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Teaser The development of simultaneous PET/MRI imaging technology is poised to reshape drug development in oncology. In this review, we highlight key advances in PET, MRI, and simultaneous PET/MRI that are driving the development of diagnostic and therapeutic strategies for glioma.

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PET, MRI, and simultaneous PET/MRI in the development of diagnostic and therapeutic strategies for glioma Simon Puttick1,y, Christopher Bell2,3,y, Nicholas Dowson2, Stephen Rose2 and Michael Fay3,4,5 1 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD 4072, Australia 2 CSIRO – The Australian e-Health Research Centre, Level 5, UQ Health Science Building, RBWH, Herston, QLD 4029, Australia 3 School of Medicine, The University of Queensland, 288 Herston Road, Herston, QLD 4006, Australia 4 Genesis Cancer Care, Lake Macquarie Private Hospital, 36 Pacific Highway, Gateshead, NSW 2290, Australia 5 Royal Brisbane and Women’s Hospital, Butterfield Street, Herston, QLD 4006, Australia

Glioma is the most aggressive brain tumour, resulting in death often within 1–2 years. Current treatment strategies involve surgical resection followed by chemoradiation therapy. Despite continuing improvements in the delivery of adjuvant therapies, there has not been a dramatic increase in survival for glioma. Molecular imaging techniques have become central in the development of new therapeutic strategies in recent years. The multimodal imaging technology of positron emission tomography/magnetic resonance imaging (PET/MRI) has recently been realised on a preclinical scale and the effect of this technology is starting to be observed in preclinical drug development for glioma. Here, we propose that PET/MRI will play an integral part in the development of new diagnostic and therapeutic strategies for glioma.

Introduction Glioma is the most commonly occurring primary brain tumour, signified by its invasive potential and increased capacity for proliferation [1]. Despite continuing advances in imaging, neurosurgical and radiation therapy techniques, prognosis remains poor, with a median survival of only 12–18 months [2–5]. The development of novel treatment strategies is integral to overcoming the significant challenge of improving the survival rate for patients with glioma. Ever since the proposal of a ‘magic bullet’ by Paul Erlich [6], the drug discovery process has been heavily focussed on delivering therapy directly to the site of disease. A plethora of molecules have been recruited to the cause, with many shown to have high specificity to a particular target. Biomacromolecules derived from nature have shown particular success and there is a wealth of literature surrounding the use of monoclonal antibodies, fragments of monoclonal antibodies, small chain variable

Corresponding author: Fay, M. ([email protected]), ([email protected]) y

Simon Puttick is a research fellow at the Australian Institute for Bioengineering and Nanotechnology at the University of Queensland. He is currently developing polymeric bionanoconjugates as targeted theranostic agents and new simultaneous PET/MRI strategies for improved diagnosis and treatment planning in oncology. His particular research interest lies in combining parametric MRI methods with novel PET acquisition methods to probe the distribution of potential therapeutic platforms with respect to diagnostic biomarkers in cancer. Christopher M. Bell is a PhD student in medical imaging at the University of Queensland in Australia. He obtained his bachelor degrees in computer software architecture and physics in 2012, graduating from the Queensland University of Technology with distinctions. During this time, he obtained numerous scholarships to investigate the biophysics of articular cartilage. He began his PhD in 2013 in partnership with the Australian e-Health Research Centre of CSIRO in Brisbane, Australia, focussing on glioma, obtaining multiple scholarships, including the prestigious Australian Postgraduate Award. Nicholas Dowson Before joining the CSIRO in Australia, Nicholas Dowson worked at Siemens Molecular Imaging in the UK. His current interests are in developing registration and kinetic analysis algorithms to extract information from medical images to assist clinical decisionmaking and improve outcomes for patients, with a particular focus on PET and oncology. He has a PhD from the University of Surrey. Stephen Rose is a science leader within the CSIRO– Digital Productivity Flagship, one of the leading Australian biomedical image analysis laboratories. He is also acting director of the newly established Herston Imaging Research Faculty (HIRF) in Queensland, Australia. Stephen is currently developing novel MRI and PET molecular imaging platforms in several neuroimaging research programs in oncology, neurodegenerative, and brain development disorders. His particular research interest lies in combining metabolic PET imaging with structural connectomics, improving our understanding of how brain injury or pathology impacts neural networks. Michael Fay is a radiation oncologist at Genesis Cancer Care in Newcastle. He is an honorary senior staff specialist at Royal Brisbane and Women’s Hospital. He is dual trained in medical and radiation oncology and undertakes research in functional imaging applied to oncology. He is the principal investigator of several clinical brain tumour trials through the TransTasman Radiation Oncology Group. He is currently completing his PhD in advanced imaging applied to brain tumour treatment and spent most of 2014 in a radiobiology lab in Tu¨bingen, Germany.

These authors contributed equally to this article.

