Imaging apoptosis in vivo using 124I-annexin V and PET

Abnormal regulation of apoptosis is an important pathogenic mechanism in many diseases including cancer. Techniques to assess apoptosis in living organisms are limited and, in the case of solid organs, restricted to histological examination of biopsy samples. We investigated the use of (124)I-annexin V, which binds to phosphatidylserine (PS) on the surface of apoptotic cells, as a potential positron emission tomography (PET) radioligand for the noninvasive measurement of apoptosis in vivo. Annexin V and a similar-sized protein, ovalbumin, were directly labelled with (124)I. We report the validation of (124)I-annexin V in vitro and in an animal model of liver apoptosis that has not previously been used to test iodinated annexin V. Also, for the first time, we report metabolite analysis of (124)I-annexin V and the correlation of (124)I-annexin V uptake with apoptotic density (AD). Sixfold more (124)I-annexin V was associated with Jurkat cells after apoptosis induction, indicating that PS binding by annexin V was preserved after iodination. (124)I-ovalbumin did not demonstrate increased uptake in apoptotic cells. In normal BDF-1 mice, the radioligand was rapidly cleared, but some in vivo dehalogenation resulted in the accumulation of activity in the thyroid and stomach content. PET images demonstrated uptake of (124)I-annexin V but not (124)I-ovalbumin in apoptotic liver lesions. In vivo (124)I-annexin V uptake, derived from PET images, correlated with histologically derived AD (r=.86, P<.01). These results demonstrate that (124)I-annexin V is localised to anti-Fas-induced apoptosis, in contrast to (124)I-ovalbumin, which did not show preferential uptake in the apoptotic liver.     

Functional comparison of annexin V analogues labeled indirectly and directly with iodine-124

We are interested in imaging cell death in vivo using annexin V radiolabeled with (124)I. In this study, [(124)I]4IB-annexin V and [(124)I]4IB-ovalbumin were made using [(124)I]N-hydroxysuccinimidyl-4-iodobenzoate prepared by iododestannylation of N-hydroxysuccinimidyl-4-(tributylstannyl)benzoate. [(124)I]4IB-annexin V binds to phosphatidylserine-coated microtiter plates and apoptotic Jurkat cells and accumulates in hepatic apoptotic lesions in mice treated with anti-Fas antibody, while [(124)I]4IB-ovalbumin does not. In comparison with (124)I-annexin V, [(124)I]4IB-annexin V has a higher rate of binding to phosphatidylserine in vitro, a higher kidney and urine uptake, a lower thyroid and stomach content uptake, greater plasma stability and a lower rate of plasma clearance. Binding of radioactivity to apoptotic cells relative to normal cells in vitro and in vivo appears to be lower for [(124)I]4IB-annexin V than for (124)I-annexin V.     

PET/CT with intravenous contrast can be used for PET attenuation correction in cancer patients

Purpose If the CT scan of a combined PET/CT study is performed as a full diagnostic quality CT scan including intravenous (IV) contrast agent, the quality of the joint PET/CT procedure is improved and a separate diagnostic CT scan can be avoided. CT with IV contrast can be used for PET attenuation correction, but this may result in a bias in the attenuation factors. The clinical significance of this bias has not been established. Our aim was to perform a prospective clinical study where each patient had CT performed with and without IV contrast agent to establish whether PET/CT with IV contrast can be used for PET attenuation without reducing the clinical value of the PET scan.Methods A uniform phantom study was used to document that the PET acquisition itself is not significantly influenced by the presence of IV contrast medium. Then, 19 patients referred to PET/CT with IV contrast underwent CT scans without, and then with contrast agent, followed by an ¹⁸F-fluorodeoxyglucose whole-body PET scan. The CT examinations were performed with identical parameters on a GE Discovery LS scanner. The PET data were reconstructed with attenuation correction based on the two CT data sets. A global comparison of standard uptake value (SUV) was performed, and SUVs in tumour, in non-tumour tissue and in the subclavian vein were calculated. Clinical evaluation of the number and location of lesions on all PET/CT scans was performed twice, blinded and in a different random order, by two independent nuclear medicine specialists.Results In all patients, the measured global SUV of PET images based on CT with IV contrast agent was higher than the global activity using non-contrast correction. The overall increase in the mean SUV (for two different conversion tables tested) was 4.5±2.3% and 1.6±0.5%, respectively. In 11/19 patients, focal uptake was identified corresponding to malignant tumours. Eight out of 11 tumours showed an increased SUV_max (2.9±3.1%) on the PET images reconstructed using IV contrast. The clinical evaluation performed by the two specialists comparing contrast and non-contrast CT attenuated PET images showed weighted kappa values of 0.92 (doctor A) and 0.82 (doctor B). No contrast-introduced artefacts were found.Conclusion This study demonstrates that CT scans with IV contrast agent can be used for attenuation correction of the PET data in combined modality PET/CT scanning, without changing the clinical diagnostic interpretation.     

In vivo imaging of insulin receptors by PET: preclinical evaluation of iodine-125 and iodine-124 labelled human insulin.

[A(14)-*I]iodoinsulin was prepared for studies to assess the suitability of labeled iodoinsulin for positron emission tomography (PET). Iodine-125 was used to establish the methods and for preliminary studies in rats. Further studies and PET scanning in rats were carried out using iodine-124. Tissue and plasma radioactivity was measured as the uptake index (UI = [cpm x (g tissue)(-1)]/[cpm injected x (g body weight)(-1)]) at 1 to 40 min after intravenous injection of either [A(14)-(125)I]iodoinsulin or [A(14)-(124)I]iodoinsulin. For both radiotracers, initial clearance of radioactivity from plasma was rapid (T(1/2) approximately 1 min), reaching a plateau (UI = 2.8) at approximately 5 min which was maintained for 35 min. Tissue biodistributions of the two radiotracers were comparable; at 10 min after injection, UI for myocardium was 2.4, liver, 4.0, pancreas, 5.4, brain, 0.17, kidney, 22, lung, 2.3, muscle, 0.54 and fat, 0.28. Predosing rats with unlabelled insulin reduced the UI for myocardium (0.95), liver (1.8), pancreas (1.2) and brain (0.08), increased that for kidney (61) but had no effect on that for lung (2.5), muscle (0.50) or fat (0.34). Analysis of radioactivity in plasma demonstrated a decrease of [(125)I]iodoinsulin associated with the appearance of labeled metabolites; the percentage of plasma radioactivity due to [(125)I]iodoinsulin was 40% at 5 min and 10% at 10 min. The heart, liver and kidneys were visualized using [(124)I]iodoinsulin with PET.     

放射性核素标记annexin V凋亡显像在肿瘤研究中的进展

肿瘤细胞凋亡过低是肿瘤形成的重要原因之一,诱导肿瘤细胞凋亡也成了目前抗肿瘤治疗的研究方向。用放射性核素标记的annexin V对细胞凋亡后暴露在细胞膜表面的体内PS(膦脂酰丝氨酸)进行体内显像,可检测早期细胞凋亡。这种体内凋亡显像方法可以监测抗肿瘤治疗的疗效、评估患者的预后,甚至可以指导肿瘤治疗方法的选择,提高治疗的质量。     

A simple method to quantitate iodine-124 contamination in iodine-123 radiopharmaceuticals

Iodine-123 (123I) produced by the 124Te(p,2n)123I reaction contains several percent 124I radionuclidic contamination at the time of imaging. Since 124I degrades the quality of the images and causes unnecessary radiation absorbed dose to the patient, it is important to know the amount present in radiopharmaceuticals at the time of administration. A simple approach is described which uses a radionuclide dose calibrator and lead shield. The sample is assayed both shielded and unshielded and the ratio of readings depends uniquely upon the percent 124I present. The technique can be adopted for any type of dose calibrator, sample container, and Pb shield, but use of the numeric constants reported here should be restricted to the specified equipment.     

c-erbB2 protein overexpression in breast cancer as a target for PET using iodine-124-labeled monoclonal antibodies.

ICR 12, one of a panel of rat monoclonal antibodies recognizing the external domain of the human c-erb B2 proto-oncogene product, (Styles, 1990) was chosen as a candidate for radiolabeling with 124I for positron emission tomography of selected patients with breast cancer. By using N-bromosuccinimide (NBS), optimal labeling conditions were established using 125I. The labeling efficiency was determined using instant thin-layer chromatography (ITLC) and gel filtration (HPLC). The antibody was then labeled with the positron emitter 124I, and a labeling efficiency of 96% and immunoreactivity of 80%-90% was obtained. The product was stable, with less than 5% of the radiolabel being eluted after six days storage in plasma at 37 degrees C. Immunolocalization studies were performed in athymic mice bearing human breast carcinoma xenografts overexpressing the c-erb B2 gene product using as controls 125I labeled isotype-matched rat antibody, and antigen-negative tumors. Good uptake of 124I-labeled ICR12 was obtained in c-erb B2 expressing tumors (up to 12% injected dose per gram at intervals up to 120 hr), with localization indices of 3.4-6.2. Tumor xenografts of 6 mm diameter were successfully imaged with high resolution at 24, 48 and 120 hr using the RMH/ICR MUP-PET camera. We suggest that 124I-labeled ICR12 is a suitable agent to image and quantify immunolocalization in patients whose tumors overexpress the c-erb B2 proto-oncogene product.     

