Recent progress in the imaging of c‐Met aberrant cancers with positron emission tomography

Abstract Tyrosine‐protein kinase Met—also known as c‐Met or HGFR—is a membrane receptor protein with associated tyrosine kinase activity physiologically stimulated by its natural ligand, the hepatocyte growth factor (HGF), and is involved in different ways in cancer progression and tumourigenesis. Targeting c‐Met with pharmaceuticals has been preclinically proved to have significant benefits for cancer treatment. Recently, evaluating the protein status during and before c‐Met targeted therapy has been shown of relevant importance by different studies, demonstrating that there is a correlation between the status (e.g., aberrant activation and overexpression) of the HGFR with therapy response and clinical prognosis. Currently, clinical imaging based on positron emission tomography (PET) appears as one of the most promising tools for the in vivo real‐time scanning of irregular alterations of the tyrosine‐protein kinase Met and for the diagnosis of c‐Met related cancers. In this study, we review the recent progress in the imaging of c‐Met aberrant cancers with PET. Particular attention is directed on the development of PET probes with a range of different sizes (HGF, antibodies, anticalines, peptides, and small molecules), and radiolabeled with different radionuclides. The goal of this review is to report all the preclinical imaging studies based on PET imaging reported until now for in vivo diagnosis of c‐Met in oncology to support the design of novel and more effective PET probes for in vivo evaluation of c‐Met.


| INTRODUCTION
c-Met is a membrane receptor protein with tyrosine kinase activity activated by its physiological ligand, namely the hepatocyte growth factor (HGF). This protein, once activated, stimulates a range of intracellular signaling pathways such as those related to proliferation, motility, and invasion/migration of cancer cells. 1 Unfortunately, this receptor's signaling is aberrantly activated and involved in the initiation and metastatic invasion of several tumor types, including colorectal cancer, prostate, gastric and gastroesophageal cancer, ovarian, renal, lung, cervical, breast, pancreatic cancers, and melanoma. [2][3][4][5][6] According to the ECIS (European Cancer Information System) cancer has a major impact on society worldwide. The three most common new cases of cancer are breast, colon/rectum, and lung/bronchus for females, and prostate, colon/rectum, and lung/bronchus for males. These types of cancer are also the ones that lead each year to the highest number of deaths. Notably, a significant number of human tumor types, including those already cited, often present the c-Met receptor overexpressed. 7 Considering only these four types of cancer, each year almost 7 million new cases worldwide are diagnosed (2 million lung cancer, 1.3 million prostate cancer, over 2 million breast cancer, and 1.8 million colon/rectum cancer); among them, 372,435 new cases of lung cancer, 388,278 of prostate cancer, 415,977 of breast cancer and 388,181 of colon/rectum cancer were reported in Europe in 2018. 8 In these cancers the overexpression of c-Met is well documented, for example, 35%-72% for lung adenocarcinoma, 38% for lung squamous cell carcinoma, 23% for hormone-refractory prostate cancer, 72% for prostate cancer bone metastases, up to 80% for breast cancer, and 10%-75% for colon/rectum cancer, [9][10][11][12] resulting in a very large target clinical population per year of millions of potential patients worldwide that could benefit from novel c-Met imaging agents. Moreover, drug resistance, increased metastasis, and poor clinical outcome are unfortunately associated with this receptor overexpression. 13,14 All of these characteristics indicate that this protein receptor is a key player in several stages of the pathology. Therefore, the real-time investigation of the expression of c-Met with positron emission tomography (PET) is likely to assist in the diagnosis and the observation of response to therapy. 15,16 Particularly, small molecules inhibitors of c-Met tyrosine kinases activity and antibodies-based pharmaceuticals with anti-c-Met activity have recently shown promising results in the clinical management of c-Met aberrant cancers. Crizotinib and cabozantinib (both small molecules inhibitors of the protein tyrosine kinase activity) were the two first c-Met-inhibitors approved by the US FDA. Specifically, crizotinib was the first approved (2011) for the management of advanced or metastatic non-small cell lung cancer (NSCLC), and cabozantinib was approved later (2012) for medullary thyroid cancer. 17 The imaging of the expression of this receptor in real time has the prospect to assist the clinical assessment of c-Met-targeted therapies by acceleration in the selection of patients and in monitoring the anti-c-Met therapies based on inhibitors of the protein. 18,19 Improved diagnostic methods for the identification of patients suitable for c-Met targeted treatment are of primary importance to improve the outcome of c-Met aberrant cancers in the clinic. Nowadays, patient selection is normally conducted by fluorescent in situ hybridization or immunohistochemistry. Both methodologies can yield quantitative information about c-Met expression, but they have critical limitations: they are not able to reflect the c-Met expression variation over time, they cannot deal with the receptor heterogeneity in different tumor sites, biopsies cannot be conducted on inaccessible sites, and they provide only a small sample of heterogeneous tissue within a single tumor, in addition to the fact that recurring biopsies can be hurtful and difficult for the patient. Considering  [27][28][29][30] and fluorescence 31,32 ) have been reported for c-Met. 33 Also, several radiolabeled antibodies, peptides, or small molecules against this protein have been used for in vivo cancer localization; however, there has not been any clinical translation of PET tracers for c-Met produced to date. In this study, we review the latest progress in the imaging of c-Met aberrant cancers with PET. Particular attention is directed on the development of PET imaging probes with a broad range of molecular sizes (HGF, antibodies, anticalines, peptides, and small molecules; Figure 1, Table 1) and incorporating different radionuclides. The goal of this review is to report all the in vivo real-time PET-based imaging studies reported until now for monitoring of c-Met in cancer, to support the design of novel and more effective PET probes for in vivo evaluation of the protein.
It is reasonable to hypothesize that, in future clinical settings, noninvasive imaging with PET and using these new tracers will support the diagnosis of c-Met overexpressed cancers and the selection/evaluation (responding and nonresponding) of patients for c-Met-targeting drugs.

| PET PROBES BASED ON THE HGF LIGAND
Due to the specificity and high binding affinity of the natural ligand for c-Met, the HGF structure was initially selected for the first imaging studies of the receptor (by MRI). 52 The only example of a PET probe based on the HGF was reported by Luo et al. 34 The reported product was a recombinant human HGF functionalized with the NOTA chelating moiety then labeled with 64 Cu for c-Met-targeted molecular imaging. 34 To synthesize this tracer, F I G U R E 1 c-Met/HGF signaling pathway and different classes of PET probes [Color figure can be viewed at wileyonlinelibrary.com] 2-S-(4-Isothiocyanatobenzyl)−1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCNBn-NOTA) was covalently bonded to rh-HGF. The resulting product 64 Cu-NOTA-rh-HGF was assessed by flow cytometry using human glioblastoma cell lines (U87-MG) with moderate c-Met expression and human breast cancer (MDA-MB-231) with lower (compared to U87-MG) c-Met expression, confirming the specific binding of the labeled HGF to c-Met overexpressing cells. U87-MG xenografts mice were used for in vivo experiments, revealing rapid tumor absorption of 64 Cu-NOTA-rh-HGF that was distinctly visible at 30 min postinjection with a peak at 9 h (6.7 ± 1.8% ID/g, percentage injected dose per gram). Contrarily, a lower absorption was reported for MDA-MB-231 mice xenografts. These results are consistent with the different expressions of the protein in the two cell lines used in the xenografts. Heating of the 64 Cu-NOTA-rh-HGF resulted in denaturation of the protein and the denatured product, termed 64 Cu-NOTAdnrh-HGF, had notably reduced uptake in U87-MG bearing mice than the unheated 64 Cu-NOTA-rh-HGF. The specificity of Cu-NOTA-rh-HGF was also established by the fact that its uptake in all other main organs was comparable between the natural and the denatured form of the tracer. As expected, the liver and kidney uptake values were similar between xenografts injected with the natural and the denatured form, because these organs represent the clearance tissues for a molecule of this size (~70 kDa). This study demonstrates the potential application of imaging agents based on the structure of the endogenous HGF for c-Met imaging. Limitation of this approach, precluding its clinical translation, could be due to the concomitant presence of endogenous HGF that will induce the classical biological effects of the HGF, that is, cell proliferation/survival, and could also promote tumor growth. Moreover, the higher concentration of the endogenous ligand could preclude the interaction of the radiolabeled ligand with c-Met with subsequent reduction of the contrast ratio of the tumor PET images. 53

| PET PROBES BASED ON ANTIBODIES AND ANTICALINES
Various monoclonal antibodies (mAbs) and anticalines targeting c-Met or its ligand (HGF), for example, DN30, rilotumumab, onartuzumab, and PRS-100, have been recently tested in both preclinical and clinical trials, with encouraging results for the management of cancer. [54][55][56] Therefore these classes of compounds have also started to attract the interest in nuclear medicine pursuing imaging agents for the membrane-targeted receptor. Zr-onartuzumab in the gastrointestinal tract, lungs, heart, blood, and muscle decreased gradually over time, with more than 50% decrement from 18 h to 5 days, whereas uptake in kidney and liver increased progressively over the studied period, remarking renal and hepatobiliary clearance. Of note, a high bone absorption up to 7% ID/g was also measured at 5-day postinjection, possibly due to free zirconium released from the tracer.
A different approach to target the HGF/c-Met pathway was investigated by van Dongen et al. in 2012, where two anti-HGF (αHGF) nanobodies, named 1E2 and 6E10, were developed to investigate HGF in vivo concentration by PET imaging (Figure 2). 37 To extend their serum half-life the two nanobodies were engineered by coupling them to an albumin-binding nanobody unit (Alb8) to obtain 1E2-Alb8 and 6E10-Alb8, then were radiolabeled with 89 Zr.
U87-MG glioblastoma xenografts were used for biodistribution studies. Both the tracers (1E2-Alb8 and 6E10-Alb8) showed decreasing blood levels over 7-day postinjection while tumor uptake levels remained relatively stable during the same period, making them suitable probes for PET scanning. The absorption in other tissues was minor than in tumor tissues, aside from kidneys that control the clearance of these proteins. In addition, the two nanobodies were evaluated for their therapeutic effect, due to their anti-HGF activity, resulting in tumor growth inhibition after treatment with 100 mg intraperitoneal injections, 3 injections per week over 5 weeks.
scFv-cysdimers H2, a human single-chain variable fragments-cys-diabody was radiolabeled with 89 Zr and studied in xenografts mouse models by Li et al. 38 The cys-diabody was labeled with the radiometal using a deferoxamine-maleimide covalently bonded to the antibody. 89 Zr-DFO-H2 cys-diabody uptake was studied in Due to their small size (less than 20 kDa), anticalins could represent a valid alternative to mAb (150 kDa) with higher tumor uptake and tissue penetration and hence potentially improved characteristics for PET tracers. 57  HCC827ErlRes tumors compared to HCC827 tumors, whereas the receptor was downregulated (minus 69 ± 9%) in HCC827 cells following the application of the Hps90 inhibitor. PET scans resulted in a 24% higher 89 Zr-onartuzumab absorption in HCC827ErlRes than in HCC827 tumors, moreover, biodistribution data showed that the uptake of the studied probe was higher in HCC827ErlRes (38.1 ± 8.4% ID/g) than in HCC827 cell lines   UM-SCC-22B tumors were clearly visible with uptakes of 4.72%, 3.83%, and 3.11% ID/g at 30, 60, and 120 min postinjection, respectively. Coadministration of unlabeled Met-pep1 was used to confirm the specificity of the tracer that resulted in decreased tumor uptake showing a high specificity. High values of organ absorption were measured in the kidneys (6.15 ± 0.71% ID/g) and in the liver (11.5 ± 0.7% ID/g), but a rapid renal clearance (65% excreted within 2 h after) suggested a mainly renal-urinary excretion of the tracer. Muscle and other organs (including intestine, stomach, bone marrow, spleen, lung, and heart), had very low uptake during all time points.
Exploiting random nonstandard peptides integrated discovery (RaPID) selection, 66 Sakai et al. recently identified HiP-8 (macrocyclic peptide, 1.6 KDa), as a novel functionally active two-chain HGF (tcHGF) 67,68 binding peptide that strongly inhibited the interaction of HFG with c-Met with subnanomolar potency. 47 Noninvasive screening/visualization and inhibition of HGF/c-Met was observed after iv administration of HiP-8 followed by PET imaging in a mouse model. HiP-8 showed a high binding affinity to HGF with a K d of 0.4 nM and a k off of 0.4 × 10 −3 s −1 (dissociation rate) was measured by surface plasmon resonance. The reported HGFinhibitory activity in cellular assays (as IC 50 ) was 8 nM. The water solubility and the pharmacokinetics of the macrocyclic peptide were improved by the inclusion of a polyethylene glycol chain to the C-terminus. The selectivity of HiP-8 to HGF was also compared with other similar proteins human growth factors, resulting in a high selectivity for HGF. Murine HGF was also evaluated and HiP-8-PEG11 displayed binding to the nonhuman grow factor with 50-fold less affinity. The use of HiP-8-PEG11 was then evaluated for PET scansion of HGF-Met activation in tumors. To achieve this, the molecule was chemically engineered and an -N 3 group was added to a lysine by using a Fmoc-Lys(N 3 )-OH, that was subsequently conjugated to the copper chelating ligand DBCO(dibenzocyclooctyne)-PEG4-CB-TE1K1P (TE1K1P is a bifunctional chelator for copper radionuclides). 69 The obtained product was then labeled with 64 Cu, resulting in a radiochemical purity ≥95% measured by radio- Based on the structure of GE-137, 70 68 Ga-EMP-100 was recently designed and used in a pioneer in-human imaging and biodistribution study. 48  Comparing the different analyzed sites, the highest uptake was reported in tumor burden at the primary site followed by bone, lymph node, and visceral metastases. All the other c-Met negative lesions were heterogeneously distributed intra-and interindividually and mostly present as lung and liver metastases. 68 Ga-EMP-100 was physiologically accumulated in the urinary bladder content and kidneys, and only moderate to low uptake was measured in the liver, spleen, pancreas, and intestine. The clinical success of the reported study warrants further research studies investigating the clinical use of 68 Ga-EMP-100 and other similar probes as a biomarker in mRCC patients.

| PET PROBES BASED ON SMALL MOLECULES
HGF, antibodies, and peptide-based PET probes all have in common that their c-Met binding sites are located in the extracellular portion of the protein; however, the catalytic activity of c-Met, that triggers the signal transduction, is activated in its intracellular domain. Thus, small molecules imaging agents that bind the receptor within its intracellular catalytic domain could provide useful knowledge about the activation status of the receptor. Also, small molecules probes, thanks to their low molecular weight and size, are highly permeable in tissue and potentially in cells, and easily cleared. The first study of such compounds for c-Met was reported by Wu et al. 49 that produced 11 C-SU11274. The designed probe is based on SU11274 is a c-Met tyrosine kinases inhibitor that was then labeled with carbon-11 generating 11 C-SU11274. The molecule was radiolabeled in a single-step reaction starting from synthesized molecule 1 ( Figure 5). The two molecules (SU11274 and 11 C-SU11274) have the same structure, so the specificity and the binding affinity of the probe are preserved. The imaging properties were evaluated by micro-PET in mice bearing H1975 (c-Met positive human NSCLC) and mice bearing H520 (c-Met negative human NSCLC). The measured tumor uptake was significantly higher for H1975 mice than H520; in particular, the one of 11

| CONCLUSIONS AND PERSPECTIVE
The frequency of c-Met receptor misregulation in cancer diseases and its known influence on the pathology evolution make this membrane protein an interesting molecular target for the design of novel diagnostic and therapeutic pharmaceuticals in several cancer types. The already approved c-Met inhibitor drugs and other Phases II and III clinical studies have shown promising results with a remarkable benefit for the patients in different types of cancers. 75 In this study, we have reviewed all the c-Met PET tracers developed up to now, from HGF-like, to mAbs, peptides, and small molecules, with each substrate type bearing unique advantages as well as limitations.
Unfortunately, only one of the reported tracers ( 68 Ga-EMP-100, Table 1) has been studied in a clinical trial with PET application and research should focus on the translation of more probes in the clinic. While zirconium-based antibodies have demonstrated good results in preclinical studies, other probes with smaller structures like peptides and anticalines have also showed good in vivo properties, with the advantages of being compatible with more popular short half-life radionuclides, for example, 18 F. 76 On the other hand, smaller organic molecules have better pharmacokinetics but they are cleared fast from the body which translates in nonoptimal probes for delayed over time imaging. A good compromise in our opinion is the family of the peptide-based tracers as exemplified by the clinical application (via fluorescence detection and recently via PET with 68 Ga-EMP-100, Table 1) of GE-137 resulting in promising impact for detection of malignant polyps and RCC. The main advantages of the peptide-based targeting moieties are that they are cheaper than antibodies and can be easily chemically modified to be radiolabeled with virtually any radionuclide making them attractive for clinical use. 77 However, PET agents based on small organic compound tyrosine-kinase inhibitors have the advantage to directly bind the intracellular binding site of the receptor and be more precise in quantifying the activation of the receptor.
Despite the scarcity of the clinical data for c-Met targeting PET probes, and only the recent first in human application of such tools, some general advantages and drawbacks for this pharmaceutical class can be summarized as commonly reported for the application of other PET radiopharmaceuticals and should be taken into account when considering the future application of c-Met PET probes. For instance, the application of PET probes other than the already discussed diagnosis and therapy monitoring is the use of PET and PET/CT for tumor ablation and guided biopsies. 78 In this field the major advantages of using a PET-guided clinical tumor ablation are to combine anatomical imaging with functional imaging. Moreover, the prolonged half-life of the used nuclides warrants continual localization of the tissue target during the clinical procedure. 79 The capability to accurately target and ablate large tumors and the possibility of estimating the success at the end of the surgery are other advantages of assisted clinical ablation by PET imaging. 79  imaging is not a primary choice for staging early stage breast cancer, it has a key role in systemic staging for this and other types of solid tumors and in comparison with MRI, PET/CT has been proved to have more specificity for breast cancer detection but less sensitivity. 80,81 Overall, PET/MRI overperforms the detection of primary breast cancer when compared to PET/CT, but the improvement is limited if in comparison with MRI only. Moreover, 18 F-FDG can localize early-stage bone marrow metastases before they appear on bone scintigraphy or CT with PET scansion. 82 Predicting the therapeutic response and optimizing it accordingly is a further application of PET imaging for breast and other solid cancers. Unfortunately, as stated in the ongoing clinical guidelines, PET imaging is not beneficial neither for early-stage breast cancer (i.e., large screening of population) in the absence of symptoms nor for the classification of clinical stages of breast cancer. 81 However, PET imaging coupled with CT and MRI adds additional information to standard diagnostic imaging techniques in the detection process of nodal disease and other metastases. Nevertheless, further optimization is still mandatory to reach advanced performances and efficiency, and novel technological advanced PET probes such as those reported in this review will have a primary role in this stage. Finally, when working with PET probes other common limitations could be: renal excretion which may limit the detection in retroperitoneum and pelvis area, short half-life nuclides (e.g., 11 C) resulting in cyclotron produced limiting availability, still limited role in primary staging and first diagnosis, target (e.g., c-Met, PSMA, or other receptors) high dependency, high bone marrow uptake, which may limit the detection of bone metastases, and significant liver and/or other tissue uptake, which may reduce the detection of metastatic disease in those areas.
The merging of accurate imaging radiotracers with PET will allow noninvasive and real-time diagnosis of specific cancer types with accurate picture of tumors and metastases and thus precise staging of the disease.
Moreover, the assessment of the studied protein expression, the uptake kinetics, and the pretherapeutic dosimetry may grant a better selection of the treatments as well as monitoring the patient response to the applied therapy and anticipate detection of recurring disease resulting in personalized medicine and radiotheranostic (e.g., probe based on 67/68 Ga). 83 The recent progress in the research field of c-Met based PET imaging here reviewed are highly encouraging, even if the optimization of a PET probe for clinical purposes still faces challenges, suggesting that this family of tracers could be useful as a diagnostic tool for patients selection and for therapeutic applications in the near future.

ACKNOWLEDGMENTS
The authors gratefully acknowledge Professor Phil Blower (Professor of Imaging Chemistry and Head of Department of Imaging Chemistry and Biology, King's College London) for helpful discussions during the manuscript drafting process. This project has received funding from the European Union's Horizon 2020 Research and Innovation Program under Grant agreement no 893784.

DATA AVAILABILITY STATEMENT
Data sharing not applicable to this article as no data sets were generated or analyzed during the current study.