Review

PET Imaging of Receptor Tyrosine Kinases inCancerWeijun Wei1,2, Dalong Ni2, Emily B. Ehlerding3, Quan-Yong Luo1, and Weibo Cai2,3,4

Abstract

Overexpression and/or mutations of the receptor tyrosinekinase (RTK) subfamilies, such as epidermal growth factorreceptors (EGFR) and vascular endothelial growth factorreceptors (VEGFR), are closely associated with tumor cellgrowth, differentiation, proliferation, apoptosis, and cellularinvasiveness. Monoclonal antibodies (mAb) and tyrosinekinase inhibitors (TKI) specifically inhibiting these RTKs haveshown remarkable success in improving patient survival inmany cancer types. However, poor response and even drugresistance inevitably occur. In this setting, the ability to detect

and visualize RTKs with noninvasive diagnostic tools willgreatly refine clinical treatment strategies for cancer patients,facilitate precise response prediction, and improve drug devel-opment. Positron emission tomography (PET) agents usingtargeted radioactively labeled antibodies have been developedto visualize tumor RTKs and are changing clinical decisions forcertain cancer types. In the present review, we primarily focuson PET imaging of RTKs using radiolabeled antibodies with anemphasis on the clinical applications of these immunoPETprobes. Mol Cancer Ther; 17(8); 1625–36. �2018 AACR.

IntroductionDue to their complex heterogeneity and tendency to spread

throughout the body, treating cancers is very challenging. Overthe past quarter century, progress in cancer biology has led tothe discovery and identification of receptor tyrosine kinases(RTK), which control many fundamental cell behaviors anddrive tumor initiation, maintenance, and progression (1).These discoveries have fueled the development of effectivetargeted therapeutics such as monoclonal antibodies (mAb)and tyrosine kinase inhibitors (TKI), shifting cancer patients'care from traditional empirical treatments to an era of person-alized treatment.

Human RTKs contain 20 subfamilies and only approximatelyhalf of the RTK families are well understood (2). RTKs areanchored in the cytoplasmic membrane, and gain-of-functionmutations and/or overexpression of these RTKs are closelyrelated to growth and proliferation in malignant tissues (2).Of them, vascular endothelial growth factor A and VEGFRs haveimplicated roles in tumor angiogenesis, and visualization ofVEGFR expression in vivo with a radiopharmaceutical may bea viable clinical option for imaging angiogenesis (3). The

epidermal growth factor receptor (EGFR) belongs to anotherfamily of RTKs and includes three other members (erbB2/HER-2, erbB3/HER-3, and erbB4/HER-4). We previously showedthat HER-kinase–targeted imaging agents would enable maxi-mum benefit (i.e., patient stratification, therapeutic responsemonitoring, and new drug development) in cancer patientmanagement (4). c-Met, another RTK for hepatocyte growthfactor (HGF), has been found overexpressed or aberrantlyactivated in a variety of cancers (5).

Although mAbs targeting the above-mentioned RTKs havebecome a standard of care for patients with certain mutation-positive tumors, the efficacy of the currently approved mAbs assingle agents is very limited; therefore, synergistic regimenscontaining conventional chemotherapeutic agents and themAbs are applied (6). In addition, drug resistance almostinvariably occurs in cancer patients treated with these thera-peutic antibodies. Traditionally, biopsy and immunohis-tochemistry are performed to determine the RTK status ofcancer tissues and to guide subsequent treatment. However,spatial expression levels of RTKs can vary over time and amonglesions (1), indicating the urgent need for novel noninvasiveapproaches to visualize RTKs' plasticity throughout the wholebody. Moreover, noninvasive methods that can select patientswho will potentially benefit from combinational therapy andpredict treatment response will greatly optimize managementstrategies for cancer patients.

In recent years, molecular imaging has rapidly developed andhas been widely used both in clinical setting and in preclinicalarenas (7–9). Single-photon emission computed tomography(SPECT) and positron emission tomography (PET) are radionu-clide molecular imaging techniques that enable noninvasiveevaluation of biochemical changes and expression of moleculartargets within living subjects. Although a SPECT probe, 111In-labeled trastuzumab, has been used in clinical trials to detectHER2-positive metastatic breast cancer (10), the sensitivity ofSPECT is several orders of magnitude lower than that of PET (11).In comparison, PET imaging is of high sensitivity and can be used

1Department of Nuclear Medicine, Shanghai Jiao Tong University Affiliated SixthPeople's Hospital, Shanghai, China. 2Department of Radiology, University ofWisconsin–Madison, Wisconsin. 3Department of Medical Physics, University ofWisconsin–Madison, Wisconsin. 4University of Wisconsin Carbone Cancer Cen-ter, Madison, Wisconsin.

W. Wei and D. Ni contributed equally to this article.

CorrespondingAuthors:WeiboCai, University ofWisconsin–Madison, Room7137,1111 Highland Avenue, Madison, WI 53705. Phone: 608-262-1749; Fax: 608-265-0614; E-mail: wcai@uwhealth.org; and Quan-Yong Luo, Department of NuclearMedicine, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600#Yishan Road, Shanghai 200233, China. E-mail: lqyn@sh163.net

doi: 10.1158/1535-7163.MCT-18-0087

�2018 American Association for Cancer Research.

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to investigate physiological andmolecularmechanismsof humandiseases (11). PET imaging of RTKs with radiolabeled mAbs,denoted as immunoPET, may provide a noninvasive method forassessing the dynamics of RTKs, selecting patients for personal-ized treatment, predicting response to RTK inhibition therapy,and facilitating drug development (7).

Most PET imaging clinical trials have been focused on usingradiolabeled FDA-approved antibodies (12), such as trastuzumabfor breast cancer and esophagogastric adenocarcinoma (EGA;refs. 13–17), cetuximab for colorectal and lung cancers (18–20), and bevacizumab for several indications (21–26). PETimaging using the positron emitters 64Cu (t1/2, 12.7 hours) and89Zr (t1/2, 78.4 hours) for antibody labeling has been extensivelystudied over the last decade (8, 27). 89Zr has a longer half-life,which generally matches the serum half-life of most mAbs andtherefore is suitable for antibody imaging (28, 29). A straightfor-

ward strategy for generating RTK-specific PET radiotracers hasmostly, but not entirely, involved the radiolabeling of therapeuticmAbs. Antibody fragments, nanobodies, and smaller moleculeinhibitors have also been investigated for in vivo visualization ofRTKs. With shorter biological half-lives, these probes are wellsuited for fast imaging protocols when labeled with rapidlydecaying radioisotopes such as 68Ga (t1/2, 68 minutes) and 18F(t1/2, 110 minutes). Our review focuses mainly on the currentresearch in the field of antibody-based immunoPET probes andhighlights potential clinical implications of immunoPET in visu-alizing RTKs and in guiding clinical decisions (Fig. 1).

PET Imaging of the HGF/c-Met PathwayDysregulation of HGF/c-Met signaling is implicated in a num-

ber of malignancies (30), and synergistic effects of EGF and HGF

Figure 1.

RTKs are deregulated in most human cancers and control many fundamental cell behaviors by regulating biochemical signals. Representative antibody-basedPET probes targeting corresponding RTKs (or pathways) are shown. MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol-3-kinase; JAK,Janus kinase; PKC, protein kinase C; PLCg , phospholipase C-g ; STAT, signal transducer and activator of transcription.

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were noted in non–small cell lung cancer (NSCLC; ref. 31).Moreover, c-Met amplification can drive HER3-dependent PI3Ksignaling, therebymediatingNSCLC resistance toEGFR inhibitors(32, 33). Currently, c-Met targeted therapy involves small-molecule inhibitors (crizotinib, capmatinib, etc.; refs. 31, 34),and antibodies (emibetuzumab, ficlatuzumab, rilotuzumab,onartuzumab, etc.; refs. 35, 36). Phase II trials in patients withadvanced NSCLC demonstrated that the addition of onartuzu-mab to the EGFR inhibitor erlotinib resulted in an improvementin progression-free and overall survival for c-Met–positivepatients (37); however, a phase III study failed to observe theadditive therapeutic effect of onartuzumab to erlotinib (38).Despite these disappointing results, suppressing the HGF/c-Metpathway may still have an antitumor effect in other primarytumors and/or overcome the resistance of molecularly targetedtherapies (30, 39). Noninvasive PET visualization of c-Metdynamics could therefore potentially facilitate patient stratifica-tion and guide c-Met–directed therapies.

Initial attempts to develop nuclear medicine imaging probesfor detecting c-Met expression used murine mAbs (40, 41). Basedon these studies, taking the step to evaluate immunoPET probesusing humanized or fully human anti-c-Met mAbs is relativelystraightforward. Using the humanized therapeutic mAb onartu-zumab, which inhibits HGF binding and therefore the HGF/METsignaling pathway (35), Jagoda and colleagues initially synthe-sized 89Zr-DFO-onartuzumab and 76Br-onartuzumab and foundthat the former probe exhibited higher uptake and tumor-to-muscle ratios in MKN-45 gastric carcinoma models (42). Li andcolleagues developed c-Met–targeting bivalent cys-diabodiesand radiolabeled one of the candidates with 89Zr. The authorsfound that uptake of 89Zr-labeled H2 cys-diabody was higher inthe gefitinib-resistant NSCLC model than in the low c-MET–expressing NSCLC model, indicating that immunoPET could beused to assess c-MET expression levels for both therapeutic anddiagnostic purposes (43). Pool and colleagues then reported thatthe readily translatable probe 89Zr-Df-onartuzumab could effec-tively discriminate erlotinib-induced c-Met upregulation inNSCLC models (44). This study herein highlighted the potentialvalue of 89Zr-Df-onartuzumab PET in assessing c-Met upregula-tion-mediated erlotinib resistance in patients with NSCLC. Moreimportantly, c-Met–directed treatment using NVP-AUY-922, aheat shock protein 90 (HSP90) inhibitor (45), could reduce89Zr-Df-onartuzumab uptake in HCC827 models (Fig. 2A). Rilo-tumumab (AMG102) is an HGF-binding antibody and can bindto and neutralize HGF, thus preventing its binding to c-Met.However, a phase III trial failed to observe the therapeutic effectsof rilotumumab in patients with c-Met–positive gastric or gastro-esophageal adenocarcinoma (46). Previously, there were no toolsto noninvasively determine the levels of HGF present in the localtumor microenvironment. To this end, 89Zr-DFO-AMG102 wasdeveloped by Price and colleagues, and this probe could selec-tively accumulate in tumors with high levels of HGF protein (Fig.2B and C; ref. 47).

Our team produced a recombinant human HGF and labeledthe agent with 64Cu. PET imaging revealed specific and prominentuptake of the tracer in c-Met–positive U87MG tumors but signif-icantly lower uptake in c-Met–negative MDA-MB-231 tumors(48). One concern for these tracers based on the HGF ligand istheir potential to stimulate tumor growth by activating c-Met(48). Burggraaf and colleagues initially developed a fluorescentlylabeled peptide (GE-137) for optical imaging of c-Met, and this

probe showed high affinity for human c-Met in 15 subjects withhigh risk of colorectal cancer (49). In addition to optical imagingin assessing c-Met (49, 50), 18F-AH113804 is a peptide-basedc-Met–specific PET imaging probe that has been used to visualizelocoregional recurrence of breast cancer in a clinical trial (51).

These results suggest thatHGF/c-Met–specific PET imagingmaycorrectly identify patients most likely to benefit from c-Met–targeted therapies or from EGFR inhibition therapy, as it has beenreported that c-Met is implicated in acquired resistance to EGFRinhibitors in NSCLC (31, 33).

PET Imaging of the VEGF/VEGFR PathwayThe VEGF/VEGFR signaling pathway plays an important role in

the regulation of angiogenesis. VEGFs bind to three associatedtransmembrane RTKs known as VEGFR-1, VEGFR-2, and VEGFR-3. Among them, VEGFR-2 is a key receptor involved in the devel-opment of blood vasculature and is an attractive target for anti-angiogenic tumor therapy (52). Approved antiangiogenic drugssuch as bevacizumab, lenvatinib, sorafenib, sunitinib, and pazo-panib target this pathway and are associated with modest survivaladvantages in certain kinds of cancers (53). Bevacizumab andramucirumab are IgG1 antibodies directed against vascular endo-thelial growth factorA (VEGF-A) andVEGFR2, respectively. Todate,clinical PET studies using 89Zr-Df-bevacizumab were performed inpatients with breast cancer, neuroendocrine tumors, renal cellcarcinoma (RCC), NSCLC, and glioma (21, 22, 26, 54, 55).

Based on fundamental preclinical studies (3, 9, 56, 57), in aclinical feasibility study, Gaykema and colleagues demonstratedthat 89Zr-Df-bevacizumab could be used to detect primary breastcancer (22). Another pilot clinical study in patients with meta-static RCC demonstrated that 89Zr-Df-bevacizumab PET visual-ized renal tumor lesions with striking heterogeneity betweenlesions, therefore reflecting differences in vascular characteristics(24). Everolimus, an inhibitor ofmammalian target of rapamycin(mTOR), also inhibits VEGF-A expression, but there are notreliable biomarkers to predict efficacy of everolimus in patientswith metastatic RCC at the moment. van Es and colleaguesinvestigated the value of 89Zr-Df-bevacizumab as a tool to identifyeverolimus efficacy in 13 patients with metastatic RCC, andreported that everolimus decreased 89Zr-Df-bevacizumab tumoruptake in 10 patients who received continuous everolimus treat-ment and achieved stable disease at 3 months (Fig. 2D; ref. 26).Jansen and colleagues reported that 89Zr-Df-bevacizumab hadpoor uptake in mice bearing diffuse intrinsic pontine glioma(DIPG; ref. 23), implying that treatment with bevacizumab inDIPG patients is justified only after 89Zr-Df-bevacizumab immu-noPET demonstrates positive VEGF expression in tumor tissue. Inspite of these disappointing preclinical data, the same teamfurther reported that, in children with DIPG, five of seven primarytumors showed focal 89Zr-Df-bevacizumab uptake while no sig-nificant uptake was seen in the healthy brain (58). A furtherstudy by Veldhuijzen van Zanten and colleagues reported that89Zr-Df-bevacizumab uptake was correlated with microvascularproliferation in a patient with DIPG and highlighted that 89Zr-Df-bevacizumab PET could be used to delineate intralesional het-erogeneity (Fig. 2E; ref. 59). These results indicate that 89Zr-Df-bevacizumab immunoPET is feasible in children with DIPG andmay help select patients with the greatest chance of benefitingfrom bevacizumab treatment. Notably, 89Zr-Df-bevacizumabcould also offer a tool to refine clinical management of patients

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with von Hippel–Lindau disease (25), and neuroendocrinetumors (55). From a clinical perspective, a patient's baselineplasma VEGF-A level is an independent prognostic factor forcertain cancer types (60), and resistance to antiangiogenic ther-apies ultimately leads to patients' relapse and poor survival (53,61). Therefore, it would be significant to develop personalizedtherapeutic strategies through immunoPET imaging of angiogen-esis biomarkers that might help select proper patients and reflectthe response of tumor cells to antiangiogenic drugs.

Ranibizumab is amAbFabderivative of bevacizumab andhas ahigher affinity for all soluble and matrix-bound human VEGF-Aisoforms than bevacizumab (62). Nagengast and colleagues ini-tially created 89Zr-Df-ranibizumab and reported that VEGF PETimaging using 89Zr-Df-ranibizumab allowed serial analysis ofangiogenic changes in ovarian tumormodels following treatmentwith the kinase inhibitor sunitinib (63). In addition, severalstudies have been performed to image tumor vasculature byvisualizing VEGFR-2. Meyer and colleagues engineered and

Figure 2.

Representative c-Met and VEGF pathway–specific PET probes. A,89Zr-Df-onartuzumab is a c-Met targeting PET probe. While in vehicle-treated HCC827 xenograftsuptake of 89Zr-Df-onartuzumab did not differ before treatment and after treatment (left), the corresponding uptake of the radiotracer in the NVP-AUY-922–treated mice decreased more than 30% after treatment (right), indicating that 89Zr-Df-onartuzumab PET may provide a powerful tool for visualizing c-Metdynamics. B, 89Zr-DFO-AMG102 is another HGF/c-Met signaling pathway–specific PET probe. PET maximum intensity projection (MIP) images in HGF and METdouble–positive U87MG tumor models at 24 and 120 hours after injection of 89Zr-DFO-AMG102. C, Corresponding MIP images in MKN45 tumor models thathad negative HGF and positive MET in the local tumor microenvironment. These results imply that 89Zr-DFO-AMG102 may act as a companion diagnostic tool forselection of patients more likely to respond to any HGF-targeted therapy. D, 89Zr-Df-bevacizumab PET MIP image before everolimus treatment showed multiplelocal and distantmetastases from a left kidney tumor. 89Zr-Df-bevacizumab PET demonstrated that tumor uptake decreased significantly after 6weeks of treatmentusing everolimus. E, In vivo performance of 89Zr-Df-bevacizumab PET in the diagnosis and localization of DIPG. Gadolinium-enhanced T1 MR images (top)showed the enhanced primary pontine glioma and metastatic gliomas (white arrows) in the right ventricular trigone and cervicomedullary junction, andall these glioma lesions were clearly visualized by 89Zr-Df-bevacizumab PET (bottom). Panels reproduced with permission from ref. 26, 47, 59, � SNMMIand ref. 44, � Springer.

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radiolabeled single-chain VEGF and further validated the feasi-bility of imaging VEGFR-1 and VEGFR-2 in an orthotopic murinetumor model (64). We synthesized and characterized a ramucir-umab-based PET imaging agent, 64Cu-NOTA-RamAb, for map-ping VEGFR-2 expression in vivo (65), and found that this tech-nology could visualize VEGFR-2 in nude mice bearing HCC4006and A549 NSCLC tumor models. Based on our results, a studyfrom Eric and colleagues further suggested that a three-time-point method could be used to assess the in vivo performance of64Cu-NOTA-RamAb (66).

PET Imaging of HER2The human epidermal growth factor receptor 2 (HER2) trans-

membrane oncoprotein is overexpressed inmany human tumors,and several HER2-targeting agents have entered clinical practice(4). However, despite this success, responses to these antibodiesand small molecules have been hampered by resistance or poorresponses. In this setting, molecular PET imaging techniques can

help select proper patients for subsequent therapy and elucidatethe underlying resistance mechanisms (4, 67–69).

Trastuzumab was the first clinically approved anti-HER2 anti-body for breast cancer patients. In preclinical studies, trastuzu-mab has been used as a targeting agent to map HER2 expressionand distribution either by PET imaging or by SPECT imaging(4, 13, 70, 71). In addition, 89Zr-Df-trastuzumab PET, ratherthan 18F-FDG PET, successfully assessed the pharmacodynamiceffects of afatinib (an EGFR-HER2 dual inhibitor) in HER2-positive gastric cancer models (Fig. 3A and B; ref. 72). In clinicalpractice, HER2 status has been determined by immunohis-tochemistry, and fluorescence in situ hybridization has beenvalidated to predict the efficacy of the HER2-targeting anti-body–drug conjugate trastuzumab emtansine. Because SPECTimaging using 111In-trastuzumab discovered new HER2-positivelesions in 13 of 15 patients with breast cancer (10), Dijkers andcolleagues developed 89Zr-Df-trastuzumab (13), and reportedthat PET imaging using this probe in breast cancer patients wasable to detect both previously known metastatic lesions and

Figure 3.

Representative HER2-, EGFR-, and HER3-targeting PET probes and their potential clinical applications. A, 89Zr-Df-trastuzumab PET specifically imagedHER2-positive NCI-N87 tumors (red circle). On the contrary, 18F-FDG and 18F-FLT PET nonspecifically detected both the HER2-positive NCI-N87 tumor (red circle)and HER2-negative MKN-74 tumor (black circle). B, Afatinib therapy in HER2-positive xenograft models reduced uptake of 89Zr-Df-trastuzumab in a time-dependentmanner, justifying the potential value of 89Zr-trastuzumabPET inmeasuring the pharmacodynamic effects of afatinib in the clinical setting.C, 64Cu-PCTA-cetuximab PET imaging clearly visualized EGFR-positive TE-8 xenograftmodels (white dotted circle).D, 177Lu-cetuximab radioimmunotherapy, rather than saline orcetuximab treatment, markedly reduced 18F-FDG uptake in TE-8 models. E, 89Zr-Df-lumretuzumab is a HER3-specific immunoPET probe. PET/CT scanningperformed 4 days after injection of 89Zr-lumretuzumab detected one lung metastasis (white arrow) from ampullary cancer. Panels reproduced with permissionfrom ref. 72, 99, � SNMMI and ref. 115, � AACR.

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lesions which had been previously undetected (73). Recently,89Zr-Df-trastuzumab PET has been investigated as a useful toolto predict response in breast cancer patients treated with trastu-zumab emtansine, and to improve the understanding of tumorheterogeneity in breast cancers (14). Indeed, the heterogeneitywithin and across tumor lesions in a single patient or amongdifferent patients is increasingly being recognized, with signif-icant therapeutic implications (74). In support of this concept,Ulaner and colleagues demonstrated that 89Zr-DFO-trastuzu-mab PET/CT detected unsuspected HER2-positive metastases inpatients with HER2-negative primary breast cancer (15, 75). It isquite difficult for conventional biopsy techniques, which gen-erally obtain samples from a limited number of lesions, toidentify these unique cohorts. This exciting findingmay facilitatebetter management of HER2-negative breast cancer patients, asabout 10% to 15% of patients with HER2-negative primarybreast cancer may still benefit from HER2-targeted therapy(76). Furthermore, early changes in 89Zr-Df-trastuzumab uptakein breast cancer metastases following treatment with heatshock protein 90 inhibitor NVP-AUY922 correlated withchanges of lesion size measured on CT images (77). In additionto 89Zr-labeled trastuzumab, 64Cu-DOTA-trastuzumab is anoth-er alternative for optimizing trastuzumab treatment (16).

HER2 is overexpressed in EGA and trastuzumab has beenapproved by the FDA to treat EGA patients with positive HER2expression (78). Considering the fact that only a subset of patientswith HER2-positive EGA respond to trastuzumab (79), O'Dono-ghue and colleagues studied the value of 89Zr-DFO-trastuzumabin assessing HER2 status in primary andmetastatic EGA (17), andreported that this imaging method detected local and metastaticlesions in 80% of the imaged patients. This study indicated that89Zr-DFO-trastuzumab PET has superior advantages over single-site biopsy, because this noninvasive imaging technology canassess variation in levels of HER2 and target engagement in boththe primary and metastatic tumor lesions simultaneously. Nota-bly, bifunctional chelators have certain impact on the in vitrostability and in vivo performance of 89Zr-labeled trastuzumab andantibody–drug conjugates (80).

Pertuzumab is another HER2-targeting mAb approved bythe FDA after a survival benefit was achieved for patients withHER2-positivemetastatic breast cancer. Importantly, pertuzumabbinds to a HER2 binding site distinct from that of trastuzumab,augmenting the binding and treatment efficacy of each other (81).Thus, it is of great importance to noninvasively detect the biodis-tribution of pertuzumab-specific binding sites when synergisticregimens containing trastuzumab and pertuzumab are given forbreast cancer patients. In this setting, Marquez and colleaguesconducted an in vivo study in breast cancer models using 89Zr-DFO-pertuzumab and observed significant tracer uptake inMDA-MB-231 xenografts (82). More importantly, pretargeting strate-gies using unlabeled trastuzumab could further enhance 89Zr-DFO-pertuzumab accumulation in tumors. Combination treat-ments with trastuzumab and pertuzumab have also shownenhanced antitumor effects in xenograft models of humanovarian cancer (83), and the addition of pertuzumab to che-motherapy in patients with platinum-resistant ovarian carci-noma demonstrated favorable trends in progression-free sur-vival (84). Based these preclinical and clinical data, our groupsynthesized 64Cu-NOTA-pertuzumab and broadened the appli-cation of the probe to detect both subcutaneous and orthotopicovarian cancer models (85). These solid data demonstrated that

64Cu-NOTA-pertuzumab/89Zr-DFO-pertuzumab are effectivetools for imaging HER2 expression. More recently, a first-in-human study in patients with HER2-positive breast cancershowed that 89Zr-DFO-pertuzumab PET/CT was safe and dem-onstrated optimal imaging 5 to 8 days after administration ofthe tracer. Additionally, 89Zr-DFO-pertuzumab PET/CT wasable to image multiple sites of HER2-positive malignancyincluding HER2-positive brain metastases (86). Further clinicalstudies are still needed to validate the efficacies of these twoprobes, especially in improving patient stratification.

Compared with intact antibodies, antibody fragments andsingle-domain antibodies (sdAb) have superior imaging charac-teristics, such as rapid clearance, high target-to-background ratios,reduced radiation dose, and engineered sites for site-specificconjugation (87, 88). However, compared with clinically avail-able antibodies, the immune responses, safety profiles, and effi-cacy of these novel sdAbs in humans are largely unknown.Positron emitter–labeled nanobodies as probes for evaluatingHER2 status have been reported (89–92). One strength of suchprobes, for example, [18F]RL-I-5F7 and [18F]RL-I-2Rs15d (89, 90),is their ability to rapidly cross the intact blood–brain barrier anddetect brain metastases from breast cancers 1 hour after injectionof the tracers. In comparison, the best time points for detectingbrain metastases using either 89Zr-Df-trastuzumab or 89Zr-DFO-pertuzumab were 4 to 5 days for the former tracer and 5 to 8 daysfor the latter following tracer administration (73, 86). Anotherstrength is the higher tumor-to-blood and tumor-to-muscle ratioswhichwill result inhigher contrast PET imaging (89, 93). Aphase Istudy from Keyaerts and colleagues showed PET imaging using a68Ga-HER2-nanobody is a safe procedure and tracer accumula-tion in HER2-positive breast carcinomametastases is high, whichwarrants further assessment in a phase II trial (94).

PET Imaging of EGFR (HER1)EGFR and its relatives (i.e., ErbB2/HER2, ErbB3/HER3, and

ErbB4/HER4) are well-known oncogenic drivers in several typesof cancers such as lung cancer, breast cancer, and glioblastoma.EGFR was among the first RTKs for which the ligand bindingmechanism was studied (95). Inhibitors for this specific RTK,including antibody therapeutics (e.g., cetuximab and panitumu-mab) and small-molecule TKIs (e.g., erlotinib, gefitinib, lapatinib,and osimertinib), have been the most successful examples ofmolecularly targeted therapies in cancers (95, 96).

Cetuximab is a chimeric EGFR-specific IgG1mAband functionsby preventing ligand activation and receptor dimerization (97).It was shown in a phase I first-in-human study that 89Zr-Df-cetuximab was safe and well tolerated (20). PET imaging using89Zr-labeled cetuximabmay also select NSCLC patients, head andneck cancer patients, and colorectal cancer patients who willpotentially benefit from cetuximab treatment (18, 19, 98). Inrecent years, theranostic approaches usingmAbs have representeda rapidly expanding component of cancer treatments (12). Songand colleagues prepared 64Cu/177Lu-labeled versions of cetuxi-mab and assessed the theranostic efficacy in an esophageal squa-mous cell carcinoma model (99). The authors elaborately foundthat 64Cu-PCTA-cetuximab immunoPET evaluated EGFR expres-sion levels in tumors, and 177Lu-cetuximab radioimmunotherapyeffectively inhibited tumor growth much more thoroughly thanthat of cetuximab alone (Fig. 3C and D). Thus, the pair of64Cu/177Lu-cetuximab may provide a tailored treatment strategy

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for EGFR-positive patients by immunoPET imaging of EGFRexpression and by selective therapeutic radiation delivery.

Panitumumab is a fully humanized mAb that binds to EGFRand is FDA approved for use in receptor-expressing colorectalcancers without KRAS mutations (100). Wei and colleaguessynthesized 89Zr-DFO-panitumumab and this probe accumulat-ed in various subcutaneous athymic nude female xenograft mod-els (101). Lindenberg and colleagues then calculated the maxi-mum dosing for effective imaging with 89Zr-DFO-panitumumabin3patients, and results from this study indicated that injection ofapproximately 1 mCi (37 MBq) of 89Zr-DFO-panitumumabintravenously was safe and detected lung metastases from colo-rectal cancer, but imaging of the included 3 patients failed todemonstrate significant radiotracer accumulation in tumors forall time points and failed to detect tumors in the liver due tophysiologic hepatic excretion of the probe (102). Future studiesmay add unlabeled panitumumab to optimize the radiotracer'suptake in tumors (103). More recently, imgatuzumab (GA201),a novel humanized anti-EGFR IgG1 isotype mAb, has beenfound to efficiently inhibit EGFR pathway activation in headand neck cancer patients and may serve as a potential probe forvisualizing EGFR-expressing tumors after radiolabeling withisotopes (104, 105).

Repebody, a novel nonantibody protein scaffold (106), hasalso been engineered to monitor EGFR expression (107, 108).One advantage of repebody-based PET imaging probes is theirsmall molecular weight (�30 kDa), which may enable earlyvisualization of various EGFR-expressing solid tumors as earlyas 1 hour after injection of the probes. Furthermore, due tosuperior circulation clearance and tissue penetration, thoseprobes could be labeled with positron emitters with shorterhalf-lives, such as 64Cu (t1/2, 12.7 hours) and 18F (t1/2, 110minutes; ref. 109).

As we previously reviewed (110), PET imaging using bispecificpeptide- and antibody-based heterodimers may demonstratehigher targeting efficacy and specificity than their monospecificpeers. We generated a bispecific immunoconjugate (denoted asBs-F(ab)2) by linking a cetuximab Fab and a TRC105 Fab (target-ing CD105), and confirmed that 64Cu-NOTA-Bs-F(ab)2 had asynergistic improvement on both affinity and specificity in visu-alizing small U87MG tumor nodules (<5 mm in diameter;ref. 111). Similar results were reported by Kwon and colleagueswho developed an immunoconjugate targeting HER2 and EGFRand verified its diagnostic efficacy in breast cancer models (112),and by Mahmood and colleagues who prepared PET probesspecific to EGFR and HER3 and monitored resistance to PI3Kand protein kinase B (PKB, also known as Akt) inhibitors usingbreast cancer models as well (66).

PET Imaging of HER3Unlike other members of the family, HER3 has relatively weak

kinase activity but still can form highly activated heterodimerswith EGFR and HER2. Activating mutations in HER3 have beenidentified in multiple cancer types and therefore HER3 is nowbeing examined as a direct therapeutic target (113). Preclinicaland clinical PET imaging has also been developed using anti-HER3 antibodies, such as lumretuzumab (114, 115), patritumab(116), and mAb3481 (117), or using peptides (118), and affi-bodies (119–121). In a pilot clinical trial that included 11participants, Lockhart and colleagues reported that although

administration of 64Cu-DOTA-patritumab is safe, the diagnosticefficacy was limited in solid tumors (116). Comparatively, 89Zr-Df-lumretuzumab PET could visualize intra- and inter patienttumor heterogeneity and target accessibility, and the most opti-mal PET conditions were found to be 4 and 7 days after admin-istration of 89Zr-Df-lumretuzumab with 100 mg cold antibody(Fig. 3E; ref. 115). In the absence of side effects of lumretuzumabin treating HER3-expressing solid tumors (122), 89Zr-Df-lumre-tuzumab may have potential value in guiding lumretuzumabtreatment of patients with HER3-positive solid tumors. Morerecently, Warnders and colleagues constructed a HER3 signal-ing–specific probe, 89Zr-MSB0010853, and reported that 89Zr-MSB0010853 PET could detect HER3-overexpressing H441NSCLC cancer models in vivo (123). Although development oftherapeutic HER3 antibodies is still in an early phase (122, 124),these preliminary results shed light on the feasibility of nonin-vasive HER3-specific PET imaging for mapping HER3 expressionand distribution of HER3 antibodies.

PET Imaging of Other RTKsApart from the above RTKs, there are 17 more RTK subfamilies

and nearly half of these RTK families are poorly understood (2).One example is the platelet-derived growth factor receptor-alpha(PDGFR-a), which has been reported to be involved in tumorangiogenesis and maintenance of several cancer types includingthyroid cancer (125–127). Importantly, PDGFR-a is associatedwith lymphnodemetastases in papillary thyroid carcinoma (PTC;ref. 127). Michael and colleagues described a PDGFRa-specificimmunoPET probe—64Cu-NOTA-D13C6—for PTC and demon-strated that PET imaging using this agent could delineate PDGFRaexpression in PTC models in vivo and could therefore potentiallyserve as a novel and promising radiotracer for imaging PDGFRa-positive metastasis from PTC (128). Another example is theinsulin-like growth factor-1 receptor (IGF-1R), which has awell-established role in malignant tumors and has become apromising target for cancer therapy and imaging (129, 130). AsR1507 is amAb directed against IGF-1R, Heskamp and colleaguesdeveloped 111In-R1507 and 89Zr-Df-R1507 and reported thatboth of the tracers could be applied to noninvasively determineIGF-1R expression in vivo in breast cancer xenografts (131). Ourgroup also developed an IGF-1R–specific PET probe (denoted as89Zr-DFO-1A2G11) and demonstrated highly specific uptake ofthe tracer in IGF-1R–positive pancreatic tumor models (132),implying the potential value of this probe in identifying patientsthat may benefit from anti–IGF-1R therapy.

In addition, RTKs are important drug targets, and severalsmall-molecule inhibitors have been developed and approvedby the FDA in addition to mAbs. Actually, development ofradiolabeled TKIs and their analogues is under preclinical andclinical translational research (133), and various radiolabeledTKIs have been developed to image tumors in both preclinicaland clinical studies for several RTKs, such as EGFR (134, 135),HER2 (136), VEGFR (137), tropomyosin receptor kinase (Trk;ref. 138), stem cell growth factor receptor (SCFR; ref. 139), andIGF-1R (129, 140, 141).

Conclusion and Future PerspectivesCurrently, the role of spatial deregulation of RTKs in tumori-

genesis may be vastly underappreciated, and the development ofhigh-resolution immunoPET techniques for detecting RTK

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activity in normal and tumor tissues will be essential for studyingthe role of spatial RTK patterning in the heterogeneous tumorenvironment. In this state-of-the-art review, we presented typicalexamples of deregulated RTKs in cancers and summarized strat-egies to prepare noninvasive PET imaging probes specific for thesetargets. The current review demonstrates that noninvasive immu-noPET could accurately delineate RTK status within a tumor oracross multiple tumors within a patient. Visualization of RTKphenotype using immunoPET could facilitate selection ofpatients for targeted therapies and response assessment/predic-tion. In addition, a greater understanding of the spatial expressionof RTKs with the help of immunoPET could affect ongoing effortsto develop therapeutic drugs targeting RTKs.

In 2017, the development of antibody therapeutics pro-ceeded at a fast pace, and this is expected to continue in thecoming years. Several RTK-targeting therapeutic antibodies[such as margetuximab, (vic-)trastuzumab duocarmazine, andDS-8201 for HER2, and depatuxizumab mafodotin for EGFR]are undergoing evaluation in late-stage clinical studies ofpatients with cancer (142). For mAb-based immunoPETprobes, 89Zr is utilized in clinical trials much more extensivelythan any other radiometals. DFO and DFO-based bifunctionalchelators are the most commonly used chelators for 89Zr-radiolabeling (143), and there are two simple protocols thatcan be followed to do 89Zr radiolabeling (144, 145). However,DFO is not an optimal chelator for 89Zr coordination becauseof 89Zr transchelation, which will cause high bone uptake of89Zr. This was exemplified by a recent study by Vugts andcolleagues, in which the authors reported that, when comparedwith DFO, the bifunctional isothiocyanate variant of desfer-rioxamine (denoted as octadentate DFO�) was advantageousfor 89Zr labeling because 89Zr-DFO�-trastuzumab hadenhanced stability in in vitro studies and superior performancein in vivo studies over 89Zr-DFO-trastuzumab (80). Therefore,there is still room for future studies to optimize radiolabelingstrategies when developing 89Zr-immunoconjugates (143).With the advent and maturation of click chemistry (146), futurestudies may further harness this powerful method to synthesizeand assess antibody-based PET imaging probes with improvedin vivo performance. In addition to singly targeted imagingprobes, antibody-based heterodimers or dual-targeting probesmay have higher targeting efficacy and superior specificity thantheir corresponding monospecific peers (110, 147).

Generally, adequate tissue penetration ability, which is inverse-ly proportional to the size of an imaging probe, and high signal-to-noise ratio,which is closely related to the circulation of a probe,are two important properties of an immunoPET probe. sdAbs,also known as nanobodies or heavy chain–only antibodies bear-ing a variable region (VHH), hit the sweet spot and have emergedas substitutes for their full-size counterparts in diagnostic ortherapeutic applications (87). Enzymaticmethods such as sortasehave been used to append metal chelators to enable labelingsdAbs with 64Cu or 89Zr (148, 149). Because a first-in-human trial

demonstrated the success of 68Ga-HER2-nanobody in a clinicalsetting (94), and it is easier for immunoPET probes derived fromsdAbs to traverse the blood–brain barrier (90), future studiesmayfurther design noninvasive means to detect RTKs through the useof sdAbs (150).

In the development of antibody-based diagnostic, therapeu-tic, or theranostic probes, we should pay attention to the factthat the immunodeficient strains of animals we use in preclin-ical studies may impact the in vivo fate and performance of theinvestigated immunoPET probes. For example, a recent studyreported that immunoPET radiotracers had inefficient tumortargeting and high off-target binding to the spleen in highlyimmunodeficient mouse models (151). This was consistentwith a previous study which reported that antibody–drugconjugates had limited antitumor activity in NSG mice(152). In this setting, pretargeting strategies may also be har-nessed for optimizing both the imaging quality and therapyeffects in the coming future (151, 153, 154).

Besides assessing RTKs and selecting proper patients forsubsequent molecularly targeted therapy using either smallmolecules or antibodies, noninvasive imaging of RTKs couldfacilitate image-guided radionuclide therapy. Actually, therapyof B-cell lymphomas with radiolabeled mAbs has producedimpressive clinical results because two radiolabeled anti-CD20antibodies, 131I-tositumomab and 90Y-ibritumomab tiuxetan,were approved by FDA more than a decade ago (155, 156).RTKs are ideal candidates for investigating and performingradioimmunotherapy (157, 158). Therefore, radiolabeled anti-bodies or antibody fragments targeting markers included in thecurrent review and other potential neoantigens may provideanother therapeutic option for patients with cancer in the era ofmolecularly targeted therapy and precision medicine.

We firmly believe that immunoPET will allow for better cancerpatient management in the clinical setting with the fast develop-ment of this field. Considering most data with immunoPETprobes havebeen generated inpreclinical stages or in small groupsof patients, further studies are still needed to push clinical trans-lation of some of these promising probes and to confirm the valueof clinically reported probes.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThisworkwas partially sponsored by the Ph.D. Innovation Fund of Shanghai

Jiao Tong University School of Medicine (No. BXJ201736) and the ChinaScholarship Council (No. 201706230067) to W. Wei, the Shanghai Key Dis-cipline of Medical Imaging (No. 2017ZZ02005) to Q.Y. Luo, the AmericanCancer Society (125246-RSG-13-099-01-CCE), and the NIH (P30CA014520)to W. Cai. E.B. Ehlerding was partially sponsored by the NIH (T32CA009206and T32GM008505).

Received February 5, 2018; revised April 19, 2018; accepted June 4, 2018;published first August 1, 2018.

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2018;17:1625-1636. Mol Cancer Ther   Weijun Wei, Dalong Ni, Emily B. Ehlerding, et al.   PET Imaging of Receptor Tyrosine Kinases in Cancer

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