Multiphase contrast-enhanced CT with highly concentrated contrast agent can be used for PET attenuation correction in integrated PET/CT imaging

Purpose  

State-of-the-art positron emission tomography/computed tomography (PET/CT) systems incorporate multislice CT technology, thus facilitating the acquisition of multiphase, contrast-enhanced CT data as part of integrated PET/CT imaging protocols. We assess the influence of a highly concentrated iodinated contrast medium (CM) on quantification and image quality following CT-based attenuation correction (CT-AC) in PET/CT.     

Combined PET/CT with iodine-124 in diagnosis of spread metastatic thyroid carcinoma: a case report

Iodine-124 positron emission tomography (PET) is a useful 3D imaging technique for diagnosis and management of thyroid diseases. The difficulty in interpretation of the PET scans with highly selective tracers, such as iodine-124, is the lack of identifiable anatomical structures, so an accurate anatomical localization of foci presenting abnormal uptake is problematic. Consequently, a combined PET/CT scanner can resolve these difficulties by co-registering PET and CT data in a single session allowing a correlation of functional and morphologic imaging. A case is presented where iodine-124 produced by a clinical cyclotron and FDG were used to acquire images with a combined PET/CT scanner for clinical staging. On the basis of the PET/CT exams the treatment of the patient was modified.     

Improved detection of cell death in vivo with annexin V radiolabeled by site-specific methods.

Labeled annexin V is widely used to detect cell death in vitro and in vivo. Nearly all studies have been done with annexin V derivatized via amine-directed bifunctional agents; it was thought that these molecules retained full bioactivity compared with unmodified protein. We now show that this assumption is incorrect by measuring the affinity of annexin V for cells in vitro by quantitative calcium titration under conditions of low membrane occupancy. METHODS: Annexin V was modified with 4 different amine-directed agents: the N-hydroxysuccinimide esters of hydrazinonicotinic acid, mercaptoacetyltriglycine, and biotin; and with fluorescein isothiocyanate. RESULTS: In all cases, the membrane-binding affinity was decreased by derivatization, even at very low average stoichiometries. A statistical model based on the Poisson distribution accurately predicted the observed heterogeneity of derivatization as a function of average derivatization stoichiometry. This model also showed that multiply derivatized forms, which are the ones most likely to have compromised bioactivity, contributed disproportionately to the binding and imaging signals. The in vitro binding assay correctly predicted in vivo uptake in a mouse liver model of apoptosis for all proteins tested. The annexin V-128 protein, labeled at a single specific site at the N terminus, showed twice as much apoptosis-specific liver uptake as did all forms of annexin V derivatized randomly via amino groups. CONCLUSION: The membrane-binding activity of annexin V is much more sensitive to amine-directed chemical modification than previously realized. New annexin V molecules labeled by site-specific methods will greatly improve sensitivity for detecting cell death in vivo.     

PET scanning of iodine-124-3F9 as an approach to tumor dosimetry during treatment planning for radioimmunotherapy in a child with neuroblastoma.

A patient with advanced neuroblastoma who had failed chemotherapy presented with a large abdominal mass and virtually total bone marrow replacement by tumor on repeated marrow biopsies. She was considered a candidate for a Phase I 131I-3F8 radioimmunotherapy trial, (MSKCC 89-141A). As a potential aid to treatment planning, a test dose of 124I-3F8 was injected and the patient was imaged over the 72 hr postinjection using two BGO based PET scanners of different designs. Time activity curves were obtained, and the cumulated activity concentration of radiolabeled 3F8 in tumor was determined. Based on MIRD, an estimated radiation absorbed dose for 131I-3F8 was 7.55 rad/mCi, in the most antibody avid lesions. Because of low uptake and unfavorable dosimetry in some bulky tumor sites, it was decided not to treat the patient with radiolabeled antibody. Positron emission tomography of 124I-labeled antibodies can be used to measure cumulated activity or residence time in tumor for more accurate estimates of radiation absorbed tumor dose from radioiodinated antibodies and can help guide management decisions in patients who are candidates for radioimmunotherapy.     

Open source software EuroForMix can be used to analyse complex SNP mixtures

A series of two- and three-person mixtures of varying dilutions were prepared and analysