Shining Damaged Hearts: Immunotherapy-Related Cardiotoxicity in the Spotlight of Nuclear Cardiology

The emerging use of immunotherapies in cancer treatment increases the risk of immunotherapy-related cardiotoxicity. In contrast to conventional chemotherapy, these novel therapies have expanded the forms and presentations of cardiovascular damage to a broad spectrum from asymptomatic changes to fulminant short- and long-term complications in terms of cardiomyopathy, arrythmia, and vascular disease. In cancer patients and, particularly, cancer patients undergoing (immune-)therapy, cardio-oncological monitoring is a complex interplay between pretherapeutic risk assessment, identification of impending cardiotoxicity, and post-therapeutic surveillance. For these purposes, the cardio-oncologist can revert to a broad spectrum of nuclear cardiological diagnostic workup. The most promising commonly used nuclear medicine imaging techniques in relation to immunotherapy will be discussed in this review article with a special focus on the continuous development of highly specific molecular markers and steadily improving methods of image generation. The review closes with an outlook on possible new developments of molecular imaging and advanced image evaluation techniques in this exciting and increasingly growing field of immunotherapy-related cardiotoxicity.


Introduction
The emerging use of novel immunotherapies in cancer treatment increases the risk of immunotherapy-related cardiovascular side effects. Cardiotoxic damage ranges from asymptomatic changes to fulminant short-and long-term complications, including cardiomyopathies, arrythmias, and vascular disease [1][2][3][4][5]. In contrast to conventional chemotherapy, the use of targeted immunotherapies has expanded the forms and presentations of cardiovascular toxicity [4]. Accompanied by increasing long-term survival rates of cancer patients, integrated management and care of cardiovascular disease through cancer and cancer therapy by cardio-oncologists become important to reduce cardiovascular side effects which affect prognosis and quality of life [6].
Immunotherapies for cancer treatment follow passive or active strategies. While in passive immunotherapy, monoclonal antibodies (mAbs) targeting growth receptors, immune checkpoint inhibition, or bispecific T cell engagement are used, active strategies contain especially chimeric antigen receptor (CAR)-T cell transfer [7,8]. All of them can be associated with immunotherapy-related cardiovascular side effects. Monoclonal antibodies

Planar Scintigraphy and Single Photon Emission Computed Tomography
Planar scintigraphy and SPECT are imaging modalities that are based on the usage of gamma emitting radionuclides [80,81]. To generate images, emitted photons are detected using gamma cameras (planar imaging) or tomography systems (SPECT). The processing of additionally acquired computed tomography images (SPECT/CT) enables an improvement of image quality by attenuation correction as well as facilitating the anatomical assignment of the molecular imaging signal [82].
The gamma emitting radionuclide that is predominantly used in routine practice is 99m Tc, as it exhibits particularly favorable properties for image generation and practicability in daily use [83]. These include, inter alia, a short half-life of 6 h as well as the possibility of obtaining the radionuclide from an inhouse generator and binding it to target molecules without the need for a radiochemistry department inside the clinic or outbound practice. In addition, for some tracers, the radionuclide 123 I is used, which has a longer half-life of about 13 h [84].
With regards to drug-related cardiotoxicity, several radiotracers for planar scintigraphy/SPECT imaging are available which can be used to visualize different parameters characterizing cardiac function and damage. These include (time-resolved) visualization of ventricle morphology, examination of myocardial perfusion, and indirect visualization of myocardial damage via imaging of the sympathetic innervation system of the heart.

Positron Emission Tomography
In contrast to planar scintigraphy and SPECT, PET imaging uses positron emitters as radionuclides. The collision of positrons and electrons in the target tissue produces two annihilation photons which are emitted at an angle of about 180 • to each other. The PET imaging technique uses only coincidence events (if two photons are detected at opposite positions of the detector rings) to generate images. Therefore, the number of random detections (arising, for example, from background radiation) is reduced and a higher image quality and lower image noise compared to SPECT imaging are achieved.
In current practice, PET is performed exclusively in hybrid imaging technique, i.e., in combination with magnetic resonance tomography (PET/MR) or computed tomography (PET/CT). Like for SPECT/CT imaging, the reasons are on the one hand technical: the additional morphological imaging modality can be used for attenuation correction, which increases image quality and enables precise quantification of tracer uptake. On the other hand, it allows for anatomical assignment of structures and the simultaneous possibility of radiological co-assessment of pathologies with or without increased tracer accumulation.
The two radionuclides that are most frequently used in clinical routine are 68 Ga and 18 F [25]. These are widely available and have some features useful for routine clinical use such as a short half-life and a high proportion of positrons in their decay process leading to a high PET image quality. For imaging of cardiotoxicity, mostly glucose metabolism PET imaging after application of 18 F-FDG has been used and also been applied to immunotherapy patients. Recently, examinations using 68 Ga-DOTATOC/DOTATATE, which bind to somatostatin receptors, or 68 Ga-labeled fibroblast activating protein inhibitors ( 68 Ga-FAPIs), which target the fibroblast activating protein (FAP), were also described. Therefore, these tracers were selected for a more detailed discussion. For all three tracers, the approaches aim at using the improved image quality of PET for an early detection of cardiotoxicity. Moreover, imaging in PET technique opens the possibility of PET/MR hybrid imaging. This can allow for combined assessment of subtle morphological and functional abnormalities that might lead to very early detection of cardiotoxicity [85].
In this review, for the most promising imaging modalities, possible benefits and application scenarios will be discussed. Due to the limited applications published to date in the field of immunotherapy-related cardiotoxicity, aspects that have been investigated when used for related questions will be transferred to this topic. The examination modalities are organized by molecular target structures and presented in descending order starting with the most promising (in the authors' opinion) modality.