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18F-FDG

18F-FDOPA

MRI Reviews  KEYNOTE REVIEW

fragments, and peptide or oligonucleic acid aptamers as targeted therapeutics. By conjugation to appropriate moieties, these highly specific molecules can be effectively transformed to target recognised pathways driving cancer [7,8]. Molecular-imaging techniques have been integral to the development of advanced therapeutics in two major capacities. First, by labelling potential therapeutics with imaging modalities, such as optical dyes, positron-emitting atoms or contrast-enhancing molecules for computerised tomography (CT; high X-ray crosssection materials) or MRI (paramagnetic materials), researchers can validate the specificity of a potential therapeutic in vivo by imaging the biodistribution of the material directly. This enables an informed development process where the effects of small, systematic changes to the material on the specificity of delivery can be monitored. Second, by directly imaging diagnostic biomarkers of cancer, the efficacy of therapy can be followed during the treatment process. The potential power of combination therapy in cancer has long been recognised [9] and, as such, a complimentary approach to imaging, multimodal imaging, offers particular power in drug discovery. By combining two imaging modalities, multiple factors relating to the efficacy of a therapeutic platform or multiple therapeutic platforms can be imaged simultaneously. The fusion of two key imaging technologies for drug discovery, PET and MRI, has recently been realised on both the clinical and preclinical scale. Here, we focus on the impact of preclinical PET, MRI, and their simultaneous acquisition on the development of novel therapeutic and diagnostic strategies for glioma.

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PET/MRI

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FIGURE 1

Comparison of tumour-to-brain contrast achievable with 18F-fluorine-2deoxy-D-glucose (18F-FDG) and 6-18F-fluoro-L-dopa (18F-FDOPA). It can be seen that the high uptake of 18F-FDG in nontumour brain results in poor tumour–to-brain contrast. Images were acquired of 12-week-old female NOD/SCID mice bearing a xenograft of 105 U87 cells injected into the right striatum following an injection of either 18F-FDG or 18F-FDOPA mixed with Magnevist injected via the lateral tail vein. Positron emission tomography (PET) and magnetic resonance imaging (MRI) images were acquired on a Bruker 7 T Clinscan interfaced with a Siemens Spectrometer running Numaris/4 VB17 with a PET ring centered at the isocentre of the magnet comprising three rings of 16 crystal blocks.

PET in glioma The use of nuclear imaging for oncology-related drug development has continued to grow, primarily because the information provided is distinctly different to that available through other imaging techniques, such as MRI and CT. The nuclear-imaging techniques of single photon emission computed tomography (SPECT) and PET are intrinsically molecular-imaging techniques because the gamma photon that is detected originates from a point source. As such, with judicious choice of tracer, both techniques can provide specific information about the biology and activity of a tumour. Moreover, with the advent of targeted therapies, PET and SPECT are ideal techniques to study the biodistribution and pharmacokinetics of new potential therapeutics. Of the two techniques, PET offers a significantly higher sensitivity (approximately two to three orders of magnitude higher) because the collimators required for detection of the SPECT signal reject a significant proportion of emitted photons [10]. The increased sensitivity of PET leads to a higher signal:noise ratio (SNR) and, therefore, either a shorter imaging time or a higher quality image. In addition, the higher sensitivity leads to a higher temporal resolution making dynamic PET studies intrinsically more feasible than dynamic SPECT. The spatial resolution of the two techniques is comparable and continues to improve with developments in detector technology [11–15] and reconstruction algorithms [16–20]. However, in most systems, PET offers a higher reconstructed spatial resolution. The high sensitivity, higher spatial resolution, and potential for dynamic imaging make PET the nuclear-imaging modality of choice in a large proportion of clinical workflows. In addition, a large number of PET tracers exhibit a

biological response suitable for probing the metabolic activity of brain structures, in particular abnormalities in the brain, such as gliomas. Furthermore, the ability to fuse PET technology with MRI offers the potential to improve the information content of the technique and is the focus of this review. 18

F-fluorine-2-deoxy-D-glucose: metabolism

By far the most common PET tracer used in the clinic today is the fluorinated glucose analogue 18F-fluorine-2-deoxy-D-glucose (18FFDG). Imaging of cancers with 18F-FDG relies on tumour cells exhibiting a higher glucose metabolism than the surrounding healthy tissues. However, glucose metabolism within normal cortical brain tissues is high, leading to poor tumour–brain contrast (Fig. 1) and making tumour boundary definitions ambiguous. In low-grade gliomas, the tumour can by ‘hypometabolic’ relative to the surrounding brain. However, glucose metabolism is often on par with normal cortical tissues, resulting in false negatives. As such, significant effort has been directed towards the development of alternatives to 18F-FDG [21], with many of these now used as biomarkers to assess the efficacy of potential therapies [22]. In much the same way that glucose metabolism is higher in tumour cells than in surrounding healthy tissue, amino acid uptake is elevated in tumour cells [23,24] relative to cortical tissue and can be linked to cell proliferation [23], a useful diagnostic marker for glioma. Radiolabelled amino acids were first reported during the 1980s as suitable PET tracers for brain tumours [25]. Since this development, several amino acid-based tracers have been shown to be useful imaging agents in glioma. www.drugdiscoverytoday.com

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O-(2-[18F]-fluorethyl)-1-tyrosine: proliferation

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The tyrosine analogue O-(2-[18F]-fluorethyl)-1-tyrosine (18F-FET) has attracted significant attention for imaging glioma because it offers high tumour–brain contrast in both high- and low-grade gliomas [26–28] and is particularly useful for assessing the prognosis of low-grade gliomas [29,30]. It is widely agreed that the uptake of 18F-FET into tumour cells is mediated by amino acid transporters, such as L-amino acid transporter 1 (LAT-1) [31], and that the increased transport of amino acid into tumour cells leads to the high uptake of 18F-FET [32,33]. It has also been shown that this process is highly stereospecific [32], indicating active transport mechanisms. A more detailed coverage of the uptake mechanisms of 18F-FET is covered in a recent review by Langen et al. [34]. It has been shown that tumour:brain ratios of 18F-FET SUVmax can be used as an effective biomarker to detect treatment response [35]. Despite widespread clinical use, the number of studies using 18 F-FET in preclinical models of glioma is limited [35–38], possibly because existing studies have only been predictive of outcome when used longitudinally [39,40]. 18

F-fluorothymidine: proliferation

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F-fluorothymidine (18F-FLT) is a PET tracer analogue of thymidine, which is rapidly transported into cells by nucleoside transporters, such as equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs), to be phosphorylated to thymidine nucleotides, one of the molecular building blocks of DNA [41]. The enzyme thymidine kinase (TK-1) is highly expressed during cell proliferation and leads to the intracellular trapping of 18F-FLT, making it an effective tracer for cell proliferation [41–44]. It has been shown that 18F-FLT uptake correlates with tumour growth [45] and that a decrease in 18F-FLT uptake following treatment corresponds with tumour size reduction as measured by MRI and reduction in bioluminescence in luciferase transfected cell models [46]. In addition, Bradbury et al. showed that 18F-FLT uptake in the tumour was high in a genetically engineered mouse model of high-grade glioma and that kinetic analysis of the time activity curves using a three-compartment, four-parameter model could yield estimates of tracer kinetic parameters that were reflective of tumour proliferation [47]. Although excellent for visualising high proliferation typical of malignant gliomas, 18F-FLT has less efficacy for low-grade glioma with a higher false positive rate compared with 6-18F-fluoro-L-dopa (18F-FDOPA) or even 18F-FDG [48]. In recurrent gliomas, 18F-FLT has been shown to predict progression survival if not overall survival [49].

[Methyl-11C]-L-methionine: proliferation neovascularisation As with 18F-FET, the methionine analogue [methyl-11C]-L-methionine (11C-MET) is taken up into tumour cells by amino acid transporters, such as LAT-1 and LAT-2. It has been shown that uptake of 11C-MET spreads beyond the tumour boundary as identified by MRI or CT [50–54]. In addition, 11C-MET uptake was an earlier marker of vessel remodeling compared with 18F-FLT in a primary line rat model of glioma [55] and there was a strong correlation between uptake of 11C-MET and vessel density [56]. In a further study in a rat model of glioblastoma, Viel et al. showed that, by using MRI-derived functional measures of vasculature in combination with 11C-MET and 18F-FLT PET, significant 308

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differences could be detected between nontreated rats and those treated with bevacizumab [56]. The relatively simple synthesis of 11 C-MET [57] makes it a popular choice for preclinical studies. However, the short half-life of 11C (20.3 min), and subsequent need for a cyclotron on site, limits the use of this tracer.

6-18F-fluoro-L-dopa: invasiveness proliferation 18

F-FDOPA is an alternative amino acid-based tracer (a product of L-tyrosine) that has shown promise for PET imaging in glioma. 18FFDOPA is also taken into the tumour by LAT-1 [58] and by dopaminergic transporters, such as dopamine active transporter (DAT), and it has been shown that regions of high uptake correspond strongly with regions of high proliferation [48,59–63]. In a study of 59 patients, it was shown that 18F-FDOPA SUVmax values could be used to discriminate between high- and low-grade tumours with a high level of specificity and that SUVmax values correlated strongly with proliferation, as measured by the Ki-67 proliferation index [64]. The preclinical use of 18F-FDOPA is limited, largely because of a limited number of centres producing the tracer. However, the strong correlations seen in clinical trials between 18F-FDOPA uptake and proliferation suggest that 18FFDOPA PET would be useful as a biomarker correlating with therapeutic efficacy in preclinical drug development. Although 18 F-FDOPA has been used in several studies [49,62,63], the main disadvantage in oncologic applications is typically elevated uptake by the basal ganglia, which can require kinetic analysis to differentiate proximal tumours [63].

[18F]fluoromisoidazole: hypoxia Hypoxia is a significant concern in the treatment of glioma because hypoxic regions are intrinsically more resistant to both chemotherapy [65,66] and radiotherapy [67]. As such, PET tracers for imaging hypoxic regions have been developed and have found increasing application in glioma imaging. One of the most promising candidates for hypoxia imaging is the small molecule tracer [18F]fluoromisonidazole (18F-FMISO). The nitro functionality on the heterocycle of 18F-FMISO has a high affinity for electrons and, as such, once inside the cell, can accept an electron from the respiration cycle to form a radical anion. In cellular environments with a high partial pressure of oxygen (normoxic tissue), the radical anion is reduced back to the parent compound before further reaction. In hypoxic environments, the radical anion persists long enough to react with other electrons in the cell to form the two-electron reduction product. This can react further with intracellular macromolecules, thus trapping the tracer in hypoxic environments and providing contrast between hypoxic and normoxic regions [68]. Oehler et al. showed that 18F-FMISO could be used as a biomarker of response to 5,6-dimethylxanthenone-4-acetic acid treatment in a colorectal cancer xenograft model [69]. There is now a wealth of data on 18F-FMISO efficacy in glioma, and the reader is directed to more-detailed reviews [68,70–73]. The main clinical disadvantage of 18F-FMISO is the slow uptake in hypoxic regions; thus, a long post-injection delay is required for the tracer to wash out of healthy tissue. As such, recent research has examined alternative hypoxia markers, such as 1-a-D-(5-Fluoro-5-deoxyarabinofuranosyl)-2-nitroimidazole (18F-FAZA) and 64Cu-diacetyl-bis-(N-N-N-N-methylthiosemicarbazone) (64Cu-ATSM).

1-a-D-(5-Fluoro-5-deoxyarabinofuranosyl)-2-nitroimidazole: hypoxia 18

F-FAZA is also increasing in popularity; it operates in a similar manner to 18F-FMISO by relying on single electron reduction of the nitro group to form a radical anion. After further reduction in low-oxygen regions, the anion forms intermediaries that bind to tissue macromolecules [74]. 18F-FAZA achieves faster clearance than 18F-FMISO because of lower lipophilicity [75,76]. The specificity of 18F-FAZA has been such that it has been proposed for dose painting during radiotherapy [77] and is discussed in several reviews [78,79]. Comparisons with 18F-FMISO have shown that18F-FAZA might have more clinical utility with faster clearance from background tissue [80], if lower overall uptake.

64

Cu-diacetyl-bis-(N-N-N-N-methylthiosemicarbazone): hypoxia

Another promising candidate for hypoxia imaging with PET is the 64 Cu based tracer 64Cu-ATSM. The mechanism for retention of 64 Cu-ATSM in hypoxic cells is largely similar to that of 18F-FMISO. 64 Cu(II)-ATSM is rapidly reduced to 64Cu(I)-ATSM upon cellular uptake and can be rapidly reoxidised by oxygen under normoxic conditions. The charged 64Cu(I)-ATSM is retained in the cell under hypoxic conditions and gives rise to contrast between hypoxic and normoxic regions [68]. Comparison between 18F-FMISO and 64CuATSM [81,82] has shown that, in the models studied, the normoxic–hypoxic contrast is superior to 18F-FMISO PET. Following the evaluation of these tracers in several models, the use of PET imaging agents for hypoxia in the evaluation of potential therapeutics is poised to increase rapidly, given the importance of targeting hypoxia in glioma therapy. One possible drawback to 64 Cu-ATSM is the observation of tumour specific uptake in some cases [83].

PET imaging of macromolecular nanomedicines Over the past two decades, PET imaging has evolved significantly and has become integral to the newly developing field of theranostics. The wide availability of multidentate ligand systems for many radiometals coupled with simple conjugation chemistries has enabled researchers to transform highly specific biological molecules and polymers into targeted radiotracers and radiotherapeutics. The appealing concept of combining an almost inexhaustible library of peptides, monoclonal antibodies, antibody fragments, and synthetic macromolecules specific to a huge range of receptors with positron-emitting radiometals, such as 68Ga, 64 Cu, and 89Zr, or with beta-emitting nuclides, such as 90Y or 177 Lu, has been widely embraced by the academic community [84–88]. Pressman and Korngold reported the ex vivo biodistribution of a radioiodinated antibody in a mouse model of Wagner osteogenic sarcoma in 1953 and showed uptake of the antibody within the tumour [89]. The field has progressed significantly over the past 60 years and there is now a wealth of literature on the use of PET to study the biodistribution and pharmacokinetics of radiolabelled antibodies, peptides, antibody fragments and variants, and polymer nanoparticles [85,90–94]. Alongside the developments in PET imaging, the combination of imaging and therapy in the same molecule, commonly termed ‘theranostics’ [95], has been gaining considerable traction. The success in using a highly specific, radiolabelled biological molecule as both imaging agent and therapeutic is evidenced by its translation into the clinic in

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several products. For instance, ibritumomab, which targets the CD20 receptor, is labelled with 111In (a SPECT imaging isotope) and 90Y (a beta-emitting therapeutic) for the diagnosis and treatment of non-Hodgkin’s lymphoma. Similarly, the peptide octreotate and various derivatives, which target the somatostatin family of receptors, are labelled with 68Ga (a PET-imaging isotope) and 177 Lu (beta-emitting therapeutic) for the diagnosis and treatment of neuroendocrine tumours. However, for planning treatments and assessing treatment outcome, anatomical imaging with good soft tissue contrast (i.e., MRI) is essential both in the clinic and on the workbench. A particular advantage of MRI simultaneously acquired with PET in the investigation of potential theranostics is the ability to overlay highresolution high-contrast anatomical data (MRI) and functional data, such as angiogenic factors [dynamic contrast-enhancedMRI (DCE-MRI)] or necrosis [diffusion-weighted-MRI (DW-MRI) or susceptibility-weighted-MRI (SW-MRI)] with the distribution of the theranostic (PET) from a single imaging session. This information is invaluable in assessing the potential efficacy of a theranostic platform and, potentially for glioma research, PET/MRI is unique in this ability.

MRI in glioma Conventional MRI MRI is currently the gold standard in glioma imaging, providing a variety of high-resolution anatomical and functional information for diagnosis and treatment planning. Conventional MRI sequences [T2-weighted spin echo (SE) and T1-weighted gradient echo (GE) and variations thereof] offer greater contrast between grey matter, white matter, and cerebral spinal fluid compared with CT, enabling delineation of structures within the brain (Fig. 2). This facilitates the analysis of the anatomical properties of a tumour, such as location and size. With the addition of a paramagnetic contrast agent, such as complexes of gadolinium (Gd) [96,97], breakages in the blood–brain barrier can be visualised using T1weighted sequences, and it has been shown that there are strong correlations between high-contrast agent uptake and necrosis [98]. The power and accuracy of preclinical trials depends, among other factors, on the identification of a reliable biomarker for drug efficacy. As with the examples in PET imaging highlighted in the previous section, there have been several studies conducted in preclinical models of glioma to establish relations between MRI phenotypes and histological or genetic phenotypes of the disease [98–103]. It has been shown that tumour burden, as measured by T2-weighted MRI, is comparable to that measured histologically [99,102,104] and that necrotic regions identified by Gd contrast-enhanced T1-weighted MRI are comparable to those measured by traditional immunohistochemistry [98]. Given the correlation between conventional MRI phenotypes and molecular phenotypes measured by traditional means, preclinical drug development studies are beginning to adopt MRI-based phenotypes as a means to assess the efficacy of treatment. A study by McConville et al. demonstrated the diagnostic and prognostic power of basic MRI sequences [104]. The authors used T2-weighted and Gd contrast-enhanced T1-weighted MRI to group mice into high- and low-grade gliomas in the Nestin Tva genetically engineered mouse model, which spontaneously develops glioblastoma. Mice were imaged weekly from 4 weeks of age and tumour volumes were delineated from the T2-weighted image. High-grade www.drugdiscoverytoday.com

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CT

T2Weighted MRI

T1Weighted MRI

Contrast-enhanced MRI

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FIGURE 2

Comparison between computerised tomography (CT) and magnetic resonance imaging (MRI) imaging for glioma. It can be seen that the diversity of MRI allows for generation of different contrasts, each of which can be used to describe the underlying biology of the tumour in addition to providing anatomical location. Images were sequentially acquired of a 12-week-old female NOD/SCID mouse bearing a xenograft of 105 U87 cells injected into the right striatum. The CT image was acquired on a Siemens Inveon PET/CT scanner with an X-ray source with the voltage set to 80 kV and the current set to 500 mA. Scans were performed using 3608 rotation with 180 rotation steps with low magnification, a binning factor of 4 and exposure time of 230 ms. MRI was performed on a Bruker 7 T Clinscan using a RARE sequence (T2 weighted MRI) and a 3D GE sequence before (T1-weighted MRI) and after injection of Magnevist via the lateral tail vein (contrast-enhanced MRI).

image reflects the dynamics of water in the microscopic environment and inferences of what is happening at the cellular level can be drawn [105]. In particular, information on cellular density can be extracted. Given that the tumour environment is highly heterogenous, regional differences in cellular density can be linked to apoptosis and necrosis within the tumour. Parametric images of the apparent diffusion coefficient (ADC), which highlight these differences, have been shown to reflect regional differences in treatment response. Moffat et al. showed that changes in DWMRI measures precluded changes in tumour volume following treatment in a rat model of glioblastoma [106] and that ADC values within the tumour increased following a high dose of carmustine, indicating necrosis within the tumour. In a previous publication, Chenevert et al. showed that ADC maps were able to discriminate between high- and low-dose treatments with carmustine and were predictive of treatment efficacy [107]. In addition, the change correlated well with the change in cellular density over time measured by histology. Sun et al. also demonstrated the use of DW-MRI in highlighting tumour heterogeneity in a U87MG mouse model of glioblastoma [108]. The authors showed by a radial analysis of the ADC map that ADC values varied from the tumour core to the periphery and into the surrounding brain tissue. The premise that a reduction in ADC can be used as a surrogate marker of tumour cellularity still requires further validation. It has recently been demonstrated in patients with highgrade glioma that tumoural regions exhibiting low ADC measures did not anatomically correlate with areas of enhanced 18F-FDOPA uptake [109]. This finding highlights that other factors, such as hypoxia and tissue compression, also need to be considered when interpreting ADC measures. It should be noted that, in the preclinical arena, DW-MRI can be relatively difficult to implement, because the higher fields used in preclinical research to improve the resolution of the image cause instabilities in the echo planar imaging (EPI) sequences often used for DW-MRI.

PW-MRI: angiogenesis tumours were identified as having twice the growth of low-grade tumours and three times the size on initial imaging. In addition, the authors showed that high-grade tumours exhibited contrast enhancement in a Gd contrast-enhanced T1-weighted image, whereas low-grade tumours did not. Furthermore, the authors used their MRI-based classification to select mice with high-grade tumours for treatment with temozolomide and to follow tumour progression post treatment.

Functional MRI The power of more complex MRI sequences has become particularly evident in neuroimaging over the past 20 years, with the use of these sequences in preclinical glioma research growing. Functional MRI sequences, such as dynamic perfusion-weighted (PW) MRI, DW-MRI, functional MRI (fMRI) and magnetic resonance spectroscopy (MRS), can provide complementary biological information to the anatomical information available from conventional MRI.

DW-MRI: cell density, apoptosis, and necrosis DW-MRI measures the diffusion properties of water in tissue and, as such, is sensitive to changes in tissue structure. The acquired 310

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Haemodynamic properties are an important biomarker for the changing biology of the tumour. Several techniques broadly encompassed by the term ‘perfusion-weighted’ MRI can provide highresolution information on tissue perfusion in vivo. DCE-MRI measures the T1-shortening effect of a contrast agent in a series of images and utilises a high temporal resolution to generate parametric maps from the change in intensity of each voxel over time. By measuring the rate of incoming blood into the imaging slice via an arterial input function (AIF) and fitting the time-series data to an appropriate kinetic model, it is possible to derive information on tumour blood volume, tumour blood flow, and the volume transfer constant Ktrans [110]. Cha et al. showed a good correlation between relative cerebral blood volume (rCBV) as measured by DCE-MRI and vessel density as measured by hematoxylin and eosin (H&E) histology, as well as CD31 immunostaining in a GL261 mouse model of glioblastoma [111]. In a further study, Veeravagu et al. showed that an increase in the DCE-MRI measure of time to peak preceded a burst in tumour growth [112]. The authors showed that the increased time to peak correlated with an increase in vessel density and increased expression of vascular endothelia growth factor (VEGF) and angiotensin 2 as measured by real-time quantitative PCR. The strong correlations between changes in functional parameters measured by

PW-MRI and functional measures of angiogenesis (vessel density and increased expression of angiogenic factors) have led researchers to turn to PW-MRI as a means to assess the efficacy of antiangiogenic treatments. Gossman et al. showed that microvascular permeability as measured by DCE-MRI was significantly lower in treatment groups compared with controls in a U87 rat model treated with an anti-VEGF antibody [113]. The reduction in microvascular permeability correlated with a reduction in tumour burden in the treatment group. Arterial spin labelling (ASL) is a second PW-MRI technique and measures the difference in signal intensity in images with and without a spin labelling pulse. Given that the technique does not require the injection of a contrast agent, it is completely non-invasive and free from any potential artefacts caused by recirculation of a tracer, equilibration of a tracer within the blood stream, or the presence of residual tracer in longitudinal measures. As such, the measurement can be repeated as frequently as needed. Sun et al. investigated the correlation between cerebral blood flow (CBF) as measured by ASL-MRI and microvascular density and functionality as measured by histological techniques [114]. The authors showed that CBF decreased from the tumour borders to the tumour core and that this correlated with the presence of dysfunctional tumour vasculature. The ability to acquire perfusion metrics simultaneously to distribution maps of a potential therapeutic is crucial in drug development. In the most basic form, information on the joint spatial distribution of vascular beds and high concentrations of potential therapeutic can inform on penetration into the tumour [115]. More complex analysis can highlight potential reasons for a lack of therapeutic efficacy; for example, low perfusion might result in no drug delivery.

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example, Wehrl et al. showed the diversity of the technique with a montage of images acquired with the PET/MRI at the University ¨ bingen [116]. A recent review by Yankeelov et al. [121] of Tu highlights the need for commercially available preclinical PET/ MRI systems to validate the use of the technology in both preclinical and clinical workflows and, hopefully, find significant problems for which simultaneous PET/MRI might be the solution. In the following section, we highlight specific cases where there is a potential advantage of PET/MRI over independent PET or MRI or combined PET/CT in preclinical research in glioma.

Increasing soft tissue contrast in the brain The most apparent advantage of PET/MRI over PET/CT is the increased soft tissue contrast offered by MRI (Fig. 2). This is particularly attractive for neurological applications where detail of the underlying anatomy of the brain is of importance. This was recently highlighted in a study by Wehrl et al. in which the authors acquired simultaneous fMRI and 18F-FDG PET images in a rat brain using the simultaneous PET/MRI system at the University of ¨ bingen [122]. The authors showed correlation between blood Tu oxygenation level dependent (BOLD) responses in fMRI and high uptake of 18F-FDG PET upon activation of the whisker pad in a rat. This experiment clearly demonstrated the potential power of PET/ MRI and highlighted several key advantages: (i) the ability to acquire data simultaneously; and (ii) the high soft tissue contrast of MRI that allows for localisation of functional signals within specific centres of the brain. For applications in glioma research, the increased soft tissue contrast of MRI allows for more in-depth analysis of the heterogeneity of the tumour environment.

Simultaneous acquisition: dual functional information

PET/MRI in glioma PET/MRI combines two extremely flexible and powerful imaging modalities. PET has the advantages of high sensitivity and numerous biologically relevant tracers with a drawback of low spatial and temporal resolution. MRI offers not only high spatial and temporal resolution to compliment PET but, as outlined above, also has a broad diversity of acquisition protocols that provide different soft tissue contrasts and highly functional information. The development of multimodal imaging systems is particularly pertinent in oncology because there are multiple diagnostic biomarkers that, when combined, are predictive of or indicate increased tumourigenesis. Four key hallmarks of cancer identified by Hanahan and Weinberg [7,8] are: (i) angiogenesis; (ii) apoptosis resistance; (iii) proliferation; and (iv) invasiveness, each of which can be imaged by either PET or MRI. A significant advantage of combined PET/ MRI systems is the ability to either image one of these hallmarks by two complimentary techniques for validation, image two or more biological factors independently and simultaneously, or image the distribution of a potential therapeutic and a biomarker of disease simultaneously. The potential for simultaneous acquisition is particularly attractive and, in contrast to PET/CT, is possible with PET/MRI systems. This has been highlighted as a significant advantage of PET/MRI in several reviews on the technology of simultaneous PET/MRI [116–121]. Given that the technology for PET/MRI is new, examples of preclinical studies utilising it are limited. Early indications of the power of PET/MRI for glioma research have been revealed in several reviews [116,119]. For

The possibility of acquiring dual functional information is one of the key advantages of PET/MRI imaging. This is demonstrated by the studies of Viel et al. [55,56] and is highlighted in Figs. 3 and 4. The images in Fig. 4 were acquired sequentially but highlight the principle of dual functional imaging with PET/MRI. The authors used multiple measures of tumourigenesis to assess the efficacy of the antiangiogenic therapy bevacizumab. It was shown that regions identified as having no viable tissue or blood vessels in immunohistochemical analysis corresponded to regions of abnormal blood flow identified by MRI. Proliferation as assessed by Ki-67 staining correlated well with 18F-FLT uptake and was significantly higher in nontreated than in treated groups. Vessel density assessed by von Willebrand factor staining was lower in treated than in nontreated rats and the average vessel size was larger in nontreated rats versus treated rats. The authors demonstrated that dual functional imaging with PET/MRI was able to provide detailed information on two key biomarkers of tumourigenesis (proliferation and angiogenesis) and could be used to gain detailed insight into the efficacy of a potential therapeutic. More importantly, the authors showed that PET/MRI-derived biomarkers correlated strongly with immunohistochemical analysis. Further examples of the power of dual functional PET/MRI have been shown outside of glioma research and should be mentioned because the experimental methods and conclusions drawn apply equally to fundamental challenges in drug development for glioma. Cho et al. conducted a study in a rat model of prostate cancer to evaluate the complimentary information available from www.drugdiscoverytoday.com

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(i) 18F-FDG

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FIGURE 3

Imaging hallmarks of cancer with positron emission tomography/magnetic resonance imaging (PET/MRI). (a) PET/MRI image of a 64Cu-labelled antibody specific to the EphA2 receptor tyrosine kinase, showing the distribution of a potential therapeutic. (b) 6-18F-fluoro-L-dopa (18F-FDOPA)PET/MRI showing a marker of proliferation and invasiveness. (c) T1-weighted MRI, showing the anatomical location and size of the tumour. (d) T2-weighted MRI showing the anatomical location and size of the tumour. (e) [18F]fluoromisonidazole (18F-FMISO) PET/MRI imaging hypoxia. (f ) Susceptibility-weighted (SW) MRI imaging necrosis. (g) Contrastenhanced (CE) MRI imaging blood–brain barrier breakdown and markers of vascular density. (h) relative cerebral blood flow (rCBF) map generated from a dynamic contrast-enhanced (DCE) MRI, imaging angiogenic factors. (i) 18F-fluorine-2-deoxy-D-glucose (18F-FDG) image showing metabolic activity. Images were acquired of 12-week-old female NOD/SCID mice bearing a xenograft of 105 U87 cells injected into the right striatum on a Bruker 7 T Clinscan interfaced with a Siemens Spectrometer running Numaris/4 VB17 with a PET ring centered at the isocentre of the magnet comprising three rings of 16 crystal blocks at the Centre for Advanced Imaging at The University of Queensland. (b, c, d, f, g and h) were acquired in a 1-h imaging session and correspond to the hematoxylin and eosin (H&E) stained histology in the centre of the figure.

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F-FMISO PET and DCE-MRI [123]. The PET and MRI acquisitions were performed separately in this study but registration of the images was made more robust by the addition of fiduciary markers to the tumour. This was essential in this case because the authors

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generated detailed statistics from voxelwise joint histograms from the two images, a process that requires accurate registration of images. The authors showed that the DCE-MRI-derived perfusion parameter Akep (an approximate measure of blood flow) was

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Transaxial sections of co-registered images of the brain of nude rats implanted with glioblastoma spheroids (columns 1–3) and nude rats implanted with glioblastoma spheroids treated with the antiangiogenic therapy bevacizumab. Anatomical images (T2-weighted, top row) and functional magnetic resonance imaging (fMRI) images (rows 2–5) calculated from T2 and T2* maps acquired pre and post injection of superparamagnetic iron oxide nanoparticles (SPIONs) were acquired independently to [methyl-11C]-L-methionine (11C-MET) positron emission tomography (PET) and 18F-fluorothymidine (18F-FLT) PET and co-registered post acquisition. Reprinted, with permission, from [56].

positively correlated with the early slope of 18F-FMISO uptake into the tumour and negatively correlated with the late slope of 18FFMISO uptake into the tumour, indicating that perfusion and hypoxia are independent characteristics of the tumour microenvironment. Bokacheva et al. also investigated the correlation

between 18F-FMISO uptake and DCE-MRI-derived parameters, in this case, the plasma to EES transfer constant Ktrans, in a rat model of colorectal cancer to assess the efficacy of cediranib. The authors showed a significant reduction in Ktrans in treated versus vehicle rats that correlated with a reduction in hypoxic volume as www.drugdiscoverytoday.com

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FIGURE 5

Dual functional [18F]fluoromisonidazole (18F-FMISO) positron emission tomography/dynamic contrast-enhanced magnetic resonance imaging (PET/DCE-MRI). It can be seen that areas of low relative cerebral blood flow (rCBF) correspond with areas of high 18F-FMISO uptake (white arrows). Images were acquired of a 12week-old female NOD/SCID mouse bearing a xenograft of 105 U87 cells injected into the right striatum. Images were acquired on a Bruker 7 T Clinscan interfaced with a Siemens Spectrometer running Numaris/4 VB17 with a PET ring centered at the isocentre of the magnet comprising three rings of 16 crystal blocks. Imaging started 120 min post injection of 18F-FMISO. Simultaneous to a 30-min PET acquisition, a DCE-MRI image series was acquired during which Magnevist was injected via a cannula placed in the lateral tail vein.

measured by 18F-FMISO PET [124]. Given that hypoxic regions represent a significant challenge in the treatment of glioma and rate of perfusion is an important parameter to understand with regards to the delivery of therapies to the tumour, dual 18F-FMISO PET/DCE-MRI could be a powerful biomarker for assessing the efficacy of therapy in glioma. Fig. 5 shows a dual functional 18F-FMISO/DCE-MRI image of a mouse bearing a U87 xenograft in the right striatum acquired on the simultaneous PET/MRI system at the Centre for Advanced Imaging at the University of Queensland. It can be seen that regions of hypoxia as measured by 18F-FMISO PET correlate with areas of low blood flow as measured by DCE-MRI.

Simultaneous acquisition: advantages in kinetic modelling Kinetic modeling of dynamically acquired PET data can give additional information on the tumour microenvironment and can potentially provide additional biomarkers of therapy response [47]. Dynamic PET data essentially comes free in any PET study because the acquired data can be reframed into appropriate time series data. In the preclinical environment where long scanning protocols (>1 h) are possible, there is the potential to extract valuable information through kinetic analysis. One particular advantage of simultaneous PET/MRI over PET alone is to use the MRI to improve the robustness of PET kinetic modelling. A key requirement of an accurate kinetic model in PET is the generation of an AIF, a function that describes the influx of tracer into the blood stream and equilibration of that tracer in the blood compartment. In the clinical environment, an AIF can be generated by sampling the blood of the patient at appropriate time intervals and measuring the level of activity (concentration of tracer) in the sample. However, in the preclinical setting, this is almost impossible because of the low total volume of blood in a mouse and the logistics of sampling from a mouse within the imaging space. In addition, if the site of disease is far away from 314

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the site of blood sampling, an inaccurate representation of the AIF might be generated. An alternative approach is to use an imagederived AIF where the activity in a large blood vessel close to the site of disease is monitored over time in the PET image [63]. The intrinsic spatial and temporal resolutions of PET somewhat hinder this approach, particularly in the preclinical setting. Partial volume effects are almost certain to cause error in the measured AIF because most mouse or rat blood vessels are smaller than a PET voxel. In addition, the PET temporal resolution usually means that the first pass of tracer is estimated in two to three data points. Given that the spatial and temporal resolution of MRI is superior to that of PET, there is the potential to use an MRI-derived AIF for the kinetic modeling of PET data. This can be achieved in two ways; either a high-resolution MR angiogram can be used to segment the major vessels, generate a vessel streamline that can then be applied to the PET image for accurate positioning of the PET region of interest (ROI) for measurement of an AIF, or a Gd-based tracer can be co-injected with the PET tracer and the AIF derived from a DCE-MRI sequence can be used for the PET kinetic modelling. The first of these possibilities was demonstrated by Fung and Carson, who used anatomical T1 MRI images to segment the carotid arteries in nine patients and then applied the segmentation to a registered dynamic H215O PET image [125]. The authors showed that this approach was valid for generating a PET image-derived AIF, but commented that partial volume effects were still an issue. Poulin et al. recently published a strategy to convert an MRI AIF to a PET AIF. The authors showed that the two could be interconverted using their methods in a F98 rat model of glioblastoma and that there was no significant effect on the kinetic modelling [126]. Evans et al. also presented methodologies to convert MRI image-derived AIFs for use in PET kinetic modelling. The authors used an automated voxel selection routine to improve reproducibility of the MRI AIF generation and showed that PET and MRI AIFs displayed similar characteristics in

the first pass and commented that the MRI AIF suffered less from partial volume effects [127]. Although work in this particular area of PET/MRI is preliminary, there is considerable promise in using an MRI image-derived AIF to improve PET kinetic modelling, something that can only be achieved with simultaneous PET/MRI. More accurate kinetic modelling in PET has potential beyond improved diagnosis. As previously mentioned, PET has been integral to the field of theranostics and in evaluating potential new drugs. Kinetic information from dynamic PET studies is particularly valuable for drug development if the drug of interest is the radiotracer.

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the ability to image multiple aspects of the cancer process in a temporally connected way has significant potential to develop novel, accurate biomarkers for disease progression and treatment response. In addition, increased simplicity in the robustness and reproducibility of radiolabelling macromolecular therapeutic platforms has enabled PET imaging to be at the forefront of macromolecular drug discovery. It is anticipated that PET/MRI will be a central imaging modality in the development of small molecule and macromolecular therapeutics because the high anatomical resolution, soft tissue contrast, and diversity of PET/MRI images is ideally suited for glioma research.

Acknowledgements Concluding remarks The examples presented here illustrate the broad potential for combined PET/MRI in preclinical glioma research. The particular strength of simultaneous acquisition in PET/MRI has been highlighted. Ultimately, the inclusion of PET/MRI as an imaging modality in drug development for glioma will rely on proven evidence of benefit. However, it is becoming more evident that

We thank the team at RAPID PET laboratories, Sir Charles Gairdner Hospital, Perth, WA, Australia for production of 64Cu. We thank the team at Queensland PET, Royal Brisbane and Women’s Hospital, Brisbane, QLD, Australia for the production of 18FFDOPA and the Australian National Imaging Facility for instrument access. This work was supported by the Australian National Health and Medical Research Council (APP1021759).

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Drug Discovery Today  Volume 20, Number 3  March 2015