signalling [67,68]. As a macromolecule, bevacizumab uptake is facilitated by BBB disruption; there is no uptake in the absence of tumour. Bevacizumab has clinically meaningful effects in glioblastoma, but a convoluted development history has been the first obstacle to efficient clinical adoption. In relapsed disease, a series of small phase II trials established overall response rates of 28e43% and 6 month progression-free survival rates of 21e43% [69e71], but without direct comparison against other agents. A meta-analysis of bevacizumab alone and in combination with irinotecan showed 6 month progression-free survival rates of 39 and 48%, respectively, but with a greater discontinuation rate in the combined arm of 6% versus 20% [72]. There was no significant difference in overall survival between the groups. Irinotecan has been an unconvincing partner, showing poor solitary activity [50,51]. Any benefits of the combination may be outweighed by toxicity, and therefore single-agent bevacizumab has been the agent of choice in relapsed disease where funding is available. In terms of dose, important given high drug costs, a further meta-analysis has found no difference in outcomes between doses of 5 mg/kg and 10e15 mg/kg, every 2 weeks [73]. Trials adding carboplatin [74,75], etoposide [76] or temozolomide [77,78] to bevacizumab have failed to show improved outcomes, but promising initial data have emerged from the Dutch BELOB study [79]. This is a randomised phase II study comparing bevacizumab versus bevacizumab plus lomustine versus lomustine alone. In the combination arm, bevacizumab was administered at 10 mg/ kg every 2 weeks and the lomustine dose was moderated to 90 mg/m2 every 6 weeks for improved tolerability. Combination treatment was associated with a 9 month survival

rate of 59% versus 43% with single-agent lomustine and 38% with single-agent bevacizumab. This combination is progressing to a phase III trial [80]. In the first-line treatment of glioblastoma (starting concurrently with temozolomide and radiotherapy), the role of bevacizumab has at least been assessed by two major phase III trials [3,4]. However, these have added both clarity and confusion. The clarity unfortunately is that adding bevacizumab does not increase survival in this diseasestage setting, although it does increase progression-free survival, from about 6e7 months to about 11 months. The confusion is that quality of life results were markedly different across the two studies. In the RTOG 0825 trial [4], bevacizumab was associated with increased symptom burden, worse quality of life and a decline in neurocognitive function. In the AVAglio trial [3], quality of life and performance status were maintained longer in the bevacizumab group and glucocorticoid usage was lower. These major discrepancies are being submitted to further analysis. Adverse effects with bevacizumab are usually confined to hypertension and proteinuria, but more serious consequences can occur, including thromboembolism, colonic perforation, haemorrhage and impaired wound healing. It has been observed in other malignancies that hypertension is a marker of response to bevacizumab [81e84], and there is some evidence for this association in glioblastoma [85]. A number of early radiological predictors of response have been proposed [86,87]; conventional response assessment is confounded by the direct reduction in contrast extravasation caused by the drug’s mechanism of action. Biomarker analysis from the first-line bevacizumab/chemoradiation trials is awaited. It has been suggested that increased glioma cell invasiveness may account for a lack of sustained benefit from bevacizumab [88e91]. Abrupt cessation of bevacizumab has been linked with rebound accelerated tumour progression, and slow tapering has been suggested [92]. Aflibercept is a recombinant fusion protein that binds VEGF and placental growth factor. In a phase II trial it showed similar imaging effects to bevacizumab, but with more modest delays to progression [93]. Of interest, however, accompanying biomarker data showed that responding patients had higher baseline levels of chemokines CCL27, CCL7, MIF and CXCL10, and an early decrease in VEGFR1þ monocytes, suggesting that inhibition of myeloid cell recruitment may be an important fifth rationale for pursuing VEGF-targeted therapy in glioblastoma [94].

Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibition Cancer drug development has generated a number of VEGFR tyrosine kinase inhibitors. Owing to target similarity, these agents often have activity against multiple other kinases and there is significant overlap with glial (as well as endothelial) biology. There has been natural interest in investigating these agents for glioblastoma. However, more conclusively than with bevacizumab, there has been very limited evidence for clinical efficacy. Trial results have been

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negative with cediranib (alone or in combination) [12], enzastaurin [11], sunitinib [95], sorafenib (alone [96] or in combination [97e99]), nintedanib [100], vatalinib [101] and vandetanib [102]. Pazopanib plus lapatinib has modest activity, possibly limited to certain glioblastoma subtypes [103]. Cabozantinib (XL184) has shown a response rate of 35% in patients naïve to anti-angiogenic therapy [104]; this agent also has activity against Met kinase, which may be contributory. Axitinib, lenvatinib, dovitinib and tivozanib are in phase II trials; regorafenib, motesanib, brivanib, linifanib and telatinib have not yet come to trial for glioblastoma. Across all cancers, arguably only renal cell carcinoma has shown consistent susceptibility to VEGFR tyrosine kinase inhibition [105]. As with bevacizumab, VEGFR tyrosine kinase inhibitors may have a residual role as steroid-sparing agents [106,107], which may become more relevant as drug costs recede. Other Investigational Agents and Clinical Trial Strategies Molecular profiling of glioblastoma has given insight into glial cell oncogenesis [108], showing that morphologically similar tumours disguise significant functional differences [109]. Initial genomic [110e112] and expression [113,114] analyses gave rise to taxonomies, including The Cancer Genome Atlas (TCGA) classification system of proneural, neural, classical and mesenchymal subtypes [114]. The subsequent addition of copy number, methylation and proteomic analyses to an expanded cohort [25] has generated further mechanistic paradigms and refinement of disease subsets. Nearly 60% of glioblastomas show abnormalities of the EGFR gene. Trials with erlotinib and gefitinib have been unsuccessful, readily explained by the absence of the kinase domain mutations that are associated with durable drug binding and efficacy [115,116], well-characterised in lung cancer. EGFR copy number variation and extracellular domain mutants (notably EGFRvIII) predominate in glioblastoma. There is evidence that lapatinib [115] and neratinib [117] have greater efficacy against EGFR extracellular mutants than other agents in this class. Clinical results with lapatinib have been underwhelming, attributed to subtherapeutic tissue concentrations [103]; neratinib has not yet come to trial. Afatinib, which is an irreversible inhibitor of both mutant and wild-type EGFR, has shown disappointing results [118]. Dacomitinib is in phase II; canertinib and AZD8931 are not reported to be in trial yet. Antibody blockade of EGFR is well established in other malignancies, but cetuximab does not appear to contribute to therapy in glioblastoma [119]. Despite promising phase II results [120], the addition of nimotuzumab to temozolomide and radiotherapy did not improve survival over temozolomide and radiotherapy alone (22.3 versus 19.6 months) [121]. Posthoc subgroup analysis found that the combination of EGFR overexpression, MGMT non-methylation and incomplete resection was associated with a significant survival advantage in favour of the addition of nimotuzumab (23.8 versus 13.8 months). Complementary inhibition of Notch [122e124] and Met [125] signalling are two of several ways

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to build upon EGFR targeting in glioblastoma and Met inhibition itself is a prime strategy for targeting glioblastoma stem cells [104,126]. Abnormalities in platelet-derived growth factor (PDGF) signalling occur in around 30% of glioblastoma [25], including ligand and receptor up-regulation and gene fusion events. This pathway is a compelling target, being a major contributor to both glial cell expansion [127,128] and angiogenesis [129]. Multiple tyrosine kinase inhibitors have potency against PDGFR. Trials in glioblastoma with imatinib [130], sunitinib [131], dasatinib [132] and nintedanib [100] have been negative. Masitinib has yet to come to trial and others (including axitinib, linifanib, motesanib, regorafenib, telatinib and brivanib) are at various stages of clinical development, as described under their VEGFR activity, above. Three intracellular signalling targets are of interest. The PI3K-Akt-mTOR axis is highly contributory to glioblastoma development. Mutations in PIK3CA and other PI3K genes are mutually exclusive of PTEN alterations; together about 60% of glioblastomas have genetic defects in this system [25]. In the absence of primary gene mutations, the pathway is activated downstream of multiple receptor tyrosine kinases, including the EGFRvIII mutant [133]. Akt activation occurs in around 85% of glioblastomas [134]. The axis is therefore an attractive therapeutic target [135], particularly as a component of combination therapy approaches. Single point intervention at mTORC1 by rapamycin paradoxically increases signalling due to loss of a negative feedback loop [136]; everolimus suffers the same concerns, but is in phase II after dose-finding with temozolomide and radiotherapy in the RTOG 0913 trial [137]. Of multiple PI3K inhibitors, BKM120 (buparlisib) is of interest as it crosses the BBB. This drug is in phase IeII trials alone and in combination with other agents. mTORC2 is a central signalling hub in the malignant glioma cell, integrating signals relating to growth factors, cell death and metabolism [138] and is therefore particularly compelling. Several agents with dual or triple inhibitory activity against mTORC2 and other components of the PI3K axis are in clinical development. However, so far there have been no reported responses to PI3K axis intervention alone in glioblastoma. Missed by sequence-based analyses of glioblastoma genomes, fusion between the FGFR and TACC genes was reported in 2012 [139]. The protein product was found to be oncogenic, with at least two effects, probably most importantly that it localises to the top of the spindle pole of mitotic cells, causing mitotic defects leading to aneuploidy. This activity was tractable by FGFR inhibition with AZD4547 in vivo. The pan-FGFR inhibitor BGJ398 has entered into phase II trial for glioblastoma with FGFR amplification, translocation or activating mutation [140]. The translocation is present in 5e7% of patients [139,141]. Lastly, V600E-mutant BRAF occurs in around 6e10% of adult glioblastoma, enriched in giant cell [142] and epithelioid [143] subtypes. The B-Raf inhibitors vemurafenib and especially dabrafenib show penetration into cerebral metastases. Following experience in melanoma, vertical combination with MEK inhibition is probably more effective than single-agent therapy [144]. Preclinical

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modelling was encouraging [145,146], but no adult trials are currently registered. Notably colorectal cancer harbouring V600E-BRAF is poorly responsive to vemurafenib alone [147] and in EGFR-expressing tumours such as colorectal cancer and glioblastoma, parallel pathway inhibition by upstream EGFR blockade may also be required [148]. Despite some promising new avenues, it should be noted that to date clinical results with signal transduction inhibitors in glioblastoma have been disappointing. There are several possible explanations. The first is that tissue drug concentrations are not reaching sufficient levels for target inhibition. Although molecules of various chemistries and size seem to be able to enter parts of the tumour where the BBB is disrupted, denoted by contrast leakage, it is increasingly clear that good CNS penetration is required to access all elements of the glioblastoma process, including cell islands behind the BBB [149]. There is evidence that alternative dosing strategies can improve brain penetration [150]; but a prerequisite for progress is that clinical trials in neurooncology routinely assess brain drug delivery, which is often lacking from protocols. The second is that there is considerable clonal heterogeneity [151,152] in glioblastoma, meaning variable target prevalence. The third is that there is hierarchical heterogeneity [153] in tumours, which can mean a variable gulf between the achievement of signalling pathway disruption and a cell death response. Fourth, redundancy [25], vertical and horizontal compensation [154,155] and direct resistance mechanisms [156] are now welldocumented sources of failure for single target interventions, and may even accelerate growth [155]. Fifth, signalling pathways are continuing to reveal new complexities. TCGA multi-modal analysis has shown that PIK3CA mutations are associated with paradoxically lower phosphoAKT protein expression [25]. In the presence of EGFRvIII, EGF is anti-proliferative at the wild-type receptor [157]. The targeted therapy paradigm may prove most likely to give clinically meaningful results if based on highly brain-penetrant, durable inhibition of multiple kinase pathways, regardless of primary gene defects: essentially a generalised cytotoxic strategy, but a challenge to trial design. Large-scale molecular dissection of primary and secondary glioblastomas has yielded at least two further therapeutic targets. The initial discovery of mutations in isocitrate dehydrogenase 1 (IDH1) [112,158], and more rarely the IDH2 isoform, led to definition of a distinct class of low-grade glioma and secondary glioblastoma [159] with the glioma CpG island methylator phenotype (G-CIMP). There are intriguing new treatment implications, including (i) direct targeting of the mutant enzyme, which is in development [160], and (ii) demethylation with decitabine or azacytidine [161,162]. Demethylators also enhance responses to chemotherapy [163,164], showing synergy with temozolomide in one model [165], although potentially counter to this effect, MGMT expression is predictably de-repressed [164]. Secondly, the pre-eminence of telomerase reactivation [25] may raise interest in use of the clinical-stage inhibitor, imetelstat, as well as indirect approaches [164]. Immunotherapy is the subject of intense investigation in glioblastoma, with over 50 trials in progress or having

recently completed internationally. IMA950 is a vaccine containing 11 generic glioblastoma tumour-associated peptides, selected from over 3000 based on therapeutic potential [166]. A phase I/II trial completed accrual ahead of schedule in 2013 and results are awaited. Rindopepimut is a 14-mer peptide vaccine eliciting responses to EGFRvIII. ACT IV is a phase III trial of rindopepimut in newly diagnosed glioblastoma, due to complete recruitment in November 2016. DCVax-L is an autologous dendritic cell plus autologous tumour lysate therapy. It has progressed the furthest in clinical trials among multiple dendritic cell therapies and a large phase III trial is recruiting in the USA and Europe, including the UK [167]. At least one UK trial of immune checkpoint inhibition accepting glioblastoma patients is due to open imminently and several are in progress in the USA. Notably the full complexities of immune/inflammatory reactions in glioblastoma remain to be described and it is not certain that effects in systemic malignancies will translate [168]. Because of the proven intratumoural heterogeneity of glioblastoma [152] there is a strong case for continued emphasis on non-selective treatment approaches. Radiotherapy dose escalation has repeatedly failed to show benefits [169,170], but biological modification of radiation’s effects may develop alternative routes to better disease control without counterproductive brain injury. The poly(ADPribose) polymerase (PARP) inhibitor olaparib increases the cytotoxicity of radiation by increasing levels of double-strand breaks [171]. Following a lead-in phase, which showed tumour penetration of the agent, the OPARATIC trial is assessing the tolerability and efficacy of adding olaparib to temozolomide and the PARADIGM trial is investigating the addition of olaparib to palliative-dose radiotherapy. ATM and ATR inhibitors are in development. Cell cycle checkpoint inhibition with PD-0332991 (palbociclib) has shown preclinical promise [172,173] and is in trial for RB-deficient glioblastoma in the USA [174]. A second current trial theme concerns autophagy inhibition. Injury responses in the CNS are biased towards tissue preservation rather than cell death. It has been hypothesised that autophagy inhibition may improve therapy effects, particularly against tumour-initiating cells [175]. A trial of hydroxychloroquine combined with palliative radiotherapy is currently recruiting in the UK. The clinical trial process in glioblastoma, given the plethora of new agents and testable hypotheses, is in need of special attention. Notably the drug development of temozolomide for glioblastoma followed observations of responses in two patients with high-grade glioma in an initial all-comers phase I trial [176]. There exists the opportunity to encourage phase I trial designs to accept more neurooncology patients, who have often been excluded for perceived rather than actual risks. Referral of patients to major phase I units is an option [177,178], essentially exposing glioblastoma opportunistically to new agents, although experienced neurooncology input is an ongoing requirement for response assessment, hypothesis refinement and specialist multi-professional care. Two main caveats in standard all-comer, advanced disease phase I trial design are that: (i) more heterogeneous, resistant disease will have evolved; and (ii) the opportunity for

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pharmacokinetic/pharmacodynamic assessments on surgical tissue is more limited. Both of these aspects at best hinder clear interpretation and worse may lead to premature agent rejection. Dedicated, commercially driven neurooncology studies are relatively uncommon in glioblastoma owing to the rarity of the disease, making investigator-led initiatives particularly important. Arguably, good CNS pharmacology should be a primary consideration in agent selection. Less arguably, it is important that CNS drug uptake and effects are comprehensively assessed in order to harvest maximal information and guide iterative drug development, including novel CNS-specific dosing strategies. Of the various stages for potential trial intervention in the natural history of glioblastoma, primary and subsequent debulking surgery present important scientific opportunities [136], but require close co-operation between oncologists and neurosurgeons, including wider adoption of elective surgery [179]. Multi-arm trial designs have gained major attention recently [180], offering more rapid hypothesis testing and agent throughput. Biomarker guidance is often promoted as a salient feature of these trials, but with a few exceptions it has yet to be shown that molecular descriptors can be matched with a full range of tailored therapies. In adult glioblastoma, MGMT methylation is an obvious dichotomiser, the unmethylated promoter predicting a poor response to temozolomide and justifying alternative radiation partners in the first-line setting. The low prevalences of actionable single driver lesions in FGFR and BRAF support a master protocol approach, recruiting many hundreds of patients, allocating targeted drug approaches where scientifically supported, and otherwise non-guided arms of other investigational agents and agent combinations with prospective evaluation of candidate biological markers and taxonomies.

Conclusions for Practice and Research Temozolomide remains the mainstay of drug treatment for glioblastoma, and there are arguments for exhaustion of temozolomide over at least 6 months of therapy [181,182] for primary and first-line relapsed disease to confirm treatment refractoriness before switching therapy. Growing evidence suggests that single-agent lomustine is a reasonable alternative to the PCV schedule [11,12], offering at least a reduction in drug-specific and general toxicities and therefore optimal dosing of the most effective agent. Anecdotally, the use of bevacizumab in glioblastoma has waned recently, possibly due to negative sentiment after the failed first-line trials, and in the UK also perhaps due to a change in high-cost cancer drug funding procedures. However, the biology of recurrent disease is different from that of primary glioblastoma (intracavitary VEGF levels are over seven-fold higher in recurrent disease [183]) and for some patients, bevacizumab can give dramatic, clinically meaningful effects in late-stage illness. For the practising clinician, is it reasonable to pursue extraordinary funding for bevacizumab for patients with advanced, pre-treated glioblastoma? In the absence of baseline predictive biomarkers, the decision currently rests on functional state,

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capacity for benefit and perhaps high corticosteroid requirement. Although the evidence base is unconvincing to non-specialist review panels, arguments can be successfully made for a limited initial number of treatment cycles alongside an ‘exceptionality’ case. Early follow-up imaging may provide a guide to ultimate benefit [86,87], and in practice clinical responses, when they occur, do so rapidly, allowing major steroid dose reduction. Issues relating to drug costs and biomarker-based patient selection remain to be resolved. The combination of bevacizumab with lomustine is a highly promising investigational schedule and will be assessed in a forthcoming European Organization for Research and Treatment of Cancer (EORTC) phase III trial. Malignant glial cells are driven by similar mechanisms to those that characterise other, more common cancers [1]. Some of these common drivers are ‘druggable’. However, more broadly it has yet to be shown that molecular descriptors, sampled from complex tumours, have a generalised capacity to predict effective treatment, due to factors such as network complexity in signalling pathways, tumour heterogeneity and cell death and microenvironmental contexts. A current challenge is to decide whether to pursue clinical investigation of the numerous remaining members of signalling pathway inhibitor classes in which other drugs have failed, and whether to devote effort to revisiting the failed drugs to properly assay and address CNS pharmacokinetic/pharmacodynamic characteristics. Without these pharmacological building blocks, the experimental space for combination therapy will be excessively large. Beyond this targeted approach, optimal treatment strategies may not directly infer from cellular oncogenic mechanisms, and attention will need to shift to more general ways to attenuate the malignant process, based on a better understanding of the organ context (including reactivated embryonic programmes) and recruitment of intra- and extracranial cells, which effectively ‘repair’ the tumour. A comprehensive description of glioblastoma biology remains a work in progress, but is the essential basis for more effective treatment paradigms.

Acknowledgments Some of the concepts in this review were discussed at a meeting of the National Cancer Research Institute (NCRI) Glioma Network in London on 3 October 2013. The following were additional contributors to this meeting: Michael Brada, Peter Collins, Oliver Hanemann, Darren Hargrave, Kathreena Kurian, Catherine McBain, Alan Melcher, Michael Olson, Paolo Salomoni, Frank Saran, Richard Shaffer, Susan Short and Tracy Warr. Abigail Evans (The Brain Tumour Charity) and Joanne Rickett (NCRI) provided project support.

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