Imaging of Glucose Consumption
For the imaging of glucose metabolism, 18 F-FDG PET is performed. With proper preparation of the patient, an increased glucose metabolism can indicate myocarditis or ischemic myocardium as well as vascular toxicity [86][87][88]. These pathologies are linked to immunotherapy, making 18 F-FDG PET imaging a suitable tool for early detection of adverse effects during therapy which stands out due to its high image quality and the possibility of detection of subtle abnormalities. Additionally, 18 F-FDG PET is often habitually performed in oncological patients for re-staging and assessment of therapy success; therefore, the possibility of a simultaneous examination of the heart with a single protocol can arise or cardiac readouts may be performed from routine oncological scans. This might allow for a specific screening for cardiotoxic effects and leads the authors to consider 18 F-FDG imaging as the most promising imaging modality in this review.
In the pathophysiological development of chemotherapy-related cardiotoxicity, cardiac glucose metabolism can be increased leading to a possible early detection by 18 F-FDG PET [89]. In the context of immunotherapy-related cardiotoxicity, several reports describe an application for investigation of immune checkpoint inhibitor-related myocarditis [11,[90][91][92][93]. These hint at a possibility of early detection and possible improvements compared to cardiac MR [94]. Moreover, 18 F-FDG is an established and commercialized PET tracer that is available at many centers.
Technically, 18 F-FDG accumulates in cardiomyocytes after uptake through GLUT receptors and intracellular retention following phosphorylation by hexokinase [25]. For suppression of the PET signal from physiological myocardial glucose consumption, adequate patient preparation must be performed that aims at shifting myocardial energy consumption to free fatty acids. Typical protocols include prolonged fasting (for at least 12 h), carbohydrate restriction (Atkins diet) for at least one day prior to scanning, fatty meals, and loading with unfractionated heparin (at about 15 min before 18 F-FDG injection) [95]. Figure 1 shows an example of a patient with metastatic malignant melanoma under adjuvant anti-PD1 immune checkpoint inhibition treatment with nivolumab. The patient underwent 18 F-FDG PET/MR as immune checkpoint inhibitor-related myocarditis was suspected due to elevated troponin. Eventually, active myocarditis was verified by PET/MR imaging showing the possibility to perform the examination in immunotherapyrelated cardiotoxicity patients.
compared to cardiac MR [94]. Moreover, 18 F-FDG is an established and commercialized PET tracer that is available at many centers.
Technically, 18 F-FDG accumulates in cardiomyocytes after uptake through GLUT receptors and intracellular retention following phosphorylation by hexokinase [25]. For suppression of the PET signal from physiological myocardial glucose consumption, adequate patient preparation must be performed that aims at shifting myocardial energy consumption to free fatty acids. Typical protocols include prolonged fasting (for at least 12 h), carbohydrate restriction (Atkins diet) for at least one day prior to scanning, fatty meals, and loading with unfractionated heparin (at about 15 min before 18 F-FDG injection) [95]. Figure 1 shows an example of a patient with metastatic malignant melanoma under adjuvant anti-PD1 immune checkpoint inhibition treatment with nivolumab. The patient underwent 18 F-FDG PET/MR as immune checkpoint inhibitor-related myocarditis was suspected due to elevated troponin. Eventually, active myocarditis was verified by PET/MR imaging showing the possibility to perform the examination in immunotherapy-related cardiotoxicity patients. Despite the advantages of 18 F-FDG PET for imaging of immunotherapy-related toxicity, some opinions point to a limited diagnostic value of the technique [96]. In addition, cardiac 18 F-FDG PET uptake can be non-specific and disregarding the laborious patient preparation protocol may prevent diagnostic validity [93]. Therefore, further systematic investigation is needed to identify patient groups in whom 18 F-FDG PET should be performed as imaging modality of choice and to review PET investigations using other tracers that may have further specific advantages for imaging of immunotherapy-related toxicity. Despite the advantages of 18 F-FDG PET for imaging of immunotherapy-related toxicity, some opinions point to a limited diagnostic value of the technique [96]. In addition, cardiac 18 F-FDG PET uptake can be non-specific and disregarding the laborious patient preparation protocol may prevent diagnostic validity [93]. Therefore, further systematic investigation is needed to identify patient groups in whom 18 F-FDG PET should be performed as imaging modality of choice and to review PET investigations using other tracers that may have further specific advantages for imaging of immunotherapy-related toxicity.

Imaging of Cardiac Remodeling and SSTR-Expression
One of these novel PET imaging modalities that can broaden the applicability of PET imaging for immunotherapy-related cardiotoxicity is the recently introduced 68 Ga-FAPI PET. Several reports describe the applicability of the technique, which is also used for oncologic imaging [97,98] and subsequent radionuclide therapy in a theranostic approach [99], for imaging of cardiac fibroblast activation as an early sign of cardiac damage that might be induced by immunotherapy-related cardiotoxicity [100][101][102]. Thus, early detection might be possible [101] using a highly specific target that does not exhibit the shortcomings of 18 F-FDG PET. Moreover, also for 68 Ga-FAPI PET the possibility of simultaneous oncological staging and cardiac assessment in a single examination emphasizes its significance as a highly promising tool. However, 68 Ga-FAPI has not yet been approved and examinations are performed as part of clinical trials or as compassionate use. Therefore, its availabil-ity is still limited to large centers and further developments have to be awaited before widespread use can be recommended.
Biologically, 68 Ga-FAPI targets FAP, a cell-surface serine protease that acts on various hormones and extracellular matrix components [103]. FAP is expressed rarely in healthy adult tissues. However, it is highly upregulated in a wide variety of cancers at sites of active tissue remodeling and is often used as a marker for pro-tumorigenic stroma (cancer mesenchymal stem cells, cancer-associated fibroblasts, sarcoma, and melanoma cells), wound healing, and fibrosis [104]. Moreover, FAP shows an overexpression in benign remodeling processes as they can occur in the context of immunotherapy-related cardiac damage [93,100,105] and after myocardial infarction [106]. After myocardial infarction, fibroblasts undergo dynamic phenotypic changes and differentiate into collagen-secreting proto-myofibroblasts, which can further differentiate into mature myofibroblasts. Activated fibroblasts migrate into the injured myocardium and contribute to tissue replacement, thereby helping to preserve the structural integrity of the infarcted heart, which elicits reactive and interstitial fibrosis and hence decreases cardiac contractility [106]. This pathophysiological process might in part be comparable to remodeling processes after immunotherapy-related damage to cardiomyocytes. Moreover, FAP is upregulated in cardiomyopathies [107]. Figure 2 shows an example of a pancreatic ductal adenocarcinoma patient under distinct chemotherapy regimens who displayed increased cardiac 68 Ga-FAPI uptake as a possible sign of chemotherapy-related cardiotoxity.
[99], for imaging of cardiac fibroblast activation as an early sign of cardiac damage that might be induced by immunotherapy-related cardiotoxicity [100][101][102]. Thus, early detection might be possible [101] using a highly specific target that does not exhibit the shortcomings of 18 F-FDG PET. Moreover, also for 68 Ga-FAPI PET the possibility of simultaneous oncological staging and cardiac assessment in a single examination emphasizes its significance as a highly promising tool. However, 68 Ga-FAPI has not yet been approved and examinations are performed as part of clinical trials or as compassionate use. Therefore, its availability is still limited to large centers and further developments have to be awaited before widespread use can be recommended.
Biologically, 68 Ga-FAPI targets FAP, a cell-surface serine protease that acts on various hormones and extracellular matrix components [103]. FAP is expressed rarely in healthy adult tissues. However, it is highly upregulated in a wide variety of cancers at sites of active tissue remodeling and is often used as a marker for pro-tumorigenic stroma (cancer mesenchymal stem cells, cancer-associated fibroblasts, sarcoma, and melanoma cells), wound healing, and fibrosis [104]. Moreover, FAP shows an overexpression in benign remodeling processes as they can occur in the context of immunotherapy-related cardiac damage [93,100,105] and after myocardial infarction [106]. After myocardial infarction, fibroblasts undergo dynamic phenotypic changes and differentiate into collagen-secreting proto-myofibroblasts, which can further differentiate into mature myofibroblasts. Activated fibroblasts migrate into the injured myocardium and contribute to tissue replacement, thereby helping to preserve the structural integrity of the infarcted heart, which elicits reactive and interstitial fibrosis and hence decreases cardiac contractility [106]. This pathophysiological process might in part be comparable to remodeling processes after immunotherapy-related damage to cardiomyocytes. Moreover, FAP is upregulated in cardiomyopathies [107]. Figure 2 shows an example of a pancreatic ductal adenocarcinoma patient under distinct chemotherapy regimens who displayed increased cardiac 68 Ga-FAPI uptake as a possible sign of chemotherapy-related cardiotoxity. Chemotherapy-related cardiotoxicity was suspected due to increased cardiac FAP-expression (emphasized in early images p.i.) and incomplete cardiac wash-out (observed in late images). PET/CT images were acquired at our institution.
Moreover, somatostatin receptor (SSTR) PET that was previously identified as promising tool for imaging of myocarditis [108] was recently applied for early detection of immune checkpoint inhibitor-related myocarditis. A case series [109] and a case report [102] describe applicability for both 68 Ga-DOTATOC and 68 Ga-DOTATATE (both tracers target SSTR but show differences in their affinity to different SSTR subtypes). Earlier detection of cardiac damage than by cardiac MR is a possible benefit and, compared to 18 F-FDG PET, no laborious patient preparation is necessary [109]. Future studies are necessary to confirm these results which hint at an identification of somatostatin receptor PET as additional modality for imaging of immunotherapy-related cardiotoxicity. However, its applicability for oncological staging is limited to somatostatin receptor expressing malignancies which precludes broad use for simultaneous staging and cardiac assessment. On the other hand, 68 Ga-DOTATOC/DOTATATE are commercialized and, therefore, available at many centers.
The small number of published reports on the applicability of non-FDG PET imaging for immunotherapy-related cardiotoxicity underlines that further systematic investigations are necessary before a widespread clinical application can be achieved. Moreover, future studies might also introduce different tracers targeting other specific molecules in the interaction of immunotherapy and myocardium and advanced methods of image evaluation.

Imaging of Myocardial Damage
A different diagnostic tool for early detection of myocardial damage that can be applied in scintigraphy, SPECT, and PET techniques is imaging of cardiac sympathetic innervation. For the commonly performed imaging in scintigraphy or SPECT technique, 123 I-meta-iodobenzylguanidine ( 123 I-MIBG) is applied, which is a norepinephrine analog and therefore a tracer for sympathetic neuron integrity and function. The dysfunction of the autonomous nervous system contributes to the pathophysiology of heart failure and 123 I-MIBG imaging has already found its way into clinical examination of heart failure patients [110,111].
As chemotherapy-induced damage to the myocardial beta-adrenergic system might contribute to the pathophysiology of cardiotoxicity [112,113] and preclinical studies hint at an earlier detection of chemotherapy-related cardiotoxicity by a decrease in 123 I-MIBG uptake than, for example, by a decline in left ventricular ejection fraction (LVEF) [114], 123 I-MIBG SPECT imaging is a promising technique for early detection of chemotherapy-related cardiotoxicity. Moreover, 123 I-MIBG is a commercialized tracer available at many centers. However, it has not yet been established for clinical routine imaging in this patient cohort.
Most of the literature on the use of 123 I-MIBG imaging for evaluating patients with heart failure is based on measurements from anterior planar scintigraphy images with cardiac uptake quantified in terms of the heart-to-mediastinum ratio (HMR) and the washout rate between early and late images [115]. 123 I-MIBG SPECT has been shown to yield complementary results to planar imaging in patients with heart failure after acute myocardial infarction [115]. Figure 3 depicts examples of physiological and pathologic 123 I-MIBG planar scintigraphy and SPECT imaging.
To date, the application of the technique has not explicitly been described for imaging of immunotherapy-related cardiotoxic effects. Given that for immunotherapy, like for chemotherapy, cardiotoxic effects to the sympathetic innervation seem possible, 123 I-MIBG imaging might also be a promising technique for early detection of cardiac damage in this patient group. Future systematic investigations are necessary to elucidate possible benefits. These might also focus on the application of imaging of cardiac innervation in PET technique using 124 I-MIBG, 18F-dihydroxyphenylalanine ( 18 F-DOPA) [116], or 18 Fflubrobenguane which [117] which shows a similar behavior to MIBG. In this way, a higher image quality can be achieved, which could also improve the possibility of early detection of immunotherapy-related cardiotoxic effects. However, these tracers are currently only used in clinical trials or small patient cohorts and are not widely available. To date, the application of the technique has not explicitly been described for imaging of immunotherapy-related cardiotoxic effects. Given that for immunotherapy, like for chemotherapy, cardiotoxic effects to the sympathetic innervation seem possible, 123 I-MIBG imaging might also be a promising technique for early detection of cardiac damage in this patient group. Future systematic investigations are necessary to elucidate possible benefits. These might also focus on the application of imaging of cardiac innervation in PET technique using 124 I-MIBG, 18F-dihydroxyphenylalanine ( 18 F-DOPA) [116], or 18 F-flubrobenguane which [117] which shows a similar behavior to MIBG. In this way, a higher image quality can be achieved, which could also improve the possibility of early detection of immunotherapy-related cardiotoxic effects. However, these tracers are currently only used in clinical trials or small patient cohorts and are not widely available.

Investigation of Myocardial Perfusion and Viability
Myocardial perfusion imaging is one of the most frequently performed molecular imaging procedures for evaluation of the heart. The examination is typically performed both in rest and after physical stress to investigate perfusion of the myocardium in conditions of reversible ischemia [118]. The comparison of stress and rest images allows detection of stress-induced perfusion defects as an indicator for coronary artery disease [119]. Moreover, myocardial viability can be evaluated [120] and the LVEF can be estimated in electrocardiogram-gated examinations [118]. Imaging can either be performed using cardiac-centered gamma cameras [118] or in SPECT(/CT) technique [121].
Typically, the 99m Tc-labeled tracers 99m Tc-sestamibi and 99m Tc-Tetrofosmin are used for myocardial perfusion imaging. These accumulate in cardiac mitochondria and are not washed out from myocardial tissue after their uptake [122][123][124][125]. Alternatively, the thallium isotope 201 Tl can be used, which mimics potassium in physiological processes (transport into myocardiocytes by Na-K-ATPase and subsequent wash-out), the uptake process correlates linearly with myocardial perfusion [122]. However, 201 Tl myocardial perfusion imaging has a higher radiation exposure to patients than 99m Tc-sestamibi and 99m Tc-Tetrofosmin [126] and can, therefore, currently be considered a second-line option. For an examination under stress conditions, either ergometric stress can be performed or . HMR: heart-to-mediastinum ratio. Images were acquired at our institution.

Investigation of Myocardial Perfusion and Viability
Myocardial perfusion imaging is one of the most frequently performed molecular imaging procedures for evaluation of the heart. The examination is typically performed both in rest and after physical stress to investigate perfusion of the myocardium in conditions of reversible ischemia [118]. The comparison of stress and rest images allows detection of stress-induced perfusion defects as an indicator for coronary artery disease [119]. Moreover, myocardial viability can be evaluated [120] and the LVEF can be estimated in electrocardiogram-gated examinations [118]. Imaging can either be performed using cardiac-centered gamma cameras [118] or in SPECT(/CT) technique [121].
Typically, the 99m Tc-labeled tracers 99m Tc-sestamibi and 99m Tc-Tetrofosmin are used for myocardial perfusion imaging. These accumulate in cardiac mitochondria and are not washed out from myocardial tissue after their uptake [122][123][124][125]. Alternatively, the thallium isotope 201 Tl can be used, which mimics potassium in physiological processes (transport into myocardiocytes by Na-K-ATPase and subsequent wash-out), the uptake process correlates linearly with myocardial perfusion [122]. However, 201 Tl myocardial perfusion imaging has a higher radiation exposure to patients than 99m Tc-sestamibi and 99m Tc-Tetrofosmin [126] and can, therefore, currently be considered a second-line option. For an examination under stress conditions, either ergometric stress can be performed or pharmacological stress can be evoked by application of vasodilators such as the cardiac adenosine receptor agonists dipyridamole, adenosine, or regadenoson or by administration of the catecholamine dobutamine prior to injection of the radiopharmaceutical. Regadenoson can be recommended in most situations because of the drug's safety and adverse event profile and the simplified study protocol compared with the other agents [127]. Figure 4 shows examples of physiological and pathological myocardial perfusion SPECT results. pharmacological stress can be evoked by application of vasodilators such as the cardiac adenosine receptor agonists dipyridamole, adenosine, or regadenoson or by administration of the catecholamine dobutamine prior to injection of the radiopharmaceutical. Regadenoson can be recommended in most situations because of the drug's safety and adverse event profile and the simplified study protocol compared with the other agents [127]. Figure 4 shows examples of physiological and pathological myocardial perfusion SPECT results.  In oncological patients, myocardial perfusion imaging can be performed for pretherapeutic risk evaluation and to assess or monitor treatment-related cardiotoxic effects during or after immunotherapy. Myocardial perfusion imaging enables evaluation of LVEF and perfusion defects in one single examination. Patients might benefit from this simultaneous assessment, as acute coronary syndrome and (worsening of) atherosclerotic vascular disease are described complications of immune checkpoint inhibitor treatment [128,129]. Furthermore, in patients receiving anti-PD1 therapy left ventricular function was impaired in response to stress [130] making stress testing a promising examination technique.
The evaluation of myocardial perfusion imaging is highly standardized, approved, and the applied tracers are commercialized and widely available. Compared to echocardiography, assessment of LVEF by myocardial perfusion imaging is less susceptible to external factors [131]; compared to cardiac MR, the technique is advantageous in costeffectiveness and patients who can or must not undergo MR imaging [131]. Therefore, myocardial perfusion imaging can potentially lead to early diagnosis and timely beginning of cardiac therapy and/or adjustment of immunotherapy, and several reports describe application of myocardial perfusion imaging in patients with immunotherapy- In oncological patients, myocardial perfusion imaging can be performed for pretherapeutic risk evaluation and to assess or monitor treatment-related cardiotoxic effects during or after immunotherapy. Myocardial perfusion imaging enables evaluation of LVEF and perfusion defects in one single examination. Patients might benefit from this simultaneous assessment, as acute coronary syndrome and (worsening of) atherosclerotic vascular disease are described complications of immune checkpoint inhibitor treatment [128,129]. Furthermore, in patients receiving anti-PD1 therapy left ventricular function was impaired in response to stress [130] making stress testing a promising examination technique.
The evaluation of myocardial perfusion imaging is highly standardized, approved, and the applied tracers are commercialized and widely available. Compared to echocardiography, assessment of LVEF by myocardial perfusion imaging is less susceptible to external factors [131]; compared to cardiac MR, the technique is advantageous in costeffectiveness and patients who can or must not undergo MR imaging [131]. Therefore, myocardial perfusion imaging can potentially lead to early diagnosis and timely beginning of cardiac therapy and/or adjustment of immunotherapy, and several reports describe application of myocardial perfusion imaging in patients with immunotherapy-related cardiotoxicity [132][133][134]. As proposed, the technique was used for monitoring of cardiac function during immunotherapy treatment and for clarification of echocardiography results. However, due to cardiac reserve, myocardial damage may occur well before a measurable decrease in LVEF. Therefore, monitoring this parameter might not be the optimal technique for early detection of patients with therapy-related cardiotoxicity [112,135] and the use of more promising examination techniques such as the previously described PET imaging modalities may be useful.
Of note, myocardial perfusion imaging can also be performed in PET technique using, for example, H 2 15 O, 13 NH 3 , or 82 Rb, with the benefit of quantitative image evaluation [127]. However, these examinations are technically demanding, and the required radiopharmaceuticals are not available in many centers. Systematic applications of myocardial perfusion PET in the context of oncologic treatment have only been described in rare reports and preclinical studies with most of them in the context of radiotherapy and none of them covering immunotherapy patients [90,[136][137][138][139][140]. In this manuscript, we therefore refrain from a detailed description of the technique which can, alternatively, be found in various previously published review articles, for example, by Driessen et al. [141] or by Nakazato et al. [142].

Assessment of Ventricular Function and Chamber Morphology
Equilibrium radionuclide angiography (ERNA) can be used to visualize chamber morphology, determine ventricular volumes, and evaluate ventricular wall motion. From electrocardiogram-gated ERNA data, systolic and diastolic volumes can be estimated and the LVEF can be calculated [143,144]. Monitoring of LVEF can be considered a clinical standard to assess the functional impact of immunotherapy-related cardiotoxicity, as heart failure was described as an adverse event of different immunotherapeutic treatment regimens including nivolumab [145], nivolumab plus ipilimumab [146], or pembrolizumab [147]. ERNA has not yet been described in immunotherapy-related cardiotoxicity patients but, for example, in breast cancer patients that are treated with the monoclonal antibody trastuzumab [148], which targets HER2/neu receptor [149]. Cardiac dysfunction, in particular congestive heart failure, is a typical adverse event in treatment with trastuzumab [131,150] and requires monitoring of LVEF.
Technically, ERNA is a blood pool scintigraphy, for which commonly patient erythrocytes are labeled with 99m Tc. Labeling can be performed in vivo, in vitro, or in a mixed in vivo/in vitro method [143]. As an alternative to labeling of erythrocytes, 99m Tc-labeled human serum albumin can be used but exhibits higher background activity [151]. The examination is typically performed in planar imaging technique. Alternatively, SPECT imaging is possible and allows for additional investigation of right ventricular function [152]. ERNA is performed in rest and after ergometric exercise to examine an adequate increase in LVEF in response to physical stress [153]. Figure 5 shows examples of physiological and pathologic ERNA results.
ERNA shows high precision and repeatability [154,155] and fewer variations depending on external circumstances than echocardiography (e.g., observer-dependent factors or quality of echogenic window) [131]. Moreover, the imaging technique is well established and widely available. Therefore, in the context of immunotherapy-related cardiotoxicity, ERNA may be particularly beneficial for pretreatment assessment and to monitor cardiac function during therapy (including post-therapeutic surveillance). An application scenario can be patients with borderline LVEF in echocardiography, in whom accurate diagnosis influences further therapy management [1,156]. Moreover, the high repeatability might facilitate systemic evaluations in multi-centric clinical trials. Compared to cardiac MR, which is also a highly accurate and sophisticated imaging technique to evaluate even small cardiac damage, advantages of ERNA include a higher cost-effectiveness [157] and the applicability to patients who must not undergo MR after implementation of cardiac devices. A drawback might be a limited applicability for early detection of cardiac damage which, from the authors' point of view, makes the previously presented (PET) imaging modalities more promising options in the field of immunotherapy-related cardiotoxicity. ERNA shows high precision and repeatability [154,155] and fewer variations depending on external circumstances than echocardiography (e.g., observer-dependent factors or quality of echogenic window) [131]. Moreover, the imaging technique is well established and widely available. Therefore, in the context of immunotherapy-related cardiotoxicity, ERNA may be particularly beneficial for pretreatment assessment and to monitor cardiac function during therapy (including post-therapeutic surveillance). An application scenario can be patients with borderline LVEF in echocardiography, in whom accurate diagnosis influences further therapy management [1,156]. Moreover, the high repeatability might facilitate systemic evaluations in multi-centric clinical trials. Compared to cardiac MR, which is also a highly accurate and sophisticated imaging technique to evaluate even small cardiac damage, advantages of ERNA include a higher cost-effectiveness [157] and the applicability to patients who must not undergo MR after implementation of cardiac devices. A drawback might be a limited applicability for early detection of cardiac damage which, from the authors' point of view, makes the previously presented (PET) imaging modalities more promising options in the field of immunotherapy-related cardiotoxicity.

Future Trends
Future developments in molecular imaging related to the cardiotoxicity of immunomodulatory drugs may include the investigation of new molecular targets. These will particularly benefit from the higher image quality of PET imaging compared to conventional planar scintigraphy or SPECT techniques. In addition, imaging technology itself is subject to continuous technical development, both on the software and on the hardware side. New developments may significantly improve the image quality and, therefore, also the possibilities of application, for example, regarding detection of low activity concentrations as they may appear in imaging of new molecular targets. In addition, higher image quality also facilitates the application of advanced image analysis techniques. These include, for example, the analysis of time-resolved dynamic data. In addition, the study of molecular imaging data using artificial intelligence techniques is spreading. These methods, on the one hand, enable the analysis of large amounts of data and, on the other hand, can allow the determination of parameters within medical images that are not accessible to the human eye.

Future Trends
Future developments in molecular imaging related to the cardiotoxicity of immunomodulatory drugs may include the investigation of new molecular targets. These will particularly benefit from the higher image quality of PET imaging compared to conventional planar scintigraphy or SPECT techniques. In addition, imaging technology itself is subject to continuous technical development, both on the software and on the hardware side. New developments may significantly improve the image quality and, therefore, also the possibilities of application, for example, regarding detection of low activity concentrations as they may appear in imaging of new molecular targets. In addition, higher image quality also facilitates the application of advanced image analysis techniques. These include, for example, the analysis of time-resolved dynamic data. In addition, the study of molecular imaging data using artificial intelligence techniques is spreading. These methods, on the one hand, enable the analysis of large amounts of data and, on the other hand, can allow the determination of parameters within medical images that are not accessible to the human eye.

New Molecular Targets
The use of novel immunotherapeutic drugs may be accompanied by the possibility of specific labeling of these compounds for PET imaging and visualization of their distribution within the human body. For some of these substances, such imaging possibilities have already been described.
One mechanism of carcinogenesis is related with the immune evasion of cancer cells. Cytotoxic T cells have negative regulators such as CTLA-4 and PD-1. The inhibition of these receptors led to the development of multiple immunotherapy bioengineered agents, some of which block PD-1 and CTLA-4 on T cells and others of which block the PD-1 ligand PD-L1 on tumor cells leading the immune system to recognize cancer cells and destroy them. Whole-body PD-1 and PD-L1 PET (imaging the targets of immune checkpoint inhibitor therapy) can be performed using various radionuclides including 18 F-BMS-98619210 (targeting PD-L1), 89 Zr-pembrolizumab, 89 Zr-atezolizumab, 89 Zr-nivolumab [158][159][160][161], or 64 Cu-labeled PD-1 and PD-L1 antibodies [162].
The pathophysiology of immunotherapy-related cardiotoxicity is not completely elucidated. Cardiomyocytes express PD1 and PD-L1 [130] and it is supposed that the disruption of the PD1-PDL1 pathway can release autoimmune reactions with immune-mediated cardiac injury and polymorphonuclear leukocyte inflammation [7]. Autoimmune-mediated cardiotoxicity involves myosin-specific T-lymphocytes leading to myocardial cell injury and myocarditis, decreased endothelial nitric oxide synthase and consequently to vascular leak syndrome. On the other hand, mAbs targeting ERBs are related to decreased repair and survival of cardiomyocytes [7]. PET imaging using radiolabeled checkpoint inhibitors might be used to identify patients at risk with high cardiac expression. It would be desirable for clinical trials with these novel PET tracers to also examine cardiac tracer accumulation and possible correlations with immunotherapy-related cardiotoxicity.
CAR-T therapy is a promising form of immunotherapy especially for hematological malignancies [163]. After a sample of a patient's T cells has been collected from the blood, the cells are re-engineered, so they sprout chimeric antigen receptors on their surface. When these CAR-T cells are reinjected into the patient, the receptors may help the T cells identify and attack cancer cells throughout the body. One major complication of CAR-T cell therapy is the cytokine release syndrome (CRS). CAR-T cells release pro-inflammatory cytokines including interferon gamma (IFNγ), interleukin (IL)-1, IL-2 receptor alpha (RA), tumor necrosis factor alpha (TNFα), and IL-6 to induce a cytotoxic response. The release of these cytokines, particularly IL-6, also plays a role in the pathogenesis of CRS, including the recruitment of additional T-lymphocytes. High levels of circulating IL-6 can also lead to myocardial stunning which may be clinically indistinguishable from septic cardiomyopathy. Furthermore, CRS results in the activation of prostaglandins, which can also impart a risk of cardiotoxic events, such as tachycardia and hypotension. It is also possible that CAR-T therapy results in a direct or "off-target" cardiotoxic injury as a result of cross-reactivity between T cells and Titin [164]. For example, in children receiving CAR-T cell therapy, a rate of approximately 20% suffered from major cardiovascular events [165].
In PET technique, direct in vivo imaging of CAR-T cells is possible. Promising preclinical data suggest that the integration of reporter genes in the CAR-T genome enables their imaging whenever needed [166,167]. For example, if the human natrium iodine symporter is used, the PET tracer tetrafluoroborate can be employed for imaging [2], which is also a promising tracer for thyroid cancer [168]. This enables visualization of the CAR-T cell distribution in the body at a given time, potentially enabling an assessment of organ toxicity and treatment efficacy. Moreover, imaging can be used to visualize the target molecules used in the UniCAR system [169,170]. To overcome the limitations of CAR-T toxicity, the UniCAR system propagates the use of adapter molecules, which link the modified T cell to the cancer cell [169]. The adapter molecule can be conjugated with a radioisotope to visualize the distribution of the UniCAR cells, potentially allowing inferences about side effects [169]. A cardiac-emphasized distribution pattern of CAR-T and UniCAR T cells could be visualized by PET imaging to allow for identification of patients at risk for immunotherapy-related toxicity. Future studies of these PET imaging modalities could also focus on cardiac side effects.
Other exciting tracers that could be used in future for imaging of immunotherapyrelated cardiotoxicity are 18 F-GP1 [171,172], which visualizes activated platelets by targeting glycoprotein IIb/IIIa of aggregated platelets and could therefore be used to show the effects of increased thrombogenic potential, and 18 F-NaF [173,174], which binds directly to arteriosclerotic plaques. Both have, until now, not yet found their way into clinical routine imaging but might be applicable for imaging of immunotherapy-related cardiotoxicity after systematic clinical investigations.

Technological Perspectives
Over the recent years, a new generation of "digital" PET systems were introduced by all large established PET manufacturers [175][176][177][178][179]. These systems use silicon photomultiplierbased detectors leading to an improved spatial and coincidence timing resolution and reduced image noise [180][181][182][183][184][185]. Possible clinical benefits include a reduction in administered activity or acquisition time [186][187][188][189][190] leading to increased patient comfort, particularly regarding pain-stricken or dyspneic patients [191] from which symptomatic patients with cardiotoxicity might also benefit. In addition, digital PET improves the detectability, especially of small structures and those with low tracer-uptake [182,[192][193][194], which may facilitate the visualization of new biomarkers. Moreover, digital PET enhances the applicability of dynamic PET, i.e., evaluation of time-resolved PET data, a technique that can particularly benefit from the improved data quality [195] and will be introduced in the next section.
Next to the application of digital detectors, a recent technical improvement in PET technology was the introduction of total-body PET systems. The extended field-of-view of these systems (≥1 m) allows simultaneous imaging of a significantly larger section of the body than standard PET systems (with a typical field-of-view of 20-30 cm). The increased number of detectors in total-body PET systems further improves sensitivity and image quality compared to "standard" digital systems [196][197][198].

Artificial Intelligence-Based Approaches and Dynamic PET
Advanced image evaluation enables the extraction of clinically relevant information that enables assessment of cardiac function and monitoring of cardiotoxicity. Two aspects of advanced image evaluation will be discussed here.
First, dynamic PET imaging enables the extraction of additional relevant information from a PET scan. Dynamic PET imaging means that the acquisition of PET images begins simultaneously with the administration of the PET tracer. The distinct analysis of timeactivity-curves by tracer kinetic modeling in different compartments which are defined within the PET images (compartmental kinetic modeling) allows for a more precise characterization of the uptake process of the investigated tracer [195,199]. From that information, extended conclusions about the investigated disease may be drawn.
For example, if an FDG PET scan is performed as dynamic acquisition, the left ventricular function can be assessed. The first pass of the tracer will be captured in dynamic mode, enabling the quantification of the contractile function. This analysis closely resembles the results of conventional ERNA [200]. Another study could show that dynamic acquisition of 11 C-Acetate-PET enables the assessment of cardiotoxicity via imaging the myocardial oxidative metabolism. The dynamic acquisitions allow the fit of a kinetic model, which can be used to extract the myocardial oxygen consumption [201]. The possibilities for dynamic imaging will expand even further with the emergence of new technologies such as total-body PET and improved detector technologies that allow for even higher temporal resolution.
Second, novel algorithmic approaches such as methods of machine learning can be used to assess the cardiac function. For example, machine learning could successfully determine the ejection fraction on echocardiograms [202]. In another study, techniques of artificial intelligence were used to predict cancer therapy-related strain alterations [203]. Similar applications might become subjects of future approaches regarding molecular imaging data for investigation of immunotherapy-related cardiotoxicity.
Until now, the application of these approaches to immunotherapy-related changes is still limited. Further studies have to evaluate the value of dynamic PET and machine learning analysis in the context of immunotherapy-related cardiotoxicity.

Conclusions
Several applications of nuclear cardiology imaging modalities in immunotherapyrelated cardiotoxicity patients have found their way into clinical diagnostics. The different examinations reflect technological advances in imaging technique and development of new tracers from blood pool scintigraphy via three-dimensional myocardial perfusion and cardiac sympathetic innervation SPECT to novel elaborate PET technologies, such as FAPI PET for imaging of cardiac remodeling. Table 1 summarizes the most promising nuclear cardiology examination methods that are presented in this review and their most important characteristics. In particular, PET imaging using 18 F-FDG, 68 Ga-FAPI, or-depending on the malignancy- 68 Ga-DOTATATE/DOTATOC can allow for simultaneous oncologic staging and cardiac assessment with the benefit of possible early detection of cardiac damage. As the number of immunotherapy patients is increasing, early detection will become even more important to identify patients at risk who demand timely beginning of therapy. For many techniques, the number of clinical applications described so far is small and further investigation is needed before general applicability. On the other hand, due to the ongoing development, the introduction of further exciting tracers, for example, to directly visualize the effect of the therapeutics on the myocardium, can be expected. These might infer further groundbreaking improvements for the application of nuclear cardiology imaging options in immunotherapy-related cardiotoxicity.