The Current Status and Future Potential of Theranostics to Diagnose and Treat Childhood Cancer

In theranostics (i.e., therapy and diagnostics) radiopharmaceuticals are used for both therapeutic and diagnostic purposes by targeting one specific tumor receptor. Biologically relevant compounds, e.g., receptor ligands or drugs, are labeled with radionuclides to form radiopharmaceuticals. The possible applications are multifold: visualization of biological processes or tumor biology in vivo, diagnosis and tumor staging, therapy planning, and treatment of specific tumors. Theranostics research is multidisciplinary and allows for the rapid translation of potential tumor targets from preclinical research to “first-in-man” clinical studies. In the last decade, the use of theranostics has seen an unprecedented value for adult cancer patients. Several radiopharmaceuticals are routinely used in clinical practice (e.g., [68Ga/177Lu]DOTATATE), and dozens are under (pre)clinical development. In contrast to these successes in adult oncology, theranostics have scarcely been developed to diagnose and treat pediatric cancers. To date, [123/131I]meta-iodobenzylguanidine ([123/131I]mIBG) is the only available and approved theranostic in pediatric oncology. mIBG targets the norepinephrine transporter, expressed by neuroblastoma tumors. For most pediatric tumors, including neuroblastoma, there is a clear need for novel and improved radiopharmaceuticals for imaging and therapy. The strategy of theranostics for pediatric oncology can be divided in (1) the improvement of existing theranostics, (2) the translation of theranostics developed in adult oncology for pediatric purposes, and (3) the development of novel theranostics for pediatric tumor-specific targets. Here, we describe the recent advances in theranostics development in pediatric oncology and shed a light on how this methodology can affect diagnosis and provide additional treatment options for these patients.


INTRODUCTION
Theranostics in nuclear medicine includes the use and application of two identical or very closely related radiopharmaceuticals for therapy and diagnosis. In oncology, tumor-specific substrates, receptor ligands, or drugs can serve as lead for theranostic development when labeled with specific radionuclides for imaging or therapy ( Figure 1A). As the molecular structure of both the diagnostic and therapeutic radiopharmaceuticals are identical, diagnostic images can become predictive for therapeutic response because the biological characteristics and binding potential of both are similar, irrespective of the radionuclide (2)(3)(4).
For diagnosis, positron emission tomography (PET) is a nuclear imaging technique that enables the visualization and quantification of molecules equipped with positron emitting radionuclides. The most used radionuclide for imaging is Fluorine-18 ( 18 F) in the form of [ 18 F]FDG. [ 18 F]FDG PET visualizes increased carbohydrate uptake in tissue, e.g., tumor tissue, and is important for diagnosis, staging and treatment monitoring. For PET tracer development, any molecule that displays tumor-specific targeting can be used, including small molecules, peptides or biologicals. Radionuclides used for PET tracer development are, among others, Carbon-11 ( 11 C) and Fluorine-18 ( 18 F) facilitating small molecule labeling, Gallium-68 ( 68 Ga) for peptide radiolabeling, and Copper-64 ( 64 Cu) or Zirconium-89 ( 89 Zr) for the labeling of monoclonal antibodies (mAbs) and other biologicals. PET imaging enables studying the distribution and kinetics of labeled molecules and the biochemical and physiological processes. Molecular imaging by means of PET can, thus, facilitate and guide cancer treatment in many ways (5,6). Currently, PET is the most sensitive technique for nuclear imaging; it requires nanomolar amounts of the radiopharmaceutical for imaging. These nanomolar amounts will not induce pharmacological effects, hold minimal risks for toxicity, and are described as the micro-dosing concept. Microdosing allows for fast translation of novel PET tracers into clinical trials in small "first-in-man" or phase 0 studies, when produced under good manufacturing practice (GMP). Single photon emission computed tomography (SPECT) is an alternative nuclear imaging technique and enables the visualization of g-emitting radionuclides and was the basis for early theranostics development, where, among others, the different radionuclides of iodine were used for imaging (e.g., Iodine-123 ( 123 I) and Iodine-131 ( 131 I)). Therapeutic radiopharmaceuticals for treatment of cancer are predominantly labeled with b-emitting radionuclides. The radionuclide 131 I, Lutetium-177 ( 177 Lu) and Yttrium-90 ( 90 Y) are frequently used for this purpose. The emitted b-particles travel 1-12 mm through tissue upon decay while losing energy and causing cytotoxic damage to the cell to induce apoptosis. Alternatively and more recently, a-emitting radionuclides, e.g., Astatine-211 ( 211 At) or Actinium-225 ( 225 Ac) were explored for therapy (7)(8)(9). The high energy deposition and a limited range of the a-particles in tissue (0.005-0.11 mm) result in very strong cytotoxic and therapeutic effects. Nowadays, a-emitting radionuclides become more widely available, research toward the development of therapeutic radiopharmaceuticals with these radionuclides is emerging, and first-in-man studies are expected in the near future.
Successful theranostics have been developed for somatostatin receptor positive neuroendocrine tumors with [ 68 Ga/ 177 (14)(15)(16). Despite the proven value of theranostics in adult oncology, its potential was minimally explored for childhood cancer and is still at its infant stage. However, many opportunities and applications present themselves. In this review, we discuss different strategies for theranostics development for childhood cancer and divided these into (1) the existing theranostics and improvement thereof, (2) theranostics developed for adult oncology and translation thereof for childhood cancer, and (3) the development of novel theranostics for specific pediatric tumor targets. By describing the recent advances in theranostics research we discuss how it can affect diagnosis and therapy for childhood cancer in the future.

CURRENT THERANOSTICS IN PEDIATRIC ONCOLOGY
[ 123/131 I]mIBG is the only theranostic currently available for routine clinical use to image and treat neuroblastoma tumors that express the norepinephrine transporter (NET). mIBG is a structural analog of the neurotransmitter norepinephrine and is actively transported into the tumor by NET. Inside the cell, mIBG is stored in the cytoplasm, mitochondria, and in vesicular monoamine transporter (VMAT)-coated and neurosecretory vesicles (17)(18)(19)(20)(21). [ 123 I]mIBG SPECT imaging is currently the standard of care to diagnose primary tumors and distant metastases in neuroblastoma and for staging and disease response evaluation after treatment. In total, approximately 95% of neuroblastoma tumors are [ 123 I]MIBG avid. The remaining 5% of tumors are either well-differentiated ganglioblastoma or very undifferentiated neuroblastoma with little or no NET transporter expression. Although [ 123 I]mIBG SPECT has a high specificity and sensitivity, it also has disadvantages being poor image resolution, long scanning times, and iodine-driven thyroid toxicity. Accompanied by imaging, [ 131 I]mIBG initially showed therapeutic effectiveness in bulky tumors (22). Subsequently, it was shown that [ 131 I] mIBG was feasible and effective in the first treatment of high-risk neuroblastoma patients (23). However, two systematic reviews failed to show a survival advantage for [ 131 I]mIBG treated patients (24,25). In two studies [ 131 I]mIBG was combined with busulfan and melphalan followed by autologous stem cell rescue. For both, acceptable toxicity in highly pretreated patients and encouraging responses were observed. This has led to the implementation of this combination for ultra-high-risk patients who failed to respond adequately during induction treatment for high-risk neuroblastoma. The current European SIOPEN VERITAS study explores the role of [ 131 I]mIBG in combination with topotecan and stem cell rescue followed by another highdose consolidation with Buslfan and Melphalan and a second stem cell rescue. The aim is to increase the survival of these ultrahigh-risk patients. In conclusion, [ 131 I]mIBG treatment is still under investigation and its definitive role has not been determined. In addition to the discussion on therapeutic response, patients receiving [ 131 I]mIBG also suffer from iodine uptake in the thyroid and increased risk for long-term thyroid dysfunction or secundary thyroid cancer. Last, after [ 131 I]mIBG administration, patients need to live in isolation for 5-7 days and strict precepts for 2-3 weeks. Despite the value of [ 123/131 I]mIBG as a theranostic, both imaging and therapy have serious disadvantages and limitations that steer the research toward novel approaches.  (26). The radionuclide 18 F is a cyclotron produced b + -emitter with a short range in vivo, resulting in a high image quality. Furthermore, PET-CT (or PET-MRI) images can be analyzed quantitatively for tracer distribution. 18 F-labeled radiopharmaceuticals are, therefore, ideal for high-resolution diagnosis, faster acquisition, and low radiation burden. Until recently, however, the production of [ 18 F]mFBG has been challenging. It requires a nucleophilic aromatic substitution of an electron-rich molecule (27,28 In addition to improved imaging, research is now focused on the development of an improved alternative for [ 131 I]mIBG therapy. 131 I is a b --emitter with a t 1/2 of 8.04 days. Furthermore, when [ 131 I]mIBG is metabolized and 131 I is released, it will accumulate in the thyroid. Therefore, the thyroid is blocked as a preventive action by administration of excess iodine to avoid undesired effects. As an alternative for 131 I, 211 At has been explored. 211 At is an a-emitter with a t 1/2 of 7.2 h and a range of 0.005-0.11 mm in tissue. These physical properties cause very strong cytotoxic and therapeutic effects. Furthermore, 211 At does not accumulate in the thyroid and potentially will not cause any undesired damage (32,33) At]mABG, which may become a practical concern. 211 At can only be produced by high-energy cyclotrons, of which a few are installed worldwide, and thereby the access is limited (34).

FROM ADULT ONCOLOGY TO PEDIATRIC ONCOLOGY
Theranostics available in routine clinical care are a rich source of potential theranostic candidates in pediatric oncology.
The somatostatin receptor (SSTR) family is one of the first discovered and most successful targets identified for which theranostics were developed. To date, 5 subtypes of SSTR (i.e., SSTR-1, 2A, 3, 4, and 5) are characterized. In particular, SSTR-2A is important with high expression levels for neuroendocrine tumors. It is involved in secretion, proliferation, and the induction of apoptosis (35 This warrants further clinical studies on imaging and treating SSTR-2A positive pediatric cancers with these theranostics in the near future ( Figure 1C) (42).
Another theranostic candidate target that was extensively explored in adult oncology is the C-X-C chemokine receptor 4 (CXCR4). The expression levels of CXCR4 and its natural ligand, CXCL12, are correlated to tumor development and metastasis A target that recently received much attention is the fibroblast activation protein a (FAP) (51). FAP is a serine protease that is selectively expressed in the stromal fibroblasts of the tumor, which is often observed for breast cancer, colon cancer, and pancreatic cancers (52). FAP expression is observed in glioblastomas and can be a valuable theranostic target for pediatric cancers. FAP-specific inhibitors (FAPI) have been developed based on quinoline scaffolds. For diagnostic purposes, promising results were obtain after radiolabeling with 68 Ga (53,54). In particular, [ 68 Ga]FAPI-04, -21, and -46 resulted in high-contrast images, and as a proof-of-concept, 28 different tumor types were visualized with [ 68 Ga]FAPI-04 (55,56). All FAPI compounds allow radiolabeling with 177 Lu too to obtain the corresponding therapeutic radiopharmaceutical. Preclinical studies with [ 177 Lu]FAPI-21 and -46 in tumorbearing mice gave promising results (Table 1) (57). As FAP is also expressed by glioblastomas, these theranostics have potential for the diagnosis and treatment of pediatric cancers.
Monoclonal antibodies (mAbs) and mAb-fragments had unprecedented impact on the treatment of cancer patients. However, clinical benefit is usually only achieved in a percentage of the patient population. The application of 89 Zr-labeled mAbs as ImmunoPET tracers has become increasingly important to visualize these compounds in vivo and assess the distribution, kinetics, and the biochemical and physiological behaviour (4,58). Nowadays, more than 75 clinical trials are ongoing with 89 Zrlabeled mAbs and the radiolabeling can be achieved via generic methods (59,60). Despite the clinical impact of ImmunoPET with [ 89 Zr]mAbs for adult oncology and other indications, ImmunoPET with available [ 89 Zr]mAbs has barely been explored for pediatric cancers. The only reported application of ImmunoPET was [ 89 Zr] bevacizumab in diffuse intrinsic pontine glioma to study vascular endothelial growth factor (VEGF) excretion and the potential to treat these patients with bevacizumab (61). Though ImmunoPET in pediatric cancer patients is not common, it should be anticipated that this methodology can also have an impact for these patients in the future.

SPECIFIC THERANOSTIC TARGETS IN PEDIATRIC ONCOLOGY
Pediatric cancers have a distinct biological profile with unique molecular targets that are not expressed in adult cancers. These targets embody unique opportunities for the diagnosis and treatment of pediatric cancers, but due to small patient populations, it remains a challenge to identify them and develop theranostics against these targets.
A target of interest for theranostic development is ganglioside D2 (GD2). GD2 is a glycosphingolipid and selective cellular marker that is expressed by neuroblastomas, osteosarcomas, and glioblastomas (62,63). Though its exact function is still not fully understood, it is assumed that it plays a crucial role in cell adhesion, migration, and tumor metastasis. Dinutuximab (Unituxin ® , United Therapeutics) is FDA approved, and Dinutuximab beta (Qarziba ® , EUSA Pharma) is EMA approved for the treatment of GD2-positive neuroblastoma tumors (64,65). Despite increased survival rates from 46% to 66%, for high-risk neuroblastoma patients, 30% of the patients will relapse independent of the GD2 expression levels (  (70). Though encouraging, clinical translation of these radiopharmaceuticals has yet to be achieved.
More recently, B7-H3 (CD276) has become a validated pediatric cancer target for immunotherapy in pontine gliomas and neuroblastomas (71,72). To date, two mAbs were developed, Enoblituzumab (MacroGenics) and Omburtamab (Y-mAbs), to treat B7-H3 positive tumors (73, 74) Based on these immunotherapeutics, attempts at the development of theranostics are reported. Especially with 8H9 (i.e., Omburtamab) multiple clinical trials are ongoing. The theranostics pair [ 124/131 I]8H9 is investigated for B7-H3 positive pontine glioma tumors and a modest survival benefit was reported (Table 1) (75,76). As specific brain tumors (e.g., gliomas) express B7-H3, it is important that passage and delivery of the radiopharmaceutical across the blood-brain barrier is achieved. As such, radiolabeled [ 124/131 I]Omburtamab is ideal to investigate drug targeting in these patients. As B7-H3 is acknowledged as a pan-tumor target, theranostics targeting B7-H3 might become of general importance for childhood cancer.

CONSIDERATIONS AND REQUIREMENTS FOR NUCLEAR MEDICINE IN CHILDHOOD CANCER
The application of theranostics for the diagnosis and treatment of childhood cancers is in its infancy. With the availability of radiopharmaceuticals and theranostics for the various adult cancers, there is a lot of potential to translate and directly apply these for childhood cancers. Clinically available SPECT/ PET tracers and therapeutic radiopharmaceuticals can directly be applied for pediatric cancers when the target is present and validated for the respective tumor type. Examples include SSTR-2A, CXCR4, and FAP-positive tumors. As childhood cancers have unique target expression profiles, with GD2 and B7-H3 as examples, novel theranostics can be developed for these yet unexplored targets. Unique target-finding programs are in place to unveil novel childhood cancer-specific biological features for which theranostics can be developed. A critical note and challenge is that target expression of cancers in general cannot always be directly correlated to positive imaging and treatment results. Preclinical research programs are, therefore, required to validate target expression and the potential of the target against which to developed theranostics.
A successfully developed theranostic that shows potential in preclinical studies warrants clinical translation. To achieve that, the theranostic needs to be produced under GMP to guarantee product quality and patient safety (77). Clinical translation of developed theranostics is relatively straightforward as procedures and GMP production facilities are widely available.

CONCLUSION
Theranostics have unprecedented value to diagnose and treat cancers. Many novel theranostics are under development and expected to enter clinical trials and care in the near future. For the diagnosis and treatment of childhood cancers, theranostics research is still in its infancy, but following the path of adult oncology, its value is promising. They are expected to become additional and valuable tools to diagnose and treat childhood cancers.

AUTHOR CONTRIBUTIONS
AP drafted the manuscript. All authors contributed to the article and approved the submitted version.