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The Role of Labeled Annexin A5 in Imaging of Programmed Cell Death: From Animal to Clinical Imaging

Posted on: Friday, 23 April 2004, 06:00 CDT

Programmed cell death plays a critical role in embryology, homeostasis and disease. However, until recently no non-invasive imaging modality has been able to visualize this process directly. Annexin A5 binds to cells undergoing programmed cell death. When labeling this protein, Annexin A5 becomes a tool for the detection of programmed cell death in vitro and in vivo. Labeled Annexin A5 has enabled our group and others to detect programmed cell death non- invasively in animals and patients. This review will highlight the development of this imaging modality in cellular and animal models. Furthermore, we "will discuss Annexin A5 imaging in human disease. We will focus on the clinical applications and their relevance, limitations and future perspectives of non-invasive imaging of programmed cell death using labeled Annexin A5.

KEY WORDS: Apoptosis - Annexin V - Cell death - Cardiovascular diseases - Oncology.

Programmed cell death, also termed apoptosis, is a crucial mechanism in embryology, homeostasis and disease. From a clinician's standpoint, disease in which programmed cell death plays a crucial role can be divided into 2 categories; one category in which the amount of programmed cell death is too low compared to cell proliferation, as can be observed in oncology. On the other end of the spectrum are the diseases characterized by a relative excess of cell death, such as transplant rejection, myocardial/cerebral infarcts and (neuro-)degenerative disease. If these rates of cell death and cell proliferation can be controlled for specific cells, than this would enable the clinician to control the disease. However, this approach to control programmed cell death would need some measure to monitor the rate of cell death. In this review we will first discuss briefly the possibilities to detect cell death, and then go into one of the proteins able to image programmed cell death, labeled Annexin A5.

Programmed cell death: basic principles

Programmed cell death can be defined as the carefully orchestrated demise of a cell due to activation of specific enzymes. A central role is played by enzymes termed the caspases (cystein aspartate specific proteases), which cause many of the morphological features associated with programmed cell death such as chromatin condensation, dissolution of the nuclear membrane, nuclear shrinkage and formation of apoptotic bodies. These apoptotic bodies are then cleared by adjacent cells and professional phagocytes.1-3 In addition, activation of caspase 3 causes one of the final steps in programmed cell death, that of fragmentation of cellular DNA, through activating a specific endonuclease. Classically, 2 pathways are defined which can lead to activation of the caspases, the intrinsic and extrinsic pathway. Both pathway react to different stimuli, which can cause the cell to initiate its cell death program. The intrinsic pathway is activated when external signals, such as binding of Fas ligand or tumor necrosis factor (TNF) to its receptor. These receptors are also termed death receptors. The binding of either Fas ligand or TNF to death receptors then causes the activation of caspase 8, which in its turn activates a cascade of caspases. This finally leads to the demise of the cell and clearance of the cell remnants by macrophages. The intrinsic pathway is activated through direct damage to the cell, such as radiation, serum starvation or reactive oxygen species. This in term leads to release of different factors from the mitochondrion, such as cytochrome C, apaf-1 and apoptosis inducing factor (AIF). Cytochrome C and apaf-1 react with caspase 9 to form the apoptosome, which trigger further downstream apoptotic events. Both pathways converge at the activation of caspase 3. However, as research progresses, evidence of cross talk between these pathways and caspase- indepenclent cell death emerges, as more regulatory mechanisms are discovered.4 Also, the classical division between apoptosis and necrosis has been abandoned. Rather, a model has been suggested with a to be a gliding scale between classic, caspase mediated apoptosis, and accidental necrosis or cell lysis. The intermediate steps are apoptosis like programmed cell death, the "caspase independent cell death" and necrosis-like programmed cell death, which seems to be mostly mitochondrially mediated.5 Interestingly, the basal mechanisms of programmed cell death have been preserved in most species, from the nematode to human cells.

In vitro detection of programmed cell death

Detection of programmed cell death can be done in a variety of ways. The first descriptions of programmed cell death focused mainly on morphological features of programmed cell death as described above.1-3 All of these events can be distinguished by routine hematoxylin and eosin (H&E) staining. Although this technique is sensitive, quantification is difficult due to interobserver variability and assessment of larger areas can be very time- consuming and tedious.3 A wide variation of techniques for the detection of apoptosis have been described since the reports of Wyllie et al. in 1980.3 Techniques for detection of apoptosis have focused on picking up specific parts of the cell death program. One of these methods relies on detection of the final stage of cell death in which the DNA is fragmented. These fragmented pieces of DNA can be detected by different techniques. The most well known of these involves staining of tissue sections by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL).6 Additio-nally, antibodies against single stranded DNA can be used for the detection of fragmented DNA. These techniques have a the disadvantage that they also detect DNA cleavage due to non- apoptotic processes, such as for example DNA repair.7 Other features of the cell death program can also be detected, for instance the presence of activated caspases, for instance caspase 3. Detection of activated caspase 3 or caspase cleavage products can be performed on tissue sections, and thereby provides clues for detection of apoptosis in addition to morphological features and stainings mentioned above. Activated caspases can also be characterized and quantified by a variety of other techniques, including immunoblotting, cleavage of synthetic substrates, affinity labeling and confocal microscopy.8

Of course, all of these techniques require samples of the tissue to be processed. Although tissue can be taken from any organism, assessment of programmed cell death will occur ex vivo. These techniques do provide very accurate information, which can in some cases be quantified. Unfortunately, these techniques are unable to follow the cell death process in time, in the living organism, unless multiple samples are taken. In addition, there is the problem of sampling error. For in vivo monitoring of programmed cell death a technique is required that is non-invasive, can be repeated several times in the same organism and is relatively rapid in its assessment. A means to achieve this goal is molecular imaging.

Molecular imaging of cell death: basic principles

Molecular imaging can be defined as the in vivo characterization and measurement of biologic processes at the cellular and molecular level. This allows assessment of molecular abnormalities that lie at the basis of a disease, instead of visualizing anatomical abnormalities occurring in an end-stage of disease. Molecular imaging of cell death tries to visualize one of the key components of homeostasis and disease, in this case the loss of cells due to programmed cell death.9

Figure 1.-Mechanism of programmed cell death detection by labeled Annexin A5. On the left side of the scheme, the cell death program is activated by, in this case, binding of TNF. This causes activation of the cell death program (lower left part), which in turn causes phosphatidylserine (PS) to externalize (lower right). The externalized PS is then a target for Annexin A5. Annexin A5 is labeled, so by detecting the label, the apoptotic cell can be identified (upper right).

Molecular imaging is based on the principle that certain molecules can be labeled and imaged in vivo, thereby targeting events in the complex environment of a living organism. To image these molecules, they generally must have a high affinity and reasonable pharmacodynamics and they have to be able to overcome biological barriers such as a vascular wall, the interstitial space and sometimes the cell membrane. Imaging modalities must be sensitive. To have a better idea of timing of events and exact localization, these techniques require a short acquisition time and a high resolution.9 The current techniques of molecular imaging of cell death are based on the externalization of phosphatidyl serine (PS), an event that takes place during the early phase of programmed cell death, and remains. Phosphatidyl serine is distributed asymmetrically across the cell membrane in normal cells. When the cell death program is activated, PS is externalized (Figure 1). Annexin A5 forms binds with a strong affinity to the externalized PS. When Annexin A5 is labeled, detection of the cells undergoing programmed cell death is possible. Depending on the label used, this can be done in tissue sections, cell cultures, but als\o in the complex environment of a living organism. Externalized phosphatidyl serine can of course be a target for other imaging molecules. A good example for this is synaptotagmin I, from which the C2 domain binds to anionic phospholipids exposed on the outer membrane of cells. Indeed, it has been shown that this imaging molecule can be used as a MRI probe when labeled with super paramagnetic ion oxide nanoparticles (USPIOs).10

Role of PS exposure in programmed cell death

PS exposure is described by many groups as one of the hallmarks of programmed cell death. Many groups have studied its function. One of the earliest reports in the function of PS exposure is a study of the group of Fadok et al.11 In their experiments, they used a thymocyte model, in which they initiate programmed cell death by stimulation of their Ag receptors. This model is actually one of the first models by Wyllie et al.1 in which programmed cell death is reported. Fadok et al.11 describe the loss of membrane PS asymmetry in thymocytes that show DNA fragmentation, a nowadays well-known feature of programmed cell death. Then they show ex vivo and in vivo that the apoptotic thymocytes are cleared by macrophages, and that this phagocytosis is PS dependent.11 Later this and other groups have shown the same principle works in all cell types.

