Christophe Lahorte

Apoptosis-detecting radioligands: current state of the art and future perspectives

This review provides a critical and thorough overview of the radiopharmaceutical development and in vivo evaluation of all apoptosis-detecting radioligands that have emerged so far, along with their possible applications in nuclear medicine. The following SPECT and PET radioligands are discussed: all forms of halogenated Annexin V (i.e. 123I-labelled, 124I-labelled, 125I-labelled, 18F-labelled), 99mTc/94mTc-labelled Annexin V derivatives using different chelators and co-ligands (i.e. BTAP, Hynic, iminothiolane, MAG3, EDDA, EC, tricarbonyl, SDH) or direct 99mTc-labelling, 99mTc-labelled Annexin V mutants and 99mTc/18F-radiopeptide constructs (i.e. AFIM molecules), 111In-DTPA-PEG-Annexin V, 11C-Annexin V and 64Cu-, 67Ga- and 68Ga-DOTA-Annexin V. In addition, the potential role and clinical relevance of anti-PS monoclonal antibodies and other alternative apoptosis markers are reviewed, including: anti-Annexin V monoclonal antibodies, radiolabelled caspase inhibitors and substrates and mitochondrial membrane permeability targeting radioligands. Nevertheless, major emphasis is placed on the group of Annexin V-based radioligands, in particular 99mTc-Hynic-Annexin V, since this molecule is by far the most extensively investigated and best-characterised apoptosis marker at present. Furthermore, the newly emerging imaging modalities for in vivo detection of programmed cell death, such as MRI, MRS, optical, bioluminescent and ultrasound imaging, are briefly described. Finally, some future perspectives are presented with the aim of promoting the development of potential new strategies in pursuit of the ideal cell death-detecting radioligand.

Quantitative Tumor Apoptosis Imaging Using Technetium-99m–HYNIC Annexin V Single Photon Emission Computed Tomography

PURPOSE Radiolabeled annexin V may allow for repetitive and selective in vivo identification of apoptotic cell death without the need for invasive biopsy. This study reports on the relationship between quantitative technetium-99m- (99mTc-) 6-hydrazinonicotinic (HYNIC) radiolabeled annexin V tumor uptake, and the number of tumor apoptotic cells derived from histologic analysis. PATIENTS AND METHODS Twenty patients (18 men, two women) suspected of primary (n = 19) or recurrent (n = 1) head and neck carcinoma were included. All patients underwent a spiral computed tomography (CT) scan, 99mTc-HYNIC annexin V tomography, and subsequent surgical resection of the suspected primary or recurrent tumor. Quantitative 99mTc-HYNIC annexin V uptake in tumor lesions divided by the tumor volume, derived from CT, was related to the number of apoptotic cells per tumor high-power field derived from terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assays performed on sectioned tumor slices. RESULTS Diagnosis was primary head and neck tumor in 18 patients, lymph node involvement of a cancer of unknown primary origin in one patient, and the absence of recurrence in one patient. Mean percentage absolute tumor uptake of the injected dose per cubic centimeter tumor volume derived from tomographic images was 0.0003% (standard deviation [SD], 0.0004%) at 1 hour postinjection (PI) and 0.0001% (SD, 0.0000%) at 5 to 6 hours PI (P =.012). Quantitative 99mTc-HYNIC annexin V tumor uptake correlated well with the number of apoptotic cells if only tumor samples with no or minimal amounts of necrosis were considered. CONCLUSION In the absence of necrosis, absolute 99mTc-HYNIC annexin V tumor uptake values correlate well with the number of apoptotic cells derived from TUNEL assays.

Nuclear medicine imaging to predict response to radiotherapy: a review

PURPOSE To review available literature on positron emission tomography (PET) and single photon emission computerized tomography (SPECT) for the measurement of tumor metabolism, hypoxia, growth factor receptor expression, and apoptosis as predictors of response to radiotherapy. METHODS AND MATERIALS Medical literature databases (Pubmed, Medline) were screened for available literature and critically analyzed as to their scientific relevance. RESULTS Studies on 18F-fluorodeoxyglucose PET as a predictor of response to radiotherapy in head-and-neck carcinoma are promising but need confirmation in larger series. 18F-fluorothymine is stable in human plasma, and preliminary clinical data obtained with this marker of tumor cell proliferation are promising. For imaging tumor hypoxia, novel, more widely available radiopharmaceuticals with faster pharmacokinetics are mandatory. Imaging of ongoing apoptosis and growth factor expression is at a very early stage, but results obtained in other domains with radiolabeled peptides appear promising. Finally, for most of the tracers discussed, validation against a gold standard is needed. CONCLUSION Optimization of the pharmacokinetics of relevant radiopharmaceuticals as well as validation against gold-standard tests in large patient series are mandatory if PET and SPECT are to be implemented in routine clinical practice for the purpose of predicting response to radiotherapy.

Biodistribution and dosimetry study of 123I-rh-annexin V in mice and humans.

Summary This study reports on the optimization of the labelling procedure of clinical grade 123I‐rh‐annexin V and on the investigation of the biodistribution and dosimetry of 123I‐rh‐annexin V, a tracer proposed for the study of apoptosis in mice and humans. Research grade 123I‐rh‐annexin V was prepared as described previously, whereas clinical grade 123I‐rh‐annexin V was prepared according to a modified IodoGen method. NMRI mice, 3–4 weeks of age, received research grade 123I‐rh‐annexin V (74.0 + 3.7 kBq/mouse) by intravenous (i.v.) injection and killed at preset time points. Afterwards, the collected organs, blood, urine and faeces were counted for radioactivity and determined as %ID/g tissue or %ID over time. Secondly, six volunteers with normal liver and kidney function underwent whole‐body scans up to 21 h after i.v. injection of clinical grade 123I‐rh‐annexin V (345 ±38 MBq). Time‐activity curves were generated for the organs of interest, e.g., thyroid, heart, liver, kidneys and whole body, by fitting the organ specific geometric mean counts, obtained from region of interest analysis of acquired images in humans. The MIRD formulation was applied to calculate the absorbed radiation doses for various organs. Clinical grade 123I‐rh‐annexin V was obtained in radiochemical yields of 87.0 + 6.5% and radiochemical purities >98%. In mice, research grade 123I‐rh‐annexin V accumulated primarily in liver, kidney, stomach and lung tissue, limiting its usefulness for imaging of ongoing apoptosis in the abdominal and thoracic region. Clearance was predominantly urinary. In humans, acquired images with the clinical grade radioligand showed low lung uptake, resulting in good imaging conditions for the thoracic region. On the other hand, delayed imaging of the abdominal region was impeded due to extensive bowel activity. The highest absorbed doses were received by the thyroid, the kidneys, the heart wall, the liver and bone surfaces. The average effective dose of 123I‐rh‐annexin V was estimated to be 0.02 mSv·MBq‐1. The amount of 123I‐rh‐annexin V required for in vivo imaging, results in an acceptable effective dose to the patient.

Synthesis and in vitro evaluation of 123I-labelled human recombinant annexin V

Annexin V was radiolabelled with iodine-123 in order to develop a SPECT-ligand for imaging atherosclerosis and apoptosis. Iodination by means of electrophylic substitution resulted in radiochemical yields up to 70% and specific activities of 7.4-92.5 MBq/microg protein. Binding experiments with blood platelets indicated that 123I-labelled annexin V remained its biological activity.

99mTc-HYNIC Annexin-V imaging of primary head and neck carcinoma.

In this study, the potential of 99mTc-HYNIC Annexin-V scintigraphy to visualize primary head and neck carcinoma was assessed and compared with computed tomography (CT) findings and histology. Eighteen patients suspected of having primary head and neck carcinoma underwent a spiral CT scan and 99mTc-HYNIC Annexin-V scintigraphy within 1 week of each other, followed by resection of the suspected lesion. Results obtained by CT and scintigraphy were compared vs. histopathology. The diagnosis was primary head and neck carcinoma in 18 patients, accompanied by lymph node involvement in seven patients. 99mTc-HYNIC Annexin-V uptake was identified in five patients on planar images and in 17 patients on tomographic images (single-photon emission computed tomography, SPECT), corresponding to the pathological regions identified by CT. In the remaining patient, CT and 99mTc-HYNIC Annexin-V scintigraphy were false negative. In 11 patients, SPECT and CT scan were concordant, identifying all primary lesions and two sites of lymph node involvement. In the six remaining patients, CT and SPECT accurately identified the primary lesion, but were discordant with regard to the existence of lymph node involvement. In five of six patients, SPECT failed to identify lymph node involvement, whereas CT scan did not. In the remaining patient, CT scan was false positive for lymph node involvement, whereas SPECT was not. In this series, 99mTc-HYNIC Annexin-V allowed for the visualization of all primary head and neck tumours identified by CT scan, but failed to identify most of the sites of lymph node involvement.

Intraobserver, interobserver, and day-to-day reproducibility of quantitative 99mTc-HYNIC annexin-V imaging in head and neck carcinoma.

RATIONALE For clinical application, a sufficient reproducibility of 99mTc-HYNIC annexin-V quantitative uptake measurements must be demonstrated to allow a study of cell-death changes induced by chemotherapy over time and intersubject. Thus, the aim of this study was to estimate the intra-, inter-, and day-to-day reproducibility of quantitative 99mTc-HYNIC annexin-V tumor uptake values in patients suffering from head and neck carcinomas. METHODS Thirteen (13) patients suffering from clinically suspected, histologically confirmed squamous head and neck carcinomas were prospectively included in the study. All patients were scheduled to undergo a spiral computed tomography scan and two 99mTc-HYNIC annexin-V scintigraphies within 3-5 days from each other, referred to as day 1 and day 2 of scintigraphy. The percentage of uptake of the injected dose of 99mTc-HYNIC annexin-V in tumor lesions on scintigrams divided by the tumor volume, as derived from CT, was determined twice within an interval of 2 weeks by observer 1 and once by observer 2 on day 1 of scintigraphy and once on day 2 of scintigraphy by observer 1. RESULTS The mean of the difference for the intra-, inter-, and day-to-day measurements were -3.4%, 2.4%, and -6%, respectively. No systematic bias was observed for the mean of the differences for the intra-, inter-, and day-to-day measurements. The respective confidence intervals for the mean of the differences of intra-, inter-, and day-to-day variability were -8.2%-1.4%, -2.9%-7.8%, and -14.7%-2.7%. CONCLUSION The reproducibility of quantitative 99mTc-HYNIC annexin-V tumor uptake measurements using a manual method appears to be acceptable for clinical use.

Annexin V detection of lipopolysaccharide-induced cardiac apoptosis.

Acute inflammatory response to lipopolysaccharide (LPS) exposure is typically associated with cardiac myocyte apoptosis, which is difficult to quantify because of heart tissue specificity. We report here that radioiodinated Annexin V (123, 125I-AnxV), a specific ligand of phosphatidylserine exposed by apoptotic cells, allows tissue detection of apoptosis in LPS-treated rat hearts. Heart 123I-AnxV uptake was significantly increased in all cardiac territories of LPS-treated rats. In contrast, 123I-human serum albumin myocardial uptake was only slightly increased in LPS-treated rat hearts, suggesting limited changes in vascular protein permeability. Autoradiography of endotoxin-treated rat heart sections with 125I-AnxV in association with deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling and caspase 3 staining allows identification of double positive cardiac myocytes. Inhibition of apoptosis by caspase inhibitors (i.e., ZVAD.fmk and DEVD.cmk) reduced 123I-AnxV myocardial uptake in LPS-treated rats. Eventually, endotoxin-treated rats displayed pathological uptake of 99mTc-annexin in the cardiac mediastinal region whereas zVAD.fmk reduced 99mTc-annexin mediastinal uptake. Our results show that radioactive 123I-AnxV signal emerging from LPS-treated rat hearts could be related to the activation of caspase-dependent apoptotic pathway in cardiac myocytes.ABBREVIATIONS-123,125I-AnxV, 123,125I-annexinV; 123,125I-HSA, human serum albumin; PS, phosphatidylserine; TUNEL, deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling