Eur Radiol (2007) 17: 2441–2457DOI 10.1007/s00330-007-0619-9 MOLECULAR IMAGING

Markus Rudin

Received: 11 August 2006Revised: 6 February 2007Accepted: 13 February 2007Published online: 6 March 2007# Springer-Verlag 2007

Imaging readouts as biomarkers or surrogateparameters for the assessment of therapeuticinterventions

Abstract Surrogate markers andbiomarkers based on imaging readoutsproviding predictive information onclinical outcome are of increasingimportance in the preclinical andclinical evaluation of novel therapies.They are primarily used in studiesdesigned to establish evidence that thetherapeutic principle is valid in arepresentative patient population or inan individual. A critical step in thedevelopment of (imaging) surrogatesis validation: correlation withestablished clinical endpoints must bedemonstrated. Biomarkers must notfulfill such stringent validationcriteria; however, they should provideinsight into mechanistic aspects ofthe therapeutic intervention (proof-of-mechanism) or document therapyefficacy with prognostic quality withregard to the long-term clinicaloutcome (proof of concept). Currentlyused imaging biomarkers provide

structural, physiological and metabol-ic information. Novel imagingapproaches annotate structure withmolecular signatures that are tightlylinked to the pathophysiology or to thetherapeutic principle. These cellularand molecular imaging methods yieldinformation on drug biodistribution,receptor expression and occupancy,and/or intra- and intercellular signal-ing. The design of novel target-spe-cific imaging probes is closely relatedto the development of the therapeuticagents and should be considered earlyin the discovery phase. Significanttechnical and regulatory hurdles haveto be overcome to foster the useof imaging biomarkers for clinicaldrug evaluation.

Keywords Surrogate . Biomarker .Imaging . Molecular imaging .Drug development

Biomarker/surrogate markers

The term surrogate, from the Latin surrogatum, meanssubstitute/substitution. In the biomedical context a surrogateendpoint substitutes for an established clinical endpoint, i.e., itis expected to predict clinical benefit based on epidemiologic,pathophysiologic or other scientific evidence [1]. Narrowingdown even further to the evaluation of novel therapies,surrogates are parameters that predict the effect of treatment inclinical studies. Such readouts might be of a structural,physiological, metabolic, cellular or molecular nature.

A critical aspect when considering the use of surrogates isvalidation: the parameters must show a tight correlation

with classical clinical endpoints requiring large-scale multi-center clinical trials. It is obvious that validation studies forsurrogate marker development are both time consumingand expensive, and may involve large patient populations.

Biomarkers are not validated to the extent of a surrogate.According to a United States Food and Drug Administra-tion (FDA) definition, a “biomarker is a characteristic that isobjectively measured and evaluated as an indicator ofnormal biological processes, pathogenic processes, orpharmacological responses to a therapeutic intervention”[2, 3]. Biomarkers are typically developed on the basis ofmechanistic considerations. Consider a drug aimed atinhibiting a target enzyme. Demonstration that the drug is

M. Rudin (*)Institute for Biomedical Engineering,University of Zürich/ETH Zürich,AIC - HCI E488.2,8093 Zürich, Switzerlande-mail: rudin@biomed.ee.ethz.chTel.: +41-44-633-7604Fax: +41-44-633-1187

M. RudinInstitute for Pharmacology andToxicology, University of Zürich,AIC-HCI E488.2,8093 Zürich, Switzerland

effectively inhibiting the enzyme would be regarded asproof of the pharmacological principle. However, it isobvious that enzyme inhibition may not necessarily predictbeneficial outcome for patients. There is a reasonablelikelihood that normalization of a biomarker (or of a bio-marker profile) might translate into an improved clinicalstatus of the patient; but there is the complementaryprobability that the predictionmight be wrong. In contrast, asurrogate would inevitably predict the therapeutic outcome.

Nevertheless, biomarkers are highly attractive for bothpreclinical and clinical drug evaluations, not necessarily forclinical phase III trials, but rather for small-scale clinicalstudies aiming at demonstrating that a therapeutic concept isalso valid in humans (proof-of-concept studies), criticalinformation in the decision making on further development.Biomarkers are used as indicators of pharmacodynamicendpoints in patients, to assess translatability from animalstudies to clinical studies correlating preclinical and clinicaldata, to establish the optimal dosing regimen and to stratifypatient populations, i.e., to identify subgroups of patientsthat are most likely to respond to treatment. Pharmacody-namic measures reflect the effects of an intervention (e.g., adrug treatment) on the organism, i.e., structural, physiolog-ical or metabolic changes imposed by treatment. In contrast,pharmacokinetics (PK) analyzes how the organism handlesa drug, i.e., how it is absorbed, distributed, metabolized andexcreted by the body.

Biomarkers in order to be predictive early markers oftherapy response have to be linked to the pathophysiologyof the disease process. Moreover, the readout should be (1)on the ‘critical path’ leading to pathological transformationsand (2) downstream of the therapeutic intervention.Otherwise, it will not be useful as an indicator of clinicaloutcome (Fig. 1). A biomarker not associated to thedominant mechanism leading to disease might well indicatetherapy response, however, would not predict clinicaloutcome. An example is inhibition of acetylcholine esterase(AChE) as symptomatic treatment in Alzheimer’s disease(AD), which can be monitored using suitable radio-labeledenzyme inhibitors. However, AChE inhibition is not diseasemodifying; the readout will not predict the long-termclinical outcome.

The Critical Path Initiative of the FDA aims ataccelerating the drug development process, which for anewmolecular entity currently takes more than 10 years andcosts about 800 million US dollars. The initiative shouldensure that ‘basic scientific discoveries translate morerapidly into new and better medical treatment by creatingnew tools to find answers about how the safety andeffectiveness of new medical products can be demonstratedin faster time frames, with more certainty, at lower cost andwith better information’ [2]. In March 2006 the FDAreleased the Critical Path Opportunity List, which com-prises six key areas; of relevance in the context of this articleis the section on new evaluation tools - biomarker anddisease models. The agency is convinced that drug

development programs will benefit from the availabilityof biomarkers, including imaging biomarkers, imagingmethods being considered key enabling technologies. TheFDA sees a critical role of biomarkers and surrogateendpoints in providing evidence (1) for validation of atherapeutic target, (2) for the elucidation of mechanism ofaction of drug candidates, (3) for demonstrating proof-of-principle of a therapeutic intervention, (4) for stratificationpatient populations, and (5) for the evaluation of therapyresponse or eventual side effects [2].

Imaging biomarkers

There are a number of advantages of gathering informationto document the disease course or evaluate therapeuticinterventions using imaging methods:

1. ‘Seeing is believing’: Capturing biological processesor consequences thereof as imaging data sets providesevidence of high documentary value that allow an‘objective’ assessment of the patient status.

Fig. 1 (Imaging) biomarkers should probe disease-relevant path-ways. Moreover, the readout should be downstream of thetherapeutic intervention (indicated by the syringe) in order topredict clinical outcome (top). For an intervention downstream ofthe checkpoint, treatment effects will not be captured (2nd row). Incase of multiple mechanisms involved in the pathophysiologicalprocess, the therapy effect will be likely missed if the biomarkermeasurement probes a different pathway than that of the interven-tion (3rd row). In the last case (bottom) the biomarker will capturethe drug effect; however, it is not the dominant mechanism leadingto disease and may therefore not predict clinical outcome

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2. Imaging methods are versatile, i.e., techniques can beused to probe a wide range of pathological conditions.Combining multiple imaging readouts might providebiomarker profiles that are of higher diagnosticrelevance than individual markers.

3. Non-invasiveness enables longitudinal studies inindividuals: The current status can thus be related tobaseline values: Measuring relative changes to areference state accounts for part of the inter-individualvariability and thus enhances statistical power.

4. Imaging results are inherently quantitative: Structuralconsequences of a disease process and treatmentefficacy can be assessed via morphometric measuressuch as the volume of a structure. When probing tissuefunction using exogenous tracers, densitometric anal-ysis is commonly used to estimate local tracerconcentrations in tissue or tissue compartments, aprerequisite for deriving quantitative physiologicalparameters. Obtaining biologically relevant quantita-tive information from an imaging data set is by nomeans straightforward; major methodological devel-opments are required.

Imaging biomarkers in clinical drug trials: structural,functional and molecular readouts in disordersof the central nervous system (CNS)

Imaging biomarkers are currently being developed for awide range of human pathologies. It is beyond the scope ofthis article to comprehensively cover all active areas ofresearch. In the following section we focus on structuraland functional imaging readouts used to characterize strokeand neuro-degeneration of the Alzheimer type (AD).

(1) Stroke/focal cerebral ischemia: Multiple aspects ofthe pathophysiological cascade of focal cerebral ischemiacan be monitored in great detail, using, e.g., MRI, from theinitial vascular occlusion to post-acute events such as theinfiltration of immune competent cells [4]. While many ofthese imaging procedures are primarily of experimentalinterest, some have matured to clinically established tools.For assessment of therapy response in stroke, the followingimaging readouts are considered relevant:

Exclusion of cerebral hemorrhage for stratification ofpatients It is widely accepted that CT and MRI can both beused to differentiate ischemic stroke from cerebral hem-orrhage, a critical distinction when considering thrombo-lytic therapy using recombinant tissue plasminogenactivator (rt-PA), which is currently the only agentapproved by the FDA for the treatment of acute stroke.Large randomized trials that had used CT to exclude brainhemorrhage showed benefit from treatment with rt-PAwhen administered within 3 h following the insult [5].Thus, structural imaging biomarkers are routinely used forpatient stratification.

Perfusion-diffusion mismatch to predict outcome A criticalparameter for predicting therapy response in focal cerebralischemia is the identification of “tissues at risk” in case ofsustained hypoperfusion. CT- or MRI-based perfusionimaging approaches allow rapid identification of areas withcompromized perfusion [6]. A second early readout ofpathology is the formation of a cytotoxic edema reflecting aredistribution of tissue water between the intra- and extra-cellular compartments due to failure of membrane pumps.These pumps are membrane-spanning proteins that arecritical for maintaining concentration gradients across cellu-lar membranes and thus the polarization of the membrane.Membrane pumps transport ions in and out of cells againstconcentration gradients, a process requiring energy. Energyfailure leads to the breakdown of ion homeostasis, inducing achange in osmolarity and thus to a redistribution of tissuewater. This results in a decrease of the apparent diffusioncoefficient (ADC) of water. ADC values are sensitive early,yet are not specific indicators of an ischemic insult [7]. Theearly ischemia markers ADC and CBF are potentially pre-dictive with regard to the final infarct volume: a significantperfusion-diffusion mismatch, a marker for the ischemicpenumbra with the ‘perfusion lesion’ being larger than thatderived from ADC maps, is a strong predictor of lesiongrowth [8]. Such information may be translated into riskmaps [9] describing the likelihood for a specific brain area tobecome infarcted in the absence of treatment. Deviation fromthis ‘outcome’ might be considered the result of a therapy.Using such imaging readouts, a patient might be used as his/her own control when assessing treatment response.

Structural versus functional readouts of therapeuticefficacy Cytoprotective therapy using a variety of pharma-cological strategies has been demonstrated to reduce thevolume of infarction in animal models of focal cerebralischemia, infarct volume (and location) being considered abiomarker of outcome [10]. The demonstration that regionsspared from becoming infarcted are functional would beconvincing evidence that infarct volume is a valid efficacybiomarker of anti-ischemic therapy. Studies in MCAoccluded rats that were treated with a calcium antagonistconfirmed the hypothesis only in part: recovery of thefunction in cortical areas spared from becoming infarctedhas been observed; however, only a fraction of the animalsshowed a fully recovered fMRI response, while theremaining animals of the drug-treated group did not recoverat all. All animals displayed a similar reduction of the infarctvolume as compared to placebo-treated animals; inparticular the somato-sensory cortex has been spared frombecoming necrotic in all drug-treated animals [11, 12]. Theresults imply that structural integrity is a necessary, yet notsufficient criterion for functional integrity. Correspond-ingly, the biomarker ‘infarct volume’ tends to overestimatedrug efficacy as far as functional (clinical) recovery isconcerned. Whether these findings can be translated toclinical stroke care remains to be shown.

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(2) Neurodegenerative diseases/Alzheimer dementia:With an aging population neuro-degenerative diseaseshave become a major health issue. There is an extensiveeffort worldwide to develop effective therapies againstdiseases such as Alzheimer dementia (AD). A major issuewith regard to accelerating clinical drug development is theslow pace of disease progression rendering the evaluation ofnovel therapies tedious, emphasizing the need for biomar-kers for AD that yield early information treatment response.Many imaging techniques have been used to analyzestructural, functional or metabolic alteration in patientssuffering from AD or from mild cognitive impairment,which is associated with a high risk to develop AD. Anumber of candidate imaging biomarkers have beenproposed. It is beyond the scope of this article to reviewall of them (see, e.g., [13]). We will focus on well-documented examples.

Assessing gross-morphological changes of the brain/atrophy Neuronal loss in AD is accompanied by bothgeneral and focal brain atrophy that can be accuratelyassessed by volumetric analysis of structural MRI data [13](Fig. 2). It has been shown that the rate of cerebral atrophy,i.e., volume loss per annum, is significantly higher in ADpatients as compared to age-matched healthy controls [14,15]. Hence, measurement of the rate of cerebral atrophymight be considered an indicator of disease severity and

thus be used to assess potential therapeutic interventionsthat might slow down progression or even cause arrest ofbrain shrinkage. Issues with this biomarker are the highdemands on accuracy of both data acquisition and analysis(image co-registration, criteria used for tissue segmenta-tion) in order to pick up small volume changes reliably andthe potentially long duration required for a study (typicallyseveral months to more than 1 year for a proof-of-conceptstudy). In addition, atrophy is a rather non-specific markerof AD. Similarly, non-specific functional imaging readoutsin dementia include markers of brain function such ascerebral blood flow (CBF) and cerebral glucose utilization,although careful analysis of spatial patterns of abnormal-ities might be more disease specific [16].

Early clinical manifestations of AD include loss ofshort-term memory and cognitive impairment. A numberof fMRI studies have been carried out in patients sufferingfrom mild cognitive impairment and AD using a variety ofstimulation paradigms (sensory input, cognitive tasks,working memory tasks). Significant region-specific altera-tions in fMRI signals have been reported; while thesechanges vary significantly in the different studies, theywere consistent in displaying alterations when comparedto age-matched controls [17]. Similar experiments havebeen carried out in transgenic mouse models of AD or ofcerebral amyloidosis. The cortical and thalamic fMRIresponses to pharmacological and sensory stimulation are

Fig. 2 Brain atrophy as struc-tural biomarker in AD. The rateof brain volume reduction issignificantly higher in patientssuffering from AD as comparedto age-matched healthy persons.Images show a cross-sectionthrough the brain of an age-matched healthy volunteer (left)and an AD patient (right)obtained from MRI. The brainvolume is extracted by segmen-tation based on intensity thresh-olds. The cross-sectional area(and correspondingly thevolume) is significantly smallerin the patient. The accuracy ofmorphometric analyses criticallydepends on the initial imagequality, in particular thecontrast-to-noise ratio, and theperformance of the registrationand segmentation processingtools. (Images: courtesyof Nitsch R, Division ofPsychiatric Research, Universityof Zürich, Switzerland)

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significantly reduced in APP23 mice over-expressing thehuman amyloid precursor protein (APP) as compared toage-matched controls. The functional impairment wasobserved before any atrophy could be measured [18].

Readouts of cholinergic transmission AD is characterizedby a progressive dysfunction of the cholinergic system andcurrent therapies aim at increasing the availability ofacetylcholine (ACh) for synaptic transmission by inhibitingacetylcholine esterase (AChE) [19]. In longitudinal studies,progressive loss of cholinergic activity could be demon-strated using AChE-binding PET ligands [20]. The sametracer has been used to demonstrate pharmacologicalefficacy: administration of AChE inhibitors has beenshown to displace the PET ligands from AchE [21].Alternatively, the cholinergic system can be probed byanalyzing post-synaptic receptors: studies with 11C-labelednicotine demonstrated up-regulation of nicotinic AChreceptors by AChE inhibitors [22]. Functional conse-quences of AChE inhibition have been documented inAD patients following treatment with the AChE inhibitorrivastigmin: Drug treatment led to a significant increase inthe fMRI response in multiple brain regions in a task-

dependent manner [23]. These results can be related to dataobtained in the rat: administration of rivastigmin prompteda region-specific hemodynamic response (increase cerebralblood volume), which was dependent on the dose of thedrug (Fig. 3; [24]). Obviously, these molecular andfunctional imaging biomarkers provide valuable mecha-nistic information, i.e., clinical proof-of-concept that AChEinhibition infact enhances cholinergic transmission in ADpatients. However, they do not unambiguously predictwhether inhibiting ACh degradation will translate into animproved clinical outcome (see Fig. 1).

Amyloid plaque load as disease indicator The histo-pathological hallmarks of AD are deposits consisting ofβ-amyloid peptides (amyloid or Aβ plaques), and neuro-fibrillary tangles composed of hyper-phophorylated tau-protein in brain parenchyma. Quantitative assessment of theparenchymal plaque load would therefore constitute anattractive biomarker for characterizing AD. Attempts tovisualize plaques using high-resolution MRI by exploitingthe intrinsic contrast of plaques to the surrounding paren-chyma both in tissue specimens from humans [25] and oftransgenic mice [26] have shown that translation of this

Fig. 3 Cholinergic activation inrat brain. Oral administration ofthe AChE inhibitor rivastigminleads to region-specificincreases of local CBV. Images(left) show baseline CBV andCBV changes following dosingwith 8 mg/kg rivastigmin. Bargraphs shows regional CBVchanges in caudate putamen,thalamus and parietal cortexwith a dose dependency in thecaudate and parietal cortex(adapted from [24])

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approach to a clinical setting is unrealistic due toprohibitively long measurement times as a result of thehigh demands on spatial resolution. An alternative approachis the selective detection of plaque-related signals byadministering Aβ -specific tracer molecules. For example,administration of one such PET tracer to AD patientsrevealed significant increases in cortical tracer binding withan inverse correlation between amyloid tracer binding andregional glucose metabolism as assessed using [18F]-fluorodeoxyglucose (FDG) as PET label [27]. Similar data havebeen obtained in genetically engineered mouse models ofAD using plaque-binding fluorescent dyes [28]; yet thelatter approach is of limited value for clinical applicationsdue to insufficient light penetration through the humanskull, but nevertheless constitutes an attractive techniquefor preclinical evaluation of AD treatments. From a clinicalperspective, plaque-selective PET ligands are the mostpromising imaging agents for visualization and quantifica-tion of the plaque load in humans [29]. However, it has beenreported that the correlation between Aβ plaque load andclinical status in AD patients is rather weak, i.e., significantplaque deposition was observed in completely asymptom-atic elderly people [30]. To what extent quantitativeassessment of plaque load constitutes a reliable biomarkerof therapy efficacy in AD is currently unclear.

Physiological and molecular markers in cancer

Morphological and physiological manifestations of diseaseand of therapeutic interventions must be preceded byaberrations at the cellular and/or molecular level. Hence,visualization and quantification of these parameters shouldincrease the sensitivity and specificity of diagnosis andprovide earlier evidence of therapy efficacy than theclassical imaging readouts. Molecular imaging, which aimsat annotating anatomical structures with molecular infor-mation, is currently developed at a rapid pace and beyonddoubt will have a major impact on patient management. Inthis section we will focus on molecular biomarkers forcancer (see Table 1).

Several hallmarks are characteristic of neoplastic tissues:excessive proliferation, increased metabolic activity, for-mation of new blood vessels, dysregulation of cellularhomeostasis and the tendency to form colonies distant fromthe primary tumor site. In addition, tumor cells may expressspecific receptors that might be utilized for tumor targeting.

(1) Targeting general tumor hallmarks: The classicreadout when assessing tumor proliferation and treatmentefficacy is tumor volume using structural imaging methodssuch as CT, MRI or ultrasound. Although clinically wellestablished, structural readouts are recognized to be poorindicators of response. This is undesirable from severalperspectives: from a drug developer’s perspective an earlyreadout is desired in order to optimize the treatmentstrategy and to reduce development cost. More importantly,

Table 1 Potential imaging biomarkers for oncology

Tumor hallmark Mechanisms Imaging biomarker Development status

Angiogenesis/vascularity Vascular permeability Dynamic contrast-enhanced MRI (DCE-MRI)using low molecular weight contrast agents

Evaluation/deployment

Vascular permeability/blood volume DCE-MRI using macro-molecularcontrast agents

Development/evaluation

Metabolism Glucose utilization (glucosetransporter and hexokinaseactivity)

[18F]-2-fluoro-2-deoxyglucosePET (FDG-PET)

Deployment

Proliferation Membrane turnover MR spectroscopy (MRS):phosphorylcholine signal

Evaluation

[18F]-fluorocholine PET (FCh-PET) Development/evaluation

DNA synthesis [18F]-fluoro-thymidine PET(FLT-PET)

Evaluation/deployment

Apoptosis Apoptotic cell body formation MRI: Apparent waterdiffusion coeffient

Evaluation

Externalized phosphatidyl-serine

99mTc-Annexin-5A Evaluation/developmentas diagnostic

Cell surface receptorover-expression

Somatostatin receptor(SSTR) binding

111In-DTPA-D-Phe-octreotide Deployment/diagnosticproduct (Octreoscan)

Estrogen receptor binding [18F]-fluoro-estradiol Identification/development

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patients may be exposed to potentially ineffective therapyfor prolonged periods of time.

Structural growth requires the synthesis of molecularbuilding blocks of cells. DNA, protein and membranephospholipid synthesis rates are therefore potential in-dicators of proliferation. Imaging approaches, and inparticular PET methods, have been developed to probeeach of these processes. PET-based approaches use radio-labeled precursor molecules such as labeled thymidineanalogues [31], amino acids [32] or choline [33, 34].

Proliferation marker Thymidine kinase 1 (TK) is a keyenzyme in DNA synthesis. It phosphorylates thymidine, aprerequisite for DNA incorporation. TK activity can bemonitored by administration of radio-labeled substrates.The thymidine analogue [18F]-3′-fluoro-3′-deoxythymi-dine (FLT) is a substrate for TK, which has beenthoroughly evaluated as a potential indicator of DNAsynthesis. FLT is taken up by cells and phosphorylated byTK, leading to intracellular trapping [35]. Tumor levels ofradio-labeled nucleotides (thymidine analogues) thenreflect DNA synthesis and, correspondingly, cell prolifer-ation [36, 37]. Clinically, this approach is highly attractive,in particular for tissues with high intrinsic glucoseutilization, where the established [18F]-2-fluoro-2-deoxy-glucose (FDG) method (see below) might be compromisedby poor signal-to-background ratios. Clinical validation ofFLT as a proliferation marker is ongoing.

Rapid cell proliferation is associated with high proteinsynthesis rates and high demands on re-supply of aminoacids. There is experimental evidence that amino acidtransport is up-regulated in experimental tumors. Labeledamino acid precursors (such as [18F]-fluorotyrosine) mightbe used to monitor protein synthesis. However, preclinicaland clinical results up to now using fluorinated amino

acids show that tissue uptake rates are dominated by slowamino acid transport across the cell membrane rather thanactual protein synthesis rates [38].

Increased proliferation rates are also associated withincreased rates of membranes synthesis, and substratesrequired for membrane synthesis constitute another po-tential proliferation markers. In fact, magnetic resonancespectroscopy (MRS) studies of tumors revealed somecharacteristic common spectral features: (i) elevatedsignals of endogenous phosphomonoester (PME) and -diester (PDE), (ii) elevated intensity of the ‘choline’ signal,and (iii) early decreases in the PME signal as predictor oftherapy response [39]. These data point to the role of PMEand PDE in membrane biosynthesis and degradationpathways. In vivo MRS is currently evaluated by severalgroups for its potential as a prognostic tool and as apotential biomarker for treatment efficacy. Alternatively,PET using radio-labeled choline derivatives, for example[11C]-choline, first evaluated for imaging of brain tumors,could be used to assess lipid membrane turnover [40].There is evidence that for tumors located in tissues withhigh intrinsic glucose utilization (e.g., brain), measure-ment of choline accumulation yields higher tumor-to-background values as compared to FDG PET imaging.

Metabolic marker/glucose utilization Measurement ofglucose utilization rates via FDG PET has evolved as asensitive diagnostic tool for the characterization of primarytumors and for the detection of metastases. FDG is takenup by cells via the glucose transporter and phosphorylatedby hexokinase to yield FDG-6-phosphate (FDG-6P). Thismetabolite is not further processed through the glycolyticchain (due to the lack of a hydroxyl group at the 2-position). Because of the phosphate group added, FDG-6Pis charged and trapped in the cell. Hence, the trapping of

Fig. 4 Effect of tyrosine kinaseinhibitor imatinib mesylate in apatient with a bowel GIST(short arrow) suffering frommultiple liver and peritonealmetastases (long arrow). 18F-FDG PET and CT studies werecarried out prior and at variousdays following drug treatment.Within 8 days after the start ofthe imatinib treatment, 18F-FDGuptake in neoplastic tissue wascompletely abolished, while asignificant reduction of the livertumor burden could be observedafter 24 weeks only (adaptedfrom [41], reproduced withpermission)

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FDG-6P by the cells reflects glucose transporter andhexokinase activity and the PET activity becomes a markerfor the glycolytic activity of the tissue of interest. There isconvincing evidence in several clinical drug trials thatchanges in glycolytic rate precede effects on the tumorvolume. For example, in patients suffering from gastro-intestinal stromal tumors treated with imatinib, an inhibitorof cKIT tyrosine kinase (and of BCR-ABL, an oncogenictyrosine kinase associated with chronic myeloid leuke-mia), glucose utilization was significantly reduced within24 h after treatment onset, while there was no effect ontumor volume for several weeks (Fig. 4) [41], indicatingthat the glucose utilization rate predicts therapy response.

Angiogenesis marker Neo-vascularization is a criticalfactor promoting tumor growth. A driving factor ofangiogenesis is hypoxia, which leads to the expressionof angiogeneic factors. Tissue, and in particular tumorhypoxia, can be imaged using PET or MRI approaches[42]. The alternative and more widely used approach tostudy tumor neovasculature uses so-called dynamic con-trast-enhanced (DCE) MRI methods, which exploits thefact that newly formed immature vessels are characterizedby increased vascular permeability. DCE-MRI measuresthe leakage of low molecular weight contrast agents suchas GdDTPA (Magnevist) or GdDOTA (Dotarem) into theextracellular space. The method is currently beingevaluated as a biomarker for evaluating anti-angiogenictherapy efficacy [43–45].

The vascular endothelial growth factor (VEGF, alsoknown as vascular permeability factor) is an angiogenicfactor induced by most solid tumors. Inhibition of VEGFreceptor signaling should be reflected by decreased vascularpermeability and potentially also reduced tumor bloodvolume. This has been demonstrated in rodent tumormodels [46, 47]. In both an orthotopic kidney tumor modeland B16 melanoma model in mice, the VEGF-R tyrosinekinase inhibitor vatalanib (PTK787) significantly reducedvascular leakage in B16 melanoma lymph node metastases,the decrease occurring within 48 h after onset of therapy.Clinical studies in patients with liver metastases yieldedcorresponding results indicating that, in fact, vascularpermeability measures may serve as a biomarker of anti-VEGF drug efficacy [48]. Vascular permeability assessmentusing DCE-MRI is meanwhile a widely evaluated biomar-ker and has become a gold standard for assessing anti-angiogenic response. The method has been used fortranslational studies with a number of compounds (vatalanib[46–48], combretastatin [49], ZD6126 [50, 51]).

While DCE-MRI is widely used for early clinicalevaluation of anti-angiogenic drugs, the approach suffersfrom shortcomings due to the use of low molecular weightcontrast agents: the tracers leak into the interstitial spacealso in mature vessels, which compromises the dynamicrange of the measurement, and display high diffusivemobility. The local concentrations of the contrast agents

and, hence, the signal enhancement at the tumor site aregoverned by perfusion rates, vascular leakage and inter-stitial diffusion. Using DCE-MRI in combination withmacromolecular contrast agents alleviates these limitations(Fig. 5): perfusion and extravasation could be more easilyseparated due to different time scales of the processes, andthe contribution of diffusion would be largely negligible[52]. Unfortunately, no macromolecular contrast agentsare currently approved for clinical use in this indication.

Apoptosis marker Programmed cell death is an essentialprocess for cell homeostasis, and down-regulation ofapoptosis is associated with excessive cellular proliferationassociated with malignancies. Induction of apoptosis is anattractive therapy concept in oncology and many cyto-statics display a pro-apoptotic component. Several imag-ing strategies for visualizing activation of the apoptoticpathway are conceivable.

Cells undergoing apoptosis redistribute aminophospho-lipids to the outer leaflet of the cell membrane [53]. Theseextracellular aminophospholipids, primarily phosphatidyl-serine, are recognized by phagocytotic cells prompting asignal for cell removal [54]. Externalized phosphatidylser-ine moieties are recognized by annexin-5A [55], whichbinds to its target with high affinity and specificity.Specific imaging probes have been designed by couplingreporter groups such as radioligands (99mTc-chelates) toannexin-5A [56]. Labeled annexin-5A has been used todemonstrate therapy-induced apoptosis in cancer patients[57] and in animal models. For example, chemotherapyusing cyclophosphamide in a murine tumor model led toinduction of apoptosis within hours after drug administra-tion as reflected by increased annexin binding [56, 58].Radiolabeled annexin-5A is currently in clinical develop-ment as a potential radiodiagnostic.

Indirect imaging approaches, sensitive to microstructur-al or metabolic changes associated with apoptosis, havealso been proposed. Apoptosis-inducing therapy results inincreases in water ADC in a rat glioma model early aftertreatment onset prior to any changes in tumor volume [59].The effect was attributed to increases in the extracellularvolume due to the formation of apoptotic bodies. Studiesin glioma patients also have revealed early increases inADC following chemotherapy, indicating that ADC valuesmight serve as potential biomarkers to assess pro-apoptotictreatment response [60].

Metastasis formation Detection of metastases is of highdiagnostic and prognostic relevance for cancer patients.Today, the gold standard imaging approach for detectionof metastasis is FDG-PET, which is characterized by highsensitivity, but poor spatial resolution. The detection limitis currently of the order of 5-mm tumor diameter [61]. Anadvantage of PET is that the whole body can be efficientlyscreened for metastasis. Limitations include radiationexposure to patients.

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MRI has emerged as an attractive alternative fordetection of lymph node metastases. The approach usesthe fact that particulate matter and pathogens that arerecognized by the immune system are cleared fromcirculation by the cells of the monocyte phagocytoticsystem (MPS). This has be exploited by developing longcirculating nanoparticles containing a core of iron oxide(small and ultra-small particles of iron oxide: SPIO andUSPIO, or mono-crystalline iron oxide nanoparticles:MION), which due to their super-paramagnetic propertieshave a strong effect on the contrast inMRI (shortening of T2

and T2* relaxation times) [62]. While healthy lymph nodetissue shows massive uptake of USPIO/MION, metastatictissue does not. There is compelling preclinical and clinicalevidence of the improved diagnostic sensitivity and spec-ificity of the approach using USPIO/MION for detection oflymph node metastases [63, 64]; approval of the approachby the regulatory authorities is expected soon.

Protease activity marker A characteristic feature of tumorsis excessive secretion of proteases, which among othereffects, degrades the extracellular matrix and enablestumor infiltration into healthy tissue. Identifying areas ofincreased protease activity would be attractive with regardto tumor detection and to assess the efficacy of protease

inhibitors. An elegant approach uses activatable or ‘smart’near-infrared fluorescent probes [65, 66] that will onlygenerate a signal upon interaction with their target. Such aprobe has been used to assess the efficacy of the matrixmetalloprotease (MMP) inhibitor prinomastat in a murinetumor model, providing direct proof-of-mechanism of thetherapeutic concept, protease inhibition [67]. For clinicalapplication, this approach is limited due to light scatteringin biological tissue, confining penetration of NIR light to afew centimeters requiring the development of endoscopicprocedures (e.g., coloscopy for imaging colorectal tumors).

The usefulness of a specific protease imaging probe istightly linked to the validity of the respective protease asdrug target. MMP inhibitors are aimed at inhibitingcellular penetration of the basement membrane and,therefore, tissue infiltration by malignant cells. Yet,MMPs have been found to also exert host-protectivefunctions such as suppression of angiogenesis or inacti-vation of chemokines. As a therapeutic strategy MMPinhibition has failed in clinical trials [68]. Currently, it isnot clear whether the trials as such failed (trial design, sideeffects, inappropriate dosing) or whether MMP inhibitorsfailed as a drug class. Imaging biomarkers might help toresolve this issue, providing relevant mechanistic informa-tion both in animals and eventually in the patient [68].

Fig. 5 DCE-MRI (a) and simulation results (b) for uptake of lowmolecular weight (left) and macromolecular contrast agent (right) insubcutaneously implanted mammary tumor in nude mice. Tissuemodel comprises three compartments: healthy tissue (h), proliferatingtumor (pt) and necrotic tumor core (nc), which in contrast to the othertwo compartments is not vascularized. Tracer transport processesconsidered in the simulation are perfusion, diffusive transport and

convective transport. Note different patterns of contrast enhancementreflecting local tissue concentrations of the two contrast agentsobserved 20 min after administration. Simulation results indicate asignificant contribution of molecular diffusion for the low molecularweight tracer (experimental MRI data: courtesy of Allegrini P,Novartis Institute of Biomedical Research Basel; Simulation results:Grimm HP, Kuttler A, Rausch M, Rudin M, unpublished)

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(2) Targeted tumor probes: Suitable targets for imagingas well as for therapeutic intervention comprise peptidereceptors that are frequently over-expressed in specificneoplastic tissue. Targets include the somatostatin recep-tors in neuroendocrine tumors, gastrin-releasing peptidereceptors in prostate and breast cancer and neuropeptide Yreceptors in breast cancer [69]. Several correspondingpeptide ligands have been radio-labeled and are beingevaluated as potential imaging probes.

Membrane-bound somatostatin receptors (SSTRs) arehighly expressed in neuroendocrine tumors and as suchrepresent attractive targets for a tumor-specific imagingprobe. Their endogenous ligand is somatostatin, a neuro-peptide with a short plasma half life. A metabolicallystabilized somatostatin analogue, octreotide, has been usedas a targeting template to which various reporter groupshave been coupled. 111In-DTPA-D-Phe-octreotide has beendeveloped to the clinically approved probe 111In-pentetreo-tide (Octroscan) for SPECT imaging. Administration ofsuch ligands leads to highly specific enrichment at thetumor site both in patients suffering from neuro-endocrinetumors [70] and in animal models thereof [71]. There areseveral other radiopeptides that are currently beingevaluated as tumor-specific imaging agents such as bom-besin or cholecystokinin, e.g., in prostate cancer [69].

Inhibition of intracellular signaling via the estrogenreceptor (ER) pathway is an established strategy in thetherapy of breast cancer [72]. This can be achieved byvarious strategies, e.g., using ER inhibitors such astamoxifen that compete with the endogenous receptorligand, by lowering the concentration of the endogenousagonist (aromatase inhibitors) or by inducting ER degra-dation [72]. Assessment of ER levels would be of highvalue for diagnostics and in particular for development ofnew therapies. Several agents are currently being evaluatedfor PET ER imaging [73, 74]. For example, [18F]-fluoro-estradiol (FES; [75]) shows great promise for assessing thefunctional ER status in breast cancer patients by PET:quantitative levels of FES uptake in primary tumorscorrelate positively with the level of ER expression [76]and allow the detection of metastatic lesions in patientswith ER-positive tumors [77, 78]. A recent study indicatedthat quantitative FES PET imaging predicts the response to

endocrine treatment in breast cancer and may help guidetreatment selection [79]. While such results are certainlyencouraging, only multi-center studies designed withadequate statistical power will demonstrate the value ofFES-PET for therapeutic decision making.

The Her-2/neu (c-erb B-2) tyrosine kinase receptor isoverexpressed in approximately 25% of human breastcancers [80]; higher expression levels have been shown tocorrelate with poor prognosis. Her-2/neu constitutes apotential target for immunotherapeutic agents such as thehumanized monoclonal antibody trastumazab (Herceptin),which has shown promising results in HER2/neu over-expressing cancers [81]. A PET imaging probe has beendeveloped using a 68Ga-chelate as a reporter moiety [82].Initial experiments were carried out using the full mono-clonal antibody with covalently linked DOTA chelatinggroups revealing excellent tumor targeting in murine tumorxenografts; however, pharmacokinetic (PK) properties ofthis probe turned out unfavorably. A probe based onantibody fragments showed improved PK characteristics atthe expense of a slightly compromised binding affinity.This probe has been used to assess the response totreatment with an inhibitor of heat shock protein 90(HSP90), which was shown to effectively and potentlyreduce levels of Her2/neu expression within hours aftertherapy onset [82], an important feature of a potentialbiomarker. HSP are stress-induced proteins that are up-regulated in many tumors. HSP are molecular chaperonsassisting proper protein folding and stabilizing unfoldedproteins, including oncogenic proteins expressed bymalignant cells; hence, inhibition of HSP by low molecularweight ligands or antibodies is currently evaluated asanticancer therapy and several of these drugs are in clinicaldevelopment [83].

Imaging biomarkers: some challenges

Imaging methods providing quantitative structural, func-tional and more recently also cellular and molecularinformation in a non-invasive manner have become valuabletools for the evaluation of drug candidates. Imaging enableslongitudinal studies in an individual allowing the evaluation

Fig. 6 (Imaging) biomarkerdevelopment chain. Biomarkersshould be available for deploy-ment when the drug enters clin-ical trials, e.g., for clinicalproof-of-concept (POC) studies,requiring timely initiation of thebiomarker development process.Development to a surrogateendpoint requires extensive va-lidation studies comparing bio-marker readouts with classicallyaccepted endpoints

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of therapy response with respect to a pre-treatment referencestate, which will benefit from a significant reduction of theinter-individual variability. Furthermore, non-invasive diag-nostic imaging will allow stratification of patient groups toachieve homogeneous cohorts enhancing the statisticalpower of a therapy evaluation study.

An important aspect in drug development is the timelyavailability of biomarkers of therapeutic efficacy (Fig. 6).This is relevant both for the drug developer and for thepatient. A considerable number of potential imagingbiomarkers are currently in preclinical or clinical evaluation(see Table 1 listing potential biomarkers for assessment ofcancer treatment), and it is likely that several of those willmake a major impact on the development of future therapies.Nevertheless, several issues have to be addressed by drugdevelopers, regulators and the imaging community to fullyexploit the potential of imaging biomarkers.

1. General versus specific biomarkers: Imaging biomar-kers may reflect alterations of a general diseasehallmark as discussed above for tumors (proliferation,metabolism, angiogenesis, apoptosis) or cerebral vas-cular disease (lesion volume, perfusion, cell swelling,metabolism), or alternatively reflect specific molecularinteractions at the level of a drug target. Imaging ofspecific biomarkers is certainly highly attractive from amechanistic point of view, and the concept ofpersonalized therapy involving a diagnostics/therapeu-tics pair has raised considerable interest recently.However, the more target specific a biomarker is, thehigher the risks associated with its development as animaging tool. As soon as the target is not furtherpursued by the therapy developer, the biomarkerbecomes obsolete. Thus, development of imagingbiomarkers will focus-at least initially-on more generalreadouts that are of relevance for a wide range ofdiseases such as the markers of tumor hallmarks alreadymentioned or markers of inflammation (e.g., antigensexpressed activated endothelium, infiltration of mono-cytes and lymphocytes). Specific biomarkers will bedeveloped as diagnostics for ‘high value’ targets, suchas the examples already discussed in the cancer area(e.g., Her-2/neu) or, e.g., a probe targeting monomericor aggregated Aβ peptide as a marker of AD, whichwould be of relevance for large patient populations.

2. Validation studies: Validation of a surrogate requirescorrelation with established clinical endpoints, e.g.,survival in a cancer study, involving large-scale clinicalstudies, in particular when targeting chronic diseases.The surrogate in order to be acceptable for regulatoryauthorities must reliably predict clinical outcome at thelevel of the individual and when considering apopulation. The prognostic value of the surrogate isgiven by its dynamic range and its variability. Thedynamic range describes the sensitivity to the diseaseprocess and the therapeutic intervention: how small a

change can be reliably detected? The ability to detectsmall changes might translate in earlier readouts ofefficacy. Factors determining surrogate variability arebiological and methodological variation. From the pointof view of multicenter trials, standardization of themeasurement process (hardware, measurement proto-cols and analysis procedures) becomes a critical issue.Few imaging readouts have been developed to thesurrogate stage so far. For example, MRI outcomemeasures have been shown to provide objectiveevidence for the clinical endpoints in multiple sclerosis(MS). Despite this, imaging readouts cannot beconsidered fully validated surrogates of clinical out-come in MS. While they capture the inflammatorycomponent of the disease, axonal loss leading toprogressive disability is not reflected. Nevertheless,when considering therapeutic interventions that aim atpreventing relapses, new Gd-enhancing and T2 lesionscan be considered a surrogate outcome measure; theseMRI readouts qualify as the primary measure forevaluating the anti-inflammatory treatment efficacy ofnovel MS drugs [84, 85].Response Evaluation Criteria In Solid Tumors (RE-CIST) were modified in 2000 to objectively character-ize the effect of treatment on tumor volume [86, 87].Based on radiological (principally CT) readouts, anti-tumor effects are classified into complete response,partial response, no change and progressive disease.This RECIST classification has become an integral partof most clinical cancer therapy studies-however, theyare not surrogates replacing the clinical endpoint,which is patient survival, but rather constitutesupportive evidence. There are instances of bothclinical benefit without tumor regression and tumorregression without clinical benefit [86].Biomarkers are not validated to the extent of surrogates.More often they reflect mechanistic aspects of thedisease process or the therapeutic intervention orprovide (early) objective evidence that an individual isresponding to treatment. Criteria for the development ofbiomarkers are mechanistic plausibility, availability ofmethods and technologies, and translatability into theclinics. Whether these readouts will predict clinicaloutcome is not their primary purpose. For example,measurement of vascular leakiness is a widely usedbiomarker for evaluating anti-angiogenic therapy. DCE-MRI provides early information about whether a patientresponds to the therapy-important information for thedrug developer. However, the readout will, in general,not predict clinical outcome (tumor regression andpatient survival).When developing an imaging biomarker for clinicaluse, important aspects to consider are technicalfeasibility (for example, the application of opticalimaging techniques in the clinics will be severelyimpaired by limited tissue penetration of light),

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performance of clinical vs. preclinical instrumentation,translatability of experimental protocols, accuracy andreproducibility of the biomarker measurement, dynamicrange of the biomarker and the availability of contrast-enhancing agents for clinical use (see, e.g., discussionabout low molecular weight and macromolecular con-trast agents for assessing vascular permeability).

3. Standardization: Avalidated (imaging) biomarker mustdeliver quantitatively consistent results independent ofthe observer, instrumentation or institution. Thisrequires standardization of the study design, acquisi-tion protocols and data analysis procedures as clinicalstudies are commonly designed as multi-center trials.

4. Biomarker profile instead of individual biomarkers: Asingle biomarker cannot capture the complex aspectsof a disease such as cancer. This has, for example, beendemonstrated in clinical studies of colorectal cancersevaluating the biomarker potential of both vascular(perfusion, blood volume) and metabolic parameters(glucose utilization) for the evaluation of therapyeffects. Treatment with the monoclonal antibodybevacizumab significantly reduced readouts reflectingvascularity as derived from perfusion CT, but did notaffect glucose utilization rates [88]. In contrast, adecrease of the glucose utilization rate was highlyindicative for treatment response to imatinib in GISTtumors [41]. The suitability of a biomarker to assesstherapy of malignant tumors depends on the tumor typeand the therapeutic principle; complementary informa-tion provided by different biomarkers will enhance thediagnostic/prognostic value.Combination of the multiple imaging modalities willallow comprehensive characterization of pathophysio-logical processes and therapeutic interventions at thereceptor level by monitoring individual signalingcascades, and finally via the morphological, physio-logical and metabolic consequences of these molecularevents. For the drug developer multimodality imagingshould become an important instrument for under-standing drug effects at the level of the wholeorganism. The combination of multiple readouts resultsin a biomarker profile, similar to proteomics andmetabonomics [89], enhancing the specificity andsensitivity of diagnostic tools. Profiles might beanalyzed using statistical tools such as a generalizedlinear model (GLM). This approach has been applied,e.g., to generate risk maps from perfusion and ADCdata in patients suffering from stroke [9]. GLM-derivedinformation can be used to evaluate therapeuticinterventions by comparing what the model predictswould happen to each voxel in the absence of therapywith the actual outcome following treatment at thelevel of individuals. Of course, this approach is notlimited to stroke studies, but constitutes a generalconcept. A methodological limitation is the basicassumption that parameters should be related in a linear

fashion, which is unlikely to be the case for a complexbiological system. More sophisticated non-linearapproaches are therefore required. Clinical parameterssuch as age [90], general health status and geneticfactors are likely to affect outcome and might beconsidered in more general statistical approaches. Theinclusion of both genotypic and phenotypic informa-tion will thus become increasingly important factors inpredictive models [9, 90]. As stated in the context withthe Critical Path Initiative, ‘a multidimensional andcontinuous model needs to replace the current singledimension, binary model of clinical effect’ [3].

5. Improved tools for quantification: It is essential totranslate primary imaging data into biomedicallyrelevant quantitative information. Extraction of mor-phometric information is, in general, straightforwardprovided that there is high enough contrast-to-noise forimage segmentation. Accurate estimation of physiolog-ical and molecular parameters is more difficult andrequires the development of tissue models of variousdegrees of sophistication. As such models are currentlylargely inadequate, imaging readouts are frequentlyexpressed by parameters derived from primary imagingdata sets, such as the area under the signal enhancementcurve during the first 90 s following tracer administra-tion in DCE-MRI studies of tumors [44], or as relativedeviations of parameter values from the normal, healthystate. This raises the principal question: how normal is‘normal’? Is the reference region a valid control?Furthermore, as already stated, biological systems arenot linear. Considering brain perfusion, there arethresholds for cell function and cell survival; hence,minimal changes in the blood flow to the tissue mayhave dramatic consequences [91]. Clearly, improvedmethods for modeling biological systems are required.

6. Regulatory issues: Currently, the development andregistration of a new diagnostic agent are comparableto the development of a novel therapeutic, despite asignificant lower market potential. As a result, there areonly a very limited number of diagnostic imagingagents currently available for large-scale clinical usage.For example, the manufacturer of ferumoxtran-10(Combidex/Sinerem) submitted a marketing authoriza-tion application (MAA) for the compound as afunctional molecular MRI imaging agent to aid in thedifferentiation of normal from metastatic lymph nodesto the European Medicines Agency (EMEA) only inDecember 2006 [92]. The product had been refused bythe FDA for the same use in 2004 despite clearevidence of efficacy. The overall development processtook more than 10 years, similar to that of atherapeutic. Of course, not all imaging biomarkersinvolve the development of novel diagnostics; never-theless, in the context of the Critical Path Initiative, itbecomes obvious that the approval process for diag-nostics tools has to be carefully analyzed, and the

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agencies seem willing to do so. A step in the rightdirection is the so-called ‘microdosing concept’ [93],which holds for tracers that are administered in sub-pharmacological doses (at least two orders of magni-tude lower than doses prompting a pharmacologicalresponse). In this case, the requirement for applicationof the tools in clinical studies would be only evaluationfor acute toxicological and safety effects of the tracer.Both regulatory agencies, the EMEA [94] and the FDA[95], support the concept as stated in their recentposition papers.

Conclusion

Imaging is increasingly being applied in the preclinical andclinical evaluation of novel therapies. Being non-invasive,imaging allows sample sizes to be reduced by comparingoutcome measures with pretreatment values (baseline),which will allow reducing trial costs significantly. Theprimary role of such studies is to establish proof-of-concept,i.e., to demonstrate that the pharmacological principle isvalid in patients.

Current largely established imaging biomarkers arebased on structural (e.g., RECIST for tumors, infarctvolume for stroke, lesion load for MS) or physiologicalreadouts (e.g., DCE-MRI and glucose utilization rates fortumors). However, it is clear that a next generation of(molecular) imaging approaches will provide specificmechanistic information tightly linked to the therapeuticpharmacological principle, the design of novel molecularimaging probes being closely related to the development ofthe therapeutic agents (111In-pentetreotide and octreotideare an example of such a pairing).

The FDA Critical Path Initiative [2], which aims atmodernization of the development process for medicalproducts, is also a milestone for the development anddeployment of biomarkers as tools for therapy evaluation.Definitions for biomarkers, surrogate endpoint and clinicaloutcome accepted by a regulatory authority set guidelinesfor drug developers. As outlined above, imaging-basedbiomarkers and surrogate endpoints may provide informa-tion on critical aspects during therapy development such astarget validation or proof-of-concept of the therapeuticprinciple. Nevertheless, there are few imaging biomarkersand even fewer surrogates based on imaging readouts thatare currently accepted by the FDA as appropriate tools inclinical trials. There are, however, a significant number ofcandidates currently in the pipeline as illustrated for cancerbiomarkers (Table 1); some of them might soon becomeavailable for clinical drug development.

Obviously there are of number of regulatory issuesassociated with the use of biomarkers in drug developmentthat need to be resolved. As already pointed out, propervalidation is an expensive and time-consuming process.

Regulatory acceptance of biomarkers that do not fulfillstringent validation criteria, but provide insight intomechanistic aspects of the therapeutic intervention, withdocumentation of biomarker usefulness in a disease-specificmanner would help advance the field [2, 3]. The main driverof the process will be the pharmaceutical industry incollaboration with academia and regulators. Development ofbiomarkers, including imaging biomarkers, will become animportant new aspect in the development of a noveltherapeutic; these tools must be ready for deploymentwhen the clinical phase is entered (Fig. 6). Clear regulatorycriteria for the development and use of such markers as wellas guidelines for efficient co-development of therapeutic/biomarker pairs are required [3].

Apart from being accepted as biomarkers or surrogateendpoints by regulatory agencies, rapid readouts onefficacy and mechanism will provide valuable informationfor early ‘go/no-go’ decision making with regard to furtherdevelopment of the drug/therapy as illustrated for treatmentof cancers with anti-angiogenic drugs [43–51] or tyrosinekinase inhibitors [41]. Positive outcomes of proof-of-concept studies provided confidence for the initiation oflarge-scale phase III trials.

While the availability of imaging biomarkers during theearly clinical phases is highly attractive and clearly favorsan early embarking in marker development, potentiallyduring the lead optimization or profiling phase, thisstrategy also carries substantial risk in view of the highattrition rates involved in the drug development pipeline.This becomes even more relevant when considering amolecular biomarker that is tightly linked to the mechanismof action of a therapeutic, i.e., a specific target. As thistarget is dropped (e.g., because of lack of efficacy orbecause of safety issues), the marker will most likelybecome obsolete, while the indication as such might still beof interest. Therefore, many of the markers that arecurrently being evaluated reflect generic properties ofpathology. For cancer imaging probes for processes such asproliferation, angiogenesis, metabolism and apoptosis arebeing evaluated by many centers, while only few probestargeting tumor-specific antigens (receptors) are beingconsidered (herceptin, octreotides). In contrast, for brainimaging a number of target-specific PET ligands probingvarious neurotransmitters systems have been described;these tracers are attractive to characterize a large variety ofCNS disorders or effects of neuro-active compounds [96].

Imaging has become an established tool in pharmaceu-tical research, in particular at the preclinical level duringlead optimization and profiling, where it is used for morethan 2 decades to characterize drug effects in animalmodels of human disease [10]. Translation of thesemethods into clinical studies, however, has been lessstraightforward than anticipated; few biomarkers arecurrently being used for clinical drug evaluations. Thereare technical reasons accounting for this deficiency: theclinical imaging toolset is more limited than that at the

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preclinical level, safety regulations are more stringent, andcontrast agents might not yet be approved for clinical use.Clinical trials are in general designed as multi-centerstudies raising issues regarding standardization of dataacquisition and analysis, which become even moreproblematic in view of the rapidly evolving imaginghardware, acquisition protocols, analysis tools and novelcontrast-enhancing principles. Also, lack of sufficient

evaluation/validation of imaging biomarkers has preventedtheir use in early clinical studies. The major stakeholders,i.e., the regulatory authorities, drug development industryand academic partners involved in the process haveidentified these issues and seemwilling to rigorously addressthem. Thus, it is to be expected that the number of imagingbiomarkers markers deployed for clinical drug evaluationwill substantially increase during the next decade.

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