Figure 2.-Annexin A5 binds to cells in all stages of the cell death program. Here, Jurkat cells are stimulated by Fas ligand. A) The early phase of apoptosis is seen. Already we can observe binding of fluorescent labeled Annexin A5 to the cell membrane. B) A later stage, in which we see formation of blebs and Annexin A5 binding. C) It is the final stage of apoptosis, the cell remnants. These also bind labeled Annexin A5.

Figure 3.-Paw of a chick embryo at day 13. Binding of labeled Annexin A5 can be observed in the interdigit space.

Figure 4.-Transverse section of a murine heart after 30 minutes of ischaemia and 90 minutes of reperfusion (I/R 30/90). Cell stain for presence of labeled Annexin A5 in the midmyocardium, a typical site in mouse infarcts.

Molecular imaging of programmed cell death using labeled Annexin A5

All imaging modalities for molecular imaging of programmed cell that have been described in the literature so far use the mechanism of PS exposure as a target. Two molecules have been described targeting this exposed PS. The molecule for targeting PS that has been studied most is Annexin A5. Annexin A5 has been studied for a longer period of time as a phospholipid binding protein. As such, it is a family member the annexins. The Annexins have structural and functional properties in common. The common functional feature is the binding of Annexins to a phospholipid surface.12 Annexin A5 binds to phospholidips in a Ca2+ dependent manner. Some authors propose a model in which Ca2+ causes a change in conformation of the binding site of Annexin A5, which can then bind to the (exposed) phosphatidylserine. In 1995 this mechanism was used to develop assay which was based on the binding of Annexin A5 to apoptotic cells.13 When Annexin A5 is labeled with a marker, such as biotin, a fluorescent label or a radioligand it can be detected by a variety of different techniques such as light- and laser-microscopy or detection of radioactivity via a [gamma]-camera.

Figure 5.-Experimental setup for in vivo imaging of fluorescent labeled Annexin A5. A stereomicroscope is fitted with a fluorescent module and a powerful CCD camera. The mouse is under narcosis, the chest is opened and the heart is kept in the plane of focus by a micromanipulator. A: stereomicroscope; B: fluorescence module; C: CCD camera; D: computer; E: heating lamp; F: heating pad; G: ventilation equipment; H: micromanipulator.

The affinity assay of Martin et al. and van Engeland et al.14, 15 showed that Annexin A5 could be used for detection of programmed cell death in vitro. In this assay an immortalized cell line, Jurkat cells, were stimulated with a potent trigger for apoptosis, Fas ligand. It was then shown that fluorescent labeled Annexin A5 bound to these cells in all phases of the cell death program as shown in Figure 2. Further work was needed to prove that this concept would also work in the complex environment of the living organism. The 1st model that was used to address this question was a mouse embryo model. In this model our group focused on formation of the digits, which takes place at stage E13 in the chick embryo. This timing was chosen because formation of the digits at day E13 depends among others on apoptosis of the cells in the interdigit webs. Before sacrifice of the organism, Annexin A5 labeled with biotin was injected. Embryos were sacrificed and embedded. When staining for biotin, uptake of the labeled Annexin A5 was seen in the interdigit webs,16 thereby proving the concept that Annexin A5 can be used for the detection of apoptosis in the complex environment of a living organism. This is shown in Figure 3. Next, the feasibility of using labeled Annexin A5 to detect programmed cell death in pathological situations was tested. A mouse model of ischaemia and reperfusion was used. In this model, the left anterior descending artery of the heart of a mouse was ligated. Prior to ligation of the artery, Annexin A5 with a biotin label was infused. The ligation of the left anterior descending artery initiates cell death distal to the site of ligation. This model mimics the situation of an acute myocardial infarction and reperfusion strategies in humans. Dumont et al. showed that this model indeed can be used to detect cell death.17 A typical example of cardiomyocytes staining for presence of Annexin A5 is seen in Figure 4. When further analyzing the cells that stained for labeled Annexin A5, they showed presence of DNA laddering, one of the previously described hallmark features of programmed cell death. In addition, this model was used to see whether a cell death inhibitor, in this case the Na-H exchange inhibitor eniporide could prevent cell loss. They showed that when mice were pretreated with the Na-H exchange inhibitor, the amount of cell death in the area at risk was markedly reduced. Thirdly, the Na- H exchange inhibitor was administered at the time of reperfusion, thereby mimicking the clinical situation. Even in this situation reduction of the infarct size was achieved, when measuring the amount of cells that had exposed PS after 30 minutes of ischaemia and 90 minutes of reperfusion.17

Figure 6.-A, B) In vivo whole heart imaging at 1/R 30/1 and 1/R 30/8. Notice the increase in intensity of signal. Via this method, the externalization of PS can be monitored in a time dependent manner, allowing real time visualization of activation of the cell death program in the beating heart.

The use of fluorescent labeled Annexin A5 in molecular imaging of programmed cell death

The concept of detection of cell death is thus functional in a cellular, in vitro context and animal, ex vivo context. However, monitoring programmed cell death in an in vivo environment could provide insight in timing of apoptotic events. This in turn could provide clues for timing of therapy, for instance determining the therapeutic interval for cell death blockers in patients with an acute myocardial infarction. For this purpose, the fluorescent labeled Annexin A5 was used in an ischaemia and reperfusion model. Again, the LAD of the mice was ligated, and re-opened after 30 minutes of ischaemia. First, the heart was taken out and placed under the operating microscope, which was fitted with a fluorescent module. The experimental setup is shown in Figure 5. The area of infarction could be easily visualized in this manner (Figure 6). When analyzing the sections, the cardiomyocytes in the mid- myocardium had exposed PS and bound Annexin A5, as was previously shown with the biotin labeled Annexin A5. These results led to the conclusion that this technique is able to visualize cardiomyocytes exposing PS that lie in the midmyocardium, beneath a layer of 1 to 3 cardiomyocytes. Next we aimed to visualize the binding of Annexin A5 in the beating murine heart. Slowing heart rate and fixating the heart could overcome problems of motion artifacts. Increasing magnification allowed us to visualize the binding of fluorescent labeled Annexin A5 at the single cell level in the beating murine heart, as shown in Figure 7.

Figure 7.-A-C) Binding of fluorescent labeled Annexin A5 to rod- like cells in vivo. These cells have 2 nuclei. B) This is typical for cardiomyocytes. When zooming in turtlier, binding of Annexin AS to single cells can be visualized in vivo, in the beating murine heart. D-F) Ex vivo confirmation of the data.

The use of radiolabeled Annexin A5 for molecular imaging of programmed cell death

Of course, these assessments of programmed cell death still require an open thorax in these test animals. The group of Blankenberg et al. then used 3 animal models of programmed cell death to see whether labeled Annexin A5 could be used for non- invasive imaging of programmed cell death. To do this, they labeled Annexin A5 with a radioactive tracer, ^sup 121^Iodine. The ^sup 121^I can be detected using a [gamma]-camera. This device detects the specific [gamma]-radiation coming from this radioisotope, and is able to localize this within an organism. It does however, have limited spatial resolution and requires a longer acquisition time when compared to fluorescent techniques. The main advantage is that this [gamma]-racliation travels through tissue, and thus non- invasive imaging is a possibility. This was first tested in 3 animals models. Annexin A5 was derivatized with hydrazinonicotamine (HYNIC) and labeled with ^sup 121^I. This conjugate was tested in fulminant hepatic necrosis model, where BALB/c mice were injected with anti-Fas antibody, an initiator of the apoptotic cascade. This causes fulminant hepatic failure due to apoptosis of the li\ver cells. After injection with anti-Fas antibody, the labeled Annexin A5 was infused. These animals showed marked increase in hepatic uptake of the labeled Annexin A5 when compared to control animals. The 2nd model used was a transplantation model, in which this group transplanted rats with heterotropic (non-compatible) cardiac allografts and hearts from syngeneic (compatible) rats. Again labeled Annexin A5 was infused and rats were imaged. In this model they showed increased uptake of Annexin A5 in the rats that were transplanted with the heterotropic cardiac allografts, when compared to controls. The 3rd model this group used was that of a murine lymphoma. In this model they also showed uptake of Annexin A5 in the lymphoma cells. all these data were confirmed histologically, in the sense that binding of Annexin A5 to the target organ was shown, and this co-localized with the previously mentioned TUNEL staining.18

After these encouraging results our group used this technique for molecular imaging of apoptosis in cardiovascular disease. In collaboration with Heidendal and the Department of Nuclear Medicine Annexin A5 was labeled with ^sup 99m^Tc. As described above, mice were subjected to ischaemia and reperfusion by transcendent ligation of their left anterior descending coronary artery. After this infarct, mice were placed under a [gamma]-camera with a pinhole collimator. Images obtained from these experiments showed uptake within the area of the heart (Figure 8).

Figure 8.-Planar image of a mouse after I/R 30/90. Arrowhead points out uptake of technetium labeled Annexin A5 in the infracted myocardium. Annexin A5 is partially metabolized in the liver (L), and secreted through the kidneys (K) and bladder (B).

Clinical imaging using ^sup 99m^Tc labeled Annexin A5

Next, this group moved on to clinical applications. After studying biodistribution in healthy volunteers, patients were imaged who presented with an acute myocardial infarction. Directly after reperfusion was obtained by percutaneous transluminal coronary angioplasty (PTCA), the labeled Annexin A5 was infused. Patients were imaged 4 to 8 hours after infusion of the radiolabeled Annexin A5. 3 Days after the PTCA, patients underwent a ^sup 201^Thallium or ^sup 99m^Tc sestaMIBI scan to evaluate the perfusion of the heart. When reconstructing the Annexin A5 images in a similar fashion as the perfusion study, it could be shown that Annexin A5 was taken up in the area of the myocardial infarction. This provided the first images of programmed cell death in patients, as shown in Figure 9.19 Additionally, it was shown that increased uptake of labeled Annexin A5 could be detected up to 4 days after the acute myocardial infarction.20 These areas of Annexin A5 uptake in myocardial infarction probably indicate preventable cell, loss, as shown in the mice study of Dumont et al.17 Arguably "rescued" cardiomyocytes can again regain their function and thereby provide a better outcome after acute myocardial infarction. Whether or not this is true in the human situation and Annexin A5 can be used as an endpoint to measure cell loss is unknown as of yet. Investigations in this area are ongoing.

Figure 9.-The left panel shows area of perfusion prior to opening of the occluded artery, as an indication of the area at risk. The right panel shows binding of Annexin A5 in the area at risk, indicating presence of programmed cell death within the area of infarction.

A 2nd application of molecular imaging of programmed cell death with radiolabeled Annexin A5 was first described in 2002 by Narula et al.21 This study focused on cardiac allograft rejection in transplant patients. In cardiac transplantation, rejection of the donor heart poses a difficult clinical problem. This grave situation can eventually be influenced by medication that suppresses the inflammatory response to the donor heart or possibly re-operation. Nevertheless these measures not uncommonly fail and the patient will die. On a cellular level, cardiac allograft rejection is histologically characterized by infiltration of monocytes in perivascular and interstitial spaces. This in turn causes myocyte necrosis and apoptosis. The common way to monitor possible rejection of the transplanted heart is to take myocardial biopsies, and scan for these morphological features. However, a myocardial biopsy is an invasive diagnostic procedure, which is not without risks. Since these processes have to be monitored over time, multiple biopsies have to be taken, adding to the risk of complications. SPECT imaging of programmed cell death with labeled Annexin A5 could possibly provide a non-invasive method of identifying patients suffering from cardiac allograft rejection. As mentioned previously, Blankenberg et al. had already proven the feasibility of this concept in an animal model of cardiac allograft rejection.18 In the study of Narula et al. all patients (n=18) underwent an Annexin A5 SPECT study, and biopsies were taken. When assessing the SPECT images, 2 blinded observers were in perfect agreement about the uptake of radiolabeled Annexin A5 in the myocardium of 5 of these patients, when reconstructing these images in a fashion that allowed a tomographical view of the left ventricle. In 3 cases focal uptake, and in 2 cases general uptake of labeled Annexin A5 was observed. Tissue sections of the myocardial biopsies were assessed by standard H&E staining, TUNEL staining and staining for activated caspasc 3. H&E staining revealed no or only limited abnormalities in the patients that showed no uptake of the radiolabeled Annexin A5. Also, there was no activation of caspase 3 seen. In the cases that showed focal uptake, more severe abnormalities were seen in the H&E staining, when scoring according to the recommendations of the International Society of Heart and Lung Transplantation (ISHLT). Also, scattered cardiomyocytes showed evidence of activation of caspase 3. The 2 patients that showed general uptake of the labeled Annexin A5 were scored as severe transplant rejection reaction according to the ISHLT guidelines, and showed many cardiomyocytes with activated caspase 3. TUNEL staining was positive in all but 2 patients. These data, together with the previously reported data on cardiac allograft rejection in animals, provide a proof of concept for detection of programmed cell death in patients with cardiac allograft rejection. This could provide the clinician with an important imaging modality to identify patients at risk, to monitor therapy and to assess efficacy of new treatment modalities such as cell death blockers in graft rejection.21, 22

