Establishing A Preclinical Multidisciplinary Board for Brain Tumors

SUMMARY

Purpose

Curing all children with brain tumors will require an understanding of how each subtype responds to conventional treatments and how best to combine existing and novel therapies. It is extremely challenging to acquire this knowledge in the clinic alone, especially among patients with rare tumors. Therefore, we developed a preclinical brain tumor platform to test combinations of conventional and novel therapies in a manner that closely recapitulates clinic trials.

Experimental Design

A multidisciplinary team was established to design and conduct neurosurgical, fractionated radiotherapy and chemotherapy studies, alone or in combination, in accurate mouse models of supratentorial ependymoma (SEP) subtypes and choroid plexus carcinoma (CPC). Extensive drug repurposing screens, pharmacokinetic, pharmacodynamic and efficacy studies were used to triage active compounds for combination preclinical trials with ‘standard-of-care’ surgery and radiotherapy.

Results

Mouse models displayed distinct patterns of response to surgery, irradiation and chemotherapy that varied with tumor subtype. Repurposing screens identified three hour infusions of gemcitabine as a relatively non-toxic and efficacious treatment of SEP and CPC. Combination neurosurgery, fractionated irradiation and gemcitabine proved significantly more effective than surgery and irradiation alone, curing one half of all animals with aggressive forms of SEP.

Conclusions

We report a comprehensive preclinical trial platform to assess the therapeutic activity of conventional and novel treatments among rare brain tumor subtypes. It also enables the development of complex, combination treatment regimens that should deliver optimal trial designs for clinical testing. Post-irradiation gemcitabine infusion should be tested as new treatments of SEP and CPC.

INTRODUCTION

Despite decades of research, the treatment of brain tumors has remained largely unchanged. These cancers are treated with an aggressive combination of neurosurgery, radiation and chemotherapy that frequently fails to cure but inflicts significant side effects (1–4). This limited progress has occurred despite an active clinical trials effort: more than 2,580 brain tumor trials are currently registered with clinicaltrials.gov; but only six drugs are approved for treatment of brain tumors, of which only two – Everolimus, an inhibitor of the Mammalian Target of Rapamycin (5) and Bevacizumab, an inhibitor of Vascular Endothelial Growth Factor A (1) – are molecular targeted treatments.

So why have we failed to identify effective new brain tumor therapies? One possibility is that the preclinical systems used to select drugs for clinical trial do not predict therapeutic activity in patients (6). This explanation is plausible when one considers that most preclinical studies are conducted in mice harboring subcutaneous brain tumor xenografts that cannot recapitulate accurately the pharmacology or biology of brain tumor treatment. Furthermore, while brain tumor patients receive complex, multimodality therapy, mice in preclinical studies usually receive drugs as monotherapies. Such studies are unlikely to predict the survival benefit of a new treatment above that afforded by standard-of-care. Prioritizing treatments with the greatest potential for clinical efficacy is especially important for rare tumors that have limited patient populations available for clinical trial.

Modifying long established treatment regimens that have evolved empirically over many years is also challenging. For example, the treatment of supratentorial ependymoma (SEP) and choroid plexus carcinoma (CPC) – two rare pediatric brain tumors – has evolved over decades to include maximum surgical resection and postoperative cranial irradiation (7–14). These treatments are effective, but evidence suggests this efficacy varies with tumor subtype. For example, whilst most SEPs containing the C11ORF95-RELA translocation (hereon, SEP-CR[+]) resist combination surgery and irradiation, the majority of SEP-CR(−) tumors are cured with this therapy (11, 15, 16). Despite these differences in treatment sensitivity, ongoing clinical trials are testing whether classic histology SEPs, regardless of molecular tumor subtype, can be cured with total tumor resection alone (NCT01096368). Thus, there is a pressing need to determine if SEP-CR(+) resists surgery, radiotherapy, or both. Such knowledge is also important if we are to combine conventional and novel therapies to better treat these tumors; however, this knowledge is unlikely to be acquired solely in the clinic, especially given the rarity of disease variants. Therefore, to better understand the response of SEP and CPC subtypes to surgery and radiation, and to design clinical trials that integrate conventional and new treatments, we established a preclinical multidisciplinary team (pMDT) with the capacity to conduct randomized, multimodality trials in mice harboring accurate models of SEP or CPC.

MATERIALS AND METHODS

Tumor cells and implants

The isolation, culture and orthotopic implantation of all mouse and human tumor cells was described previously (15–18). The nomenclature, species, tumor type, driver oncogene and implanted cell number of each xenograft and allograft are provided in Supplementary Table S1. All cells were maintained as in vivo grafts and confirmed by ELISA as mycoplasma negative prior to and following in vitro studies. All animal studies were approved by the Animal Care and Usage Committees at St Jude Children's Research Hospital and the University of Cambridge. As discussed in detail in Supplementary Methods, host mice for all allografts and xenografts were CD-1 nude mice (strain code: 086, Charles River). All preclinical surgery, radiation and chemotherapy studies were performed among randomized cohorts of mice harboring tumors with ≥1e10⁷ photons/sec bioluminescence (16). Tumor progression and treatment response was assessed clinically and by weekly bioluminescence (16). Mice displaying signs of excessive clinical morbidity (≥20% weight loss and/or neurological impairment) were euthanized.

Preclinical neurosurgery, radiotherapy and chemotherapy

Following baseline bioluminescence imaging, mice were appropriately anaesthetized, a craniotomy fashioned over the site of maximum bioluminescence, and tumors resected using a small suction tip. Post-operative hemostasis was achieved with thrombin-soaked gel foam prior to skin closure. Mice were re-imaged in the immediate post-operative period, monitored on heating pads, and treated for three days with ibuprofen supplemented drinking water, dexamethasome (0.6 mg/kg/6 hours) and mannitol (100 mg/kg/6 hours). 54Gy of radiotherapy was delivered to appropriately anaesthetized mice as 2Gy/day fractions via an orthovoltage irradiator or image-guided rodent irradiator (SARRP, Xstrahl). Drugs were delivered via tail vein bolus injections or using Alzet pumps (2001D, mean pumping rate ~ 8.0 µL/h; loaded with 150 mg/mL gemcitabine solution prepared in 50:50 PEG300: propylene glycol; Supplementary Methods; Supplementary Table S2). For combination surgery-radiotherapy or surgery-radiotherapy-chemotherapy studies, mice were rested for 72 hours in between therapeutic modalities.

Pharmacokinetic and pharmacodynamics studies

Pharmacokinetic studies are described in detail in Supplementary Methods. Briefly, blood samples were collected from euthanized mice via cardiac stick into tubes containing tetrahydrouridine (THU, final concentration 150 µg/mL). Plasma was separated and samples were stored at −80°C until analysis. Intracranial microdialysis studies were performed as described previously (19). A guide cannula (MD-2255, BASi, West Lafayette, IN) and allografted tumor cells were implanted stereotactically in the cortex of immunocompromised mice. Once tumors formed, a pre-calibrated microdialysis probe (MD-2211, BASi; 38 KDa MWCO membrane) was implanted through the microdialysis guide cannula and perfused with artificial cerebrospinal fluid (aCSF; 0.5 µL/min). Mice were dosed with gemcitabine and plasma samples collected via retro-orbital bleeds. Drug levels were measured using a validated high performance liquid chromatography-mass spectrometry method. Tumor cell proliferation and apoptosis were assessed by immunohistochemical quantification of Ki67 and Caspase 3, respectively (Supplementary Methods).

In vitro drug testing

High-throughput screens (HTS) were performed by seeding tumor cells in 384-well plates as described in Supplemental Methods and previously (19). Each plate included dilution series of test compounds (8.3 µM to 0.5 nM), DMSO only negative controls and cyclohexamide or bortezomib single point (0.5 µM) and dose-response (0.5 µM to 0.01 nM) positive controls. Cell number was determined in each well using the Cell Titer Glo reagent (Promega). All assays were conducted in triplicate. Wash-out studies were similarly performed to assess the minimum time-concentration exposure required to inhibit cell growth by 50% by replacing drug-containing medium with fresh medium 1, 3, 6, 10, 24, or 72 hours after dosing. Tumor cell apoptosis was assessed by fluorescence-activated cell sorting (FACS) to detect Annexin V staining (apoptosis) and DAPI staining (DNA integrity).

RESULTS

Preclinical multidisciplinary brain tumor board

We recruited from our clinical MDT, a pMDT comprising statisticians, biologists, chemists, pharmacologists and clinicians. The pMDT met weekly to design, conduct and review preclinical studies that closely recapitulate multimodality clinical trials (Figure 1). Trial statisticians ensured appropriate randomization of tumor bearing animals and statistical powering of study arms; neurosurgeons performed all mouse neurosurgery; radiation oncologists prescribed and delivered fractionated radiotherapy to mice; clinical pharmacologists and oncologists guided trial drug doses and schedules; and radiologists and small animal imaging specialists evaluated treatment response. The pMDT adhered to strict, pre-agreed, standard operating procedures that dictated the progress of therapies through the preclinical pipeline (Figure 1). Preclinical trial data was accessible to all pMDT members in real-time via a centralized electronic mouse medical record.

Figure 1. Composition of the preclinical Multi-Disciplinary Tumor Board and the multistep approach taken to develop new treatment approaches.

BBB=blood brain barrier; R=randomization.

Using cross-species functional genetic screens we previously generated a series of orthotopic, genetic mouse (m) and human xenograft (x) models that recapitulate the histology, transcriptome and growth of SEP-CR(+), SEP-CR(−) and CPC tumors (mSEP-CR[+], xSEP-CR[+], mSEP-CR[−]^RTBDN, mSEP-CR[−]^EPHB2, mCPC; Supplementary Table S1) (15–18). Since clinical trials frequently employ magnetic resonance imaging (MRI) to assess treatment response, we first confirmed that bioluminescence (our preferred method of imaging) and MRI provide equivalent measures of tumor volume in our mouse models (R²=0.96, p<0.0001; Figure 2A,B). Armed with these data and the survival rates of 294 tumor bearing mice, pMDT statisticians then employed the Wilcoxon rank-sum test and Noether’s power formula to design studies with a >83% power to detect a significant survival difference between animals receiving test or control treatment.

Figure 2. Preclinical brain imaging and neurosurgery.

A, Concurrent MRI (top) and bioluminescence (bottom) imaging of the same mouse with a SEP-CR(+) tumor. B, Correlation of MRI and bioluminescence imaging of the same cohort of five mice with SEP-CR(+) tumors. C, Preclinical mouse neurosurgical protocol. D–H, (left) bioluminescence measurement of tumor growth. In parenthesis are the days (d) when treated tumor volume was significantly (p<0.05, non-parametric) less than control. (Right) survival curves of mice with indicated animal numbers and tumor types treated with surgery or control. P value=Log Rank relative to control.

Pre-clinical neurosurgery

To test the therapeutic value of surgery in our models, we established cohorts of mice harboring mSEP-CR(+), xSEP-CR(+), mSEP-CR(−)^RTBDNa, mSEP-CR(−)^RTBDNb or mCPC as described previously (15, 16, 18). Mice bearing equivalent sized tumors were then randomized to undergo microscope-guided tumor resection by neurosurgeons or anesthesia alone (Figure 2C). Gross total resection (≤10% residual post-operative bioluminescence) was achieved in 100% (n=14/14), 64% (n=9/14), 51% (n=24/47), 71% (n=15/21) and 43% (n=13/30) of mice harboring mSEP-CR(+), xSEP-CR(+), mSEP-CR(−)^RTBDNa, mSEP-CR(−)^RTBDNb or mCPC, respectively; recapitulating the total resection rates of these tumors in children (Figures 2D–H) (11–14). Surgical resection of mSEP-CR(+) and xSEP-CR(+) produced only transient, significant reductions in tumor volume and these tumors regrew rapidly following total resection, resulting in no overall survival advantage (Figure 2D,E). In contrast, total resection produced sustained, significant reductions in the volume of mSEP-CR(−)^RTBDNa and mSEP-CR(−)^RTBDNb and increased the survival of mice harboring these tumors, curing some animals (Figure 2F,G). Thus, the relatively poor prognosis of patients with SEP-CR(+) may in part reflect the failure of surgery to control these tumors (7). Gross total resection is generally regarded as optimal therapy of CPC, although this has not been demonstrated definitively because the disease is so rare (12, 13). In support of this notion, total resection significantly reduced tumor burden for around one week and marginally, but significantly, extended the survival of mice with mCPC (Figure 2H).

Pre-clinical fractionated radiotherapy

Post-operative cranial irradiation has been a mainstay of ependymoma therapy for decades and is used to treat some patients with CPC (7–9, 11–13). To test the efficacy of radiotherapy in our mouse models, we randomized mice with equally sized mSEP-CR(+), xSEP-CR(+), mSEP-CR(−)^RTBDNa, mSEP-CR(−)^RTBDNb, or mCPC to receive 27 daily fractions of 2Gy cranial irradiation (mimicking that given to patients) or mock treatment (Figure 3A). In stark contrast to surgery, radiotherapy significantly impaired the growth of all SEP-CR(+) and SEP-CR(−) models relative to controls for between 2 and 10 weeks, resulting in a significant survival advantage for treated mice (Figure 3B–E). Notably, regrowth of mSEP-CR(+) and mSEP-CR(−)^RTBDN was observed before the end of radiotherapy; suggesting the emergence of resistant clones, potentially explaining why this treatment ultimately failed. Conversely, and in agreement with the limited radio-sensitivity of infant CPC, radiation only transiently impaired mCPC growth and had no therapeutic efficacy against this tumor (Figure 3F).

Figure 3. Preclinical fractionated surgery and irradiation.

A, Preclinical mouse radiation protocol. B–F, (left) bioluminescence measurement of tumor growth. In parenthesis are the days (d) when treated tumor volume was significantly (p<0.05, non-parametric) less than control. (Right) survival curves of mice with indicated tumor types treated with fractionated irradiation or control. G, Preclinical combination mouse surgery and radiotherapy protocol. H–J, Bioluminescence measures of tumor growth (left) and survival curves (right) of mice with the indicated tumor types treated with surgery with or without fractionated irradiation (P values are Log Rank relative to control. Comparisons between treatments are Log Rank not significant (ns), P<0.005(**), P<0.0005(***), P<0.00005(****).

Having evaluated the efficacy of surgery and radiotherapy independently, we conducted a series of combination studies to determine the benefit of combining these modalities (Figure 3G). Although surgery alone did not benefit mice harboring mSEP-CR(+), post-operative irradiation significantly prolonged tumor control in these animals relative to radiotherapy alone, resulting in cures for almost half of all treated mice (Figure 3H). In contrast, surgical resection of xSEP-CR(+) did not prolong the survival of mice with this tumor relative to those treated with irradiation alone; possibly reflecting the rapid regrowth of these tumors following surgical debulking, resulting in a shorter period of tumor control overall (Figure 3I). However, combination surgery and irradiation significantly impaired the growth of mSEP-CR(−)^RTBDNa relative to surgery alone and extended the survival of mice with these tumors beyond that achieved with either therapy alone (Figure 3J). Combination surgery and radiotherapy was not attempted in mCPC since this tumor resisted both treatments.

Repurposing of chemotherapy

Having established the value of surgery and radiotherapy among our models of SEP and CPC, and shown that the pattern of response to these treatments approximates that observed in patients, we looked to see if our models might be useful for developing chemotherapies. Using an integrated in vitro and in vivo screen that we deployed previously to identify potential brain tumor treatments for clinical trial, we screened 114 drugs that are FDA approved or currently in clinical trial (19, 20). mSEP-CR(−)^RTBDNb and mCPC cells were chosen for these studies since they represent relatively responsive and resistant tumor types, respectively. In line with their relative resistance to treatments, 40 drugs inhibited the proliferation of mSEP-CR(−)^RTBDNb cells by 50% (IC50) at concentrations ≤1µM after 72 hours in vitro compared with only 26 drugs against mCPC cells: 22 of these drugs had IC50 ≤1µM against both cell types (p<0.0001, Fisher’s exact for overlap; Figure 4A).

Figure 4. Preclinical repurposing of chemotherapies.

A, Repurposing screen of 114 FDA approved and/or clinical trial drugs. Heatmap (left) reports 72 hour IC50 against the indicated cell type; grey bars (middle) indicate FDA approved drugs; graph (right) reports number of completed trials of each drug. Arrows denote drugs selected for further study in (B). B, ‘Washout studies’: heatmaps report IC50 values after timed exposures of the indicated cell types to drug. C–D, (top) bioluminescence measurement of tumor growth. In parenthesis are the days (d) when treated tumor volume was significantly (p<0.05, non-parametric) less than control. (Bottom) survival curves of mice with indicated tumor types treated with the indicated drug monotherapy. Comparisons between treatments are Log Rank not significant (ns) and P<0.0005(***).

Thirteen drugs with IC50 values ≤1µM at 72 hours were then subjected to ‘washout’ studies to determine the minimum concentration-time exposure required to inhibit cell proliferation (Figure 4B). Exposure to <1µM of cabazitaxel, pralatrexed, gemcitabine, panobinostat, carfilzombib or vosaroxin for just one hour inhibited the proliferation of both mSEP-CR(−)^RTBDNb and mCPC cells by >50%; chaetocin was similarly active against mCPC while the IC50 of acivicin after one hour exposure almost achieved 50% inhibition in the ≤1µM range. Published pharmacokinetic data indicated that cabazitaxel, gemcitabine, pralatrexed and pemetrexed would penetrate the central nervous system (CNS) and provided appropriate doses and scheduling for vosaroxin, chaetocin and acivicin (20–32). Therefore, to select which of these drugs might be suitable for further preclinical development, the pMDT designed and conducted a series of monotherapy preclinical trials in mice harboring mSEP-CR(−)^RTBDNb or mCPC. The goal of these studies was to look for any evidence of antitumor activity (growth and/or survival). Doses and schedules of each drug were designed to mimic those achievable in patients. We also conducted a monotherapy study of cladribine as a ‘negative control’ compound since this drug was relatively inactive in vitro and was predicted not to penetrate the CNS. Of all agents tested, only gemcitabine (120mg/kg intravenous bolus) displayed significant activity against both mSEP-CR(−)^RTBDNb and mCPC: this treatment was the only monotherapy to significantly impair the growth of mSEP-CR(−)^RTBDNb and to prolong the survival of mice with these tumors; therefore this drug was selected for further repurposing studies (Figure 4C,D). Vosaroxin – a topoisomerase II inhibitor causing site-selective DNA damage – also produced a modest but significant survival advantage for mice harboring mSEP-CR(−)^RTBDNb tumours (Figure 4C). These data underscore that drugs with relatively potent activity in vitro may lack efficacy in vivo when administered at clinically relevant doses. In light of the considerable activity of gemcitabine, we selected this drug for further preclinical development.

Optimization of gemcitabine therapy

Gemcitabine can be administered as an intravenous bolus or infusion, resulting in very different pharmacokinetic profiles (33). Therefore, we treated mice bearing mSEP-CR(−)^RTBDNb or mCPC with various gemcitabine regimens and simultaneously measured concentrations of the drug in plasma and brain tumor extracellular fluid (tECF) using intratumoral microdialysis. Mice were treated initially with two clinically relevant gemcitabine regimens: 60mg/kg intravenous (iv) bolus that is active against a mouse model of Group 3 medulloblastoma; or continuous three hour infusion via subcutaneous Alzet pumps (19, 20). 60mg/kg iv bolus gemcitabine produced a plasma AUC_0–6hr of 25.9 µM*hr that is equivalent to that observed in children treated with 1200 mg/m² (Figure 5A) (34). The tumor to plasma partition coefficient for unbound gemcitabine (K_p,uu) at this dose was 0.51 and 0.18 for mice bearing mSEP-CR(−)^RTBDNb and mCPC tumors, respectively. The gemcitabine concentration in tECF produced by this regimen, only remained above the in vitro washout IC50 of each tumor type for less than 3 hours (compare Figures 4B and 5A). In contrast, 3 hour infusions of gemcitabine produced plasma exposures of 95.1 ± 24.1 µM*hr – equivalent to treating children with 2000 mg/m² – and in both models maintained a tECF concentration above the IC50 in washout studies for ≥7 hours (compare Figures 4B and 5B). To determine if these in vivo exposures produce the anti-tumor cell effects predicted in vitrowe harvested tumors from mice at 3, 8, 24 and 48 hours following initiation of gemcitabine therapy and estimated levels of tumor cell proliferation and apoptosis. Three hour infusions of gemcitabine induced significantly greater and more sustained levels of tumor cell apoptosis in mSEP-CR(−)^RTBDNb and mCPC than did 60mg/kg intravenous bolus treatment, and 3 hour infusions produced a more significant and sustained reduction in tumor cell proliferation, although this was only observed in mSEP-CR(−)^RTBDNb (Figure 5C; Supplementary Figure 1).

Figure 5. Pharmacokinetic, toxicity and efficacy studies of gemcitabine.

Plasma and tECF concentration-time plots in the indicated tumor types treated with 60mg/kg bolus (A) or 3 hour infusion (B) gemcitabine. C, Graphs showing induction of tumor cell apoptosis measured by cleaved Caspase 3 immunohistochemistry in mice with the indicated brain tumors treated with 60mg/kg or 3 hour infusion of gemcitabine. D, Toxicity determined by weight loss in mice treated with the indicated doses and regimens of gemcitabine (in C, D: *, p<0.05; **, p<0.005; ***, p<0.0005, Mann-Whitney), E–J, (left) bioluminescence measurement of tumor growth in mice treated with indicated dose and schedule of gemcitabine. In parenthesis are the days (d) when treated tumor volume was significantly (p<0.05, non-parametric) less than control. (Right) survival curves of the same mice shown left. P value=Log Rank relative to control.

As a final step to select the optimal dose and schedule of gemcitabine for preclinical assessment, we further expanded the repertoire of gemcitabine regimens to assess the relative activity of 200mg/kg bolus and 6 hour infusions (n≥10 mice per cohort; Figure 5D–F). Of all regimens tested, 3 and 6 hour infusions of gemcitabine were most efficacious, producing similar degrees of tumor growth suppression and enhanced overall survival; however, 3 hour infusions proved the least toxic. Additional 3 hour gemcitabine infusion monotherapy trials identified significant active against mSEP-CR(+), xSEP-CR(+) and a mSEP-CR(−) model driven by EPHB2 (Figure 5G–I) (17). Three hour gemcitabine infusions were more efficacious than combination cisplatin/cyclophosphamide or cisplatin/etoposide/vincristine that approximates ‘standard of care’ chemotherapy regimens that have been tested against ependymoma and CPC, respectively, in the clinic (Figure 5J,K) (7, 35). Therefore, we selected 3 hour infusions of gemcitabine for our final phase of preclinical repurposing.

Combining gemcitabine and conventional therapy

The efficacy of gemcitabine monotherapy in our model systems suggests it may have value as an adjuvant therapy in the clinic. Therefore, the pMDT designed a combination study aimed at testing the value of adding 3-hour gemcitabine infusions to ‘standard-of-care’ surgery and fractionated radiotherapy (Figure 6A). With regard to ependymoma, we focused on SEP-CR(+) disease since this tumor type is the most aggressive form of SEP. The mSEP-CR(+) rather than xSEP-CR(+) model was chosen for these studies since these models displayed similar responses to surgery, radiation and gemcitabine as monotherapies, but the more rapid growth profile of mSEP-CR(+) enabled completion of these large combination studies in a timely manner. Mice were treated with GTR followed by 54Gy fractionated irradiation and then 3 weeks of consecutive 3 hour gemcitabine infusions. This treatment was tolerated remarkably well. Although the average tumor burden of mice receiving gemcitabine was lower than that of animals treated with surgery and irradiation alone, this difference was not significant (Figure 6A); however, the addition of gemcitabine doubled the median survival (183 days) of mice relative to those treated with surgery and irradiation alone (96 days), and cured 50% of animals (p<0.00001; Figure 6B). These data underscore the need to assess both tumor volume and animal survival as response metrics to preclinical therapy since tumor imaging in small animals may operate at the limits of resolution. We next assessed the value of adding serial post-operative, 3-hour gemcitabine infusions to the treatment of mCPC (Figure 6C,D). As observed previously, gross total tumor resection alone produced a modest but significant survival advantage for mice harboring mCPC (median survival surgery=34 days vs control=22 days; P<0.003; Figure 6D); and gemcitabine therapy alone markedly extended the survival of mice with these tumors (median survival gemcitabine=42 days vs control=22 days; P<0.0001; Figure 6D). Notably, no significant difference in survival was observed between mice undergoing surgery or gemcitabine therapy alone; however, surgical resection followed by gemcitabine significantly extended survival above that of animals receiving surgery alone (median survival surgery alone=44 vs surgery+gemcitabine=46.5 days; P<0.0001; Figure 6D). Together, these data suggest that 3 hour infusions of gemcitabine may add therapeutic value to ‘standard-of-care’ surgery and radiation in the treatment of SEP and may improve the results of surgical resection of CPC. We propose that these regimens should be tested in the clinic.

Figure 6. Combination surgery, fractionated irradiation, and post-irradiation gemcitabine therapy.

Bioluminescence measures of tumor growth (A) and survival curves (B) of mice with mSEP-CR(+) treated with surgery and radiation alone or surgery, radiation and 3 hour infusions of gemcitabine. Bioluminescence measures of tumor growth (C) and survival curves (D) of mice with mCPC treated with surgery and 3 hour infusions of gemcitabine alone or surgery and gemcitabine. Figures in paraenthesis in bioluminescence plots are the days (d) when treated tumor volume was significantly (p<0.05, non-parametric) less than control.

DISCUSSION

The past decade has witnessed a revolution in our understanding of human cancer. The integration of genomic and developmental biology has shown that morphologically similar cancers comprise subtypes, driven by different genetic alterations, which likely arise within distinct cell lineages (36). These data help explain why cancers once regarded histologically as homogeneous diseases display discrepant behaviors. For example, medulloblastoma and ependymoma are now known to include subtypes with extraordinarily good (e.g., WNT-medulloblastoma and SEP-CR[−]) or bad (Group 3-medulloblastoma with MYC amplification and SEP-CR[+]) prognosis (11, 37). This knowledge could pinpoint patients who might be cured with less toxic therapy, as well as poor prognosis patients who need new treatments. Indeed, clinical trials of decreased radiotherapy are ongoing among patients with WNT-medulloblastoma (NCT01878617). But integrating understanding of tumor biology into established clinical practice is enormously challenging and requires a number of assumptions that are often made without knowledge of subtype-specific treatment efficacy. For example, reducing radiotherapy for children with WNT-medulloblastoma assumes that this therapy, rather than surgery or chemotherapy, is relatively redundant. And trials of new treatments for ‘poor prognosis’ tumors often assume that relatively ineffective conventional therapies should be retained; this approach runs the risk of increasing toxicity unnecessarily.

So how can we integrate new understanding of cancer biology and therapy into empirical treatment regimens that have developed over decades? It is unlikely that we will achieve this through clinical trials alone, especially among patients with rare disease variants: the small populations of patients with these tumors limit the number of drugs and regimens that can be tested in a timely manner. The preclinical platform described here provides an evidence-based approach to guide clinical trials for rare brain tumor subtypes. It is important to note that this platform is not designed to replace or reduce the clinical trial platform; but rather to better triage drugs so that clinicians can focus on novel regimens with the greatest potential to cure. Key features of our approach include the use of accurate mouse models of human brain tumors and the coordinated engagement of clinical and research professionals in regular pMDT discussions; greatly facilitating the co-development of clinically relevant preclinical trials.

Maximal surgical resection of ependymoma followed by irradiation is consistently associated with a better patient outcome regardless of primary tumor site (8, 10, 11, 14). This observation has led to the widespread notion that SEPs have a high probability of cure with surgery alone, and underpins Arm 1 of an ongoing Children’s Oncology Group study in which children achieving a gross total resection of classic histology SEP receive no further treatment (NCT01096368; ACNS0831). But in our studies, surgery alone had no therapeutic value in the treatment of mSEP-CR(+) or xSEP-CR(+), only benefiting mice with mSEP-CR(−)^RTBDNa. However, total resection of mSEP-CR(+) did markedly improve the efficacy of irradiation, curing a significant number of animals and also improved the survival of mice with mSEP-CR(−)^RTBDNa. These data underscore the important point that treatments can display surprising interactions, producing high cure rates when used in combination that not are not apparent when the treatments are used individually. Our data also support the notion that irradiation is a highly effective treatment of SEP, and suggest that avoiding radiotherapy for all patients with totally resected SEP, regardless of subtype, may be inappropriate. Rather, as a minimum, we recommend the prospective evaluation of SEP molecular subtype in ACNS0831 to ensure that SEP-CR(+) patients are not undertreated. Reverse translation of these clinical data will be critical to validate the predictions made by our preclinical system. This later point is particularly important because our model system is likely to be predictive but not infallible. Indeed, in contrast to mSEP-CR(+), total resection of xSEP-CR(+) did not improve the efficacy of radiotherapy, indicating that further iteration between preclinical and clinical work will be required to understand the ependymoma-subtype specific relevance of combination treatment.

The effectiveness of pre-irradiation surgery in our models also supports the widely held notion that cytoreductive surgery increases radiosensitivity and chemotherapy by removing therapy-resistant, hypoxic, and highly-proliferative tumor cores (38). These data might also explain why resection and irradiation is more effective than partial resection and irradiation among patients with posterior fossa subtype-A ependymoma – another aggressive disease subtype (14). Thus, our models provide an opportunity to explore the biological basis of cytoreductive surgical efficacy. Our models may also facilitate the identification, isolation and study of radiation resistant tumor clones, since our imaging studies revealed regrowth of mSEP-CR(+), xSEP-CR(+) and mSEP-CR(−) prior to completion of radiotherapy.

In contrast to our SEP models, radiotherapy proved ineffective against mCPC. The radio-resistance of mCPC may reflect the Tp53 null status of these tumors, since this gene mediates cell death mechanisms in irradiated cells (39). Notably, 60% of human CPCs contain mutant Tp53: these tumors also tend to be radio-resistant, clinically aggressive and to develop in infants (40, 41). It is interesting that our mCPC model is initiated in embryonic choroid plexus; therefore, these tumors likely model radio-resistant, aggressive and TP53-mutant infant CPC (18).

Although chemotherapy has been evaluated in ependymoma and CPC, its role remains controversial with only limited benefit reported (7, 13). These data are in keeping our observations that most drugs displaying potent activity against our models in vitrofailed to produce therapeutic benefit in vivo. This notion is also supported by our observation that mouse models of SHH-medulloblastoma – a more chemosensitive disease – responded to treatments that were ineffective against mSEP and mCPC e.g., pemetrexed and 60mg/kg bolus gemcitabine (20, 42). Our preclinical in vitro and in vivo pipeline did identify 3 hour infusions of gemcitabine as a potential new treatment of SEP and CPC. This regimen generated tECF concentrations above the in vitro IC50 for ≥7 hours and proved more effective against mSEP-CR(+) and mCPC than combination conventional chemotherapy regimens with reported activity in patients (7, 35). Thus, we suggest that 3 hour infusions of gemcitabine should be tested in patients with SEP and CPC.

Fewer than 150 adults and children with all variants of SEP and CPC are available for enrollment on clinical trials in the United States each year, severely limiting studies of new treatments (43). Indeed, it is widely agreed that the rarity of CPC poses an almost insurmountable hurdle to the efficient development of new treatments through clinical trial (13). For example, the only multi-centre CPC clinical trial conducted to date was initiated in 2000 (CTP-SIOP-2000), but 17 years later the results of this trial are yet to be published. Our preclinical system provides an alternative, evidence-based approach to prioritize combination regimens for the clinic, potentially avoiding years of trials of ineffective therapies. Of particular note, by recapitulating surgery, irradiation and chemotherapy our approach allows for preclinical trials of multiple doses, delivery routes and schedules of novel chemotherapies in the context of standard of care treatment. In this regard, sequential total tumor resection, fractionated radiotherapy and 3 hour gemcitabine infusions doubled the median survival of mice with mSEP-CR(+) relative to surgery and irradiation alone, curing half of all animals. Our studies also provide evidence that combination surgery and gemcitabine infusion therapy may benefit the treatment of CPC. These data are in keeping with the activity of gemcitabine in other chemo-resistant cancers including pancreatic cancer (44, 45). We therefore recommend that gemcitabine infusions might prove effective as post-surgery and irradiation chemotherapy. Furthermore, since gemcitabine may also serve as a radiosensitizer, we are currently exploring the timing of gemcitabine treatment relative to irradiation and whether gemcitabine may be added to conventional treatment regimens in younger patients.

While our model system provides a promising tool to prioritize complex combination treatment regimens for clinical trial, the accuracy of these predictions remains to be assessed. It is a hard reality that most cancer treatments that are effective in animal models, fail in patients (46, 47). Indeed, while our models closely replicate the morphology and transcriptome of the corresponding human tumors, they are maintained in immunocompromised hosts and therefore cannot account for contributions of the host immune system to tumor biology and treatment. Thus, preclinical platforms such as the one presented here require careful iterative study with clinical translation to be validated and refined. This important ongoing process further underscores the value of convening pMDT teams comprising laboratory and clinical oncology professionals.

TRANSLATIONAL RELEVANCE.

Existing drug development pipelines have failed to bring new treatments to children with brain tumors. A lack of faithful preclinical models has prevented the discovery and prioritization of potential new therapies, and the rarity of these diseases presents an insurmountable hurdle for drug development through clinical trial alone. Therefore, we established a preclinical multidisciplinary tumor board comprising biologists, statisticians, pharmacologists and clinicians to conduct preclinical studies that mimic the clinic. Mouse models included those of specific ependymoma and choroid plexus carcinoma subtypes – two rare pediatric brain tumors. In contrast to previous brain tumor preclinical platforms, our approach enables the testing of potential new treatments of very rare tumors, in the context of ‘standard-of-care’ neurosurgery and fractionated irradiation. This approach enables assessment of the potential therapeutic ‘value added’ of candidate treatments and thereby prioritise novel treatment combinations for clinical trial.