Figure 10.-A) Echocardiographic picture of a large tumor, originating from the left ventricular wall and moving into the aorta (bottom light). B) A dual isotope scan. Left panel shows normal perfusion of the left ventricle. Right panel shows uptake of Annexin A5 within the area of the left ventricle. C) Histological examinations of this sarcoma. 1 trough 5 shown respectively binding of Annexin A5 to cells, presence of activated caspase 3 (2) and co- localization (3). D) Another intracardiar rumor, but this time no uptake could be detected on dual isotope imaging (E). Histology revealed a myxoma (F) without binding of Annexin AS to cells or presence of activated caspase 3.

This study touches slightly on the subject of programmed cell death imaging in inflammation. This subject was further evaluated by the group of Blankenberg et al., in an animal model of myocarditis, in which they showed enhanced uptake of radiolabeled Annexin A5 in the hearts of animals with an active myocarditis. This myocarditis was triggered by an immunization of rats by infusing porcine cardiac myosin. The animals formed antibodies against the myosin, and developed myocarditis, subsequently. Although the imaging in this model was done ex vivo, a correlation was shown between severity of the myocarditis and the amount of Annexin A5 uptake.23 Additionally, similar results had been obtained in an animal model of rheumatoid arthritis.24

Taken together, these animal models and human studies provide evidence for the assumption that molecular imaging of programmed cell death using radio-labeled Annexin A5 might provide diagnostic and therapeutic clues in disease states that are characterized by an excess of cell death, such as graft rejection in other transplanted organs and, as previously mentioned, myocardial infarction. In addition, this imaging modality might be useful in the evaluation of cell death blocking drugs, such as caspase inhibitors, for use in the clinical situation.

In contrast, oncology focuses on the lack of adequate cell loss, which can be influenced using chemotherapeutical agents. Mochizuki et al.25 recently published an intriguing study in which they introduced hepatoma cells in the calf of rats. Tumor cells were left to grow for 11 days. After 11 days a single dose of chemotherapy was infused in one group, the controls received normal saline. Hereafter ^sup 99m^technetium labeled Annexin A5 was administered. When performing radioisotope imaging, the treatment group showed significant increase in Annexin A5 uptake, when compared to controls. Results were normalized for body weight and tumor size. The results of increased apoptosis were confirmed by histological analysis, performing standard H&E staining and TUNEL staining.25 This finding could point to the fact that Annexin A5 imaging after a single close of chemotherapy can evaluate effectiveness of the therapy.

Clinical Annexin A5 imaging in tumors was clone in 2 rare cases of endocardial tumors. Endocardial tumors have an incidence of 0.02% to 0.3%, and most endocardial tumors, about 70-90%, are benign.26- 29 However, they do pose a difficult diagnostic problem for the clinician. "Classic" imaging modalities such as computer tomography (CT), magnetic resonance imaging (MRI) and echocardiography provide excell\ent data on localization, size, shape, haemodynamic consequences and pericardial ingrowth of the tumor. These techniques are, however, unable to inform the clinician about the nature and biology of the tumor. Taking a biopsy could perhaps add to making the diagnosis in these tumors, but this carries a very high risk of embolie complications. So although anatomical information about these tumors can be obtained, there is no information about the biology of the tumor. It is a well-known fact that in malignant tumors high proliferation and cell death rates are found, in sharp contrast to benign tumors. We tested the hypothesis of imaging these tumors with labeled Annexin A5 and if this could be used to differentiate between benign and malignant tumors. For orientation purposes, a dual isotope imaging technique was used. ^sup 201^Thallium and labeled Annexin A5 were infused, and imaged simultaneously. Combining the images of both compounds allowed us to localize the possible Annexin A5 uptake within the area of the left ventricle. The 1st case was a patient presenting with collapse and progressive dyspnea. An echocardiographic image of the tumor is shown in Figure 10A. Using the dual isotope imaging technique showed marked uptake of the labeled Annexin A5 was seen within the area of the tumor. After surgery, the tumor was placed under the [gamma] camera again, showing radioactive uptake. Histologically the tumor turned out to be an undifferentiated sarcoma. Immunohistochemistry showed binding of Annexin A5 to the membranes of tumor cells, which had activated caspase 3 (Figures 10B, C).30 This provides evidence of programmed cell death within this tumor, which was detected by imaging with radiolabeled Annexin A5. The 2nd case presented with similar symptoms. Again, a dual isotope imaging technique was used, which showed no uptake of labeled Annexin A5 within the area of the tumor. The tumor showed hardly any radioactive uptake when placed under the camera. Histology revealed a myxoma, a benign intracardiac tumor. No Annexin A5 or activated caspase 3 was detected in tumor tissue. These data are shown in Figures 10D, F. Taken together, these cases point out that the Annexin A5 imaging protocol might be used to study the biology of tumors that are accessible for biopsy taking.29

Abovementioned data show that Annexin A5 imaging in oncology is feasible in animal and human tumors, and therefore cell death in tumors can be assessed. The animal work of Mochizuki 25 shows that effectiveness of chemotherapy can be evaluated after a single dose of chemistry. Currently it is unknown if the same principle can be applied in clinical medicine. If so, this would be an extremely valuable addition to conventional imaging techniques, because this could lead to more accurate, individualized therapy, and better survival. Currently, clinical trials are under way to study this phenomenon in patients, performing planar imaging before and after a single dose of chemotherapy (unpublished data).

Future direction

Imaging using single photon emission tomography has a somewhat limited spatial resolution. A high-resolution nuclear imaging technique that has shown its use in clinical medicine is PET imaging. Although no animal or patient data are available, due to the higher energy of positron emitters (511 keV), detectability of lesions are expected to be better than using single photon emission tomography. A possibility to enhance appreciation of localization of uptake is to combine a nuclear imaging technique, either planar nuclear, SPECT or PET with a more anatomical technique, such as CT and MRI. Possibly, this could prove to provide the best of both worlds, i.e. the biological information with sensitive nuclear techniques and the anatomical high resolution imaging from either CT or MRI. These dual imaging setups have become commercially available in the SPECT/CT combination. Although exact co-localization of images of both techniques is still challenging, we feel that this could be a valuable addition, especially in larger animals and humans, where resolution of SPECT imaging alone is poor. Improvement of attenuation correction techniques could also aid in improving both resolution and target to background ratio. These possibilities require further investigation and open avenues to more accurate and sensitive detection of programmed cell death. This in turn would allow study of programmed cell death in disease where changes occur in relatively small quantities of cells, for instance unstable lesions in coronary arteries, cell loss in idiopathic dilated cardiomyopathy or detection of small tumors that have only limited amounts of PCD. Molecular imaging will allow accurate and early diagnosis of disease, and monitor early changes in the process, ultimately leading to better understanding of disease and better treatment of patients.

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B. L. J. H. KIETSELAER 1, L. HOFSTRA 1, E. A. W. J. DUMONT 1, C. P. M. REUTELINGSPERGER 2, G. A. K. HEIDENDAL 3

1 Department of Cardiology

University Hospital of Maastricht, Maastricht, The Netherlands

2 Department of Biochemistry

University of Maastricht, Maastricht, The Netherlands

3 Department of Nuclear Medicine

University Hospital of Maastricht, Maastricht, The Netherlands

Address reprint requests to: B. L. J. H. Kietselaer, MD, University Hospital Maastricht, Department of Cardiology, Maastricht, The Netherlands. E-mail: B.kietselaer@cardio.azm.nl

Copyright Edizioni Minerva Medica Dec 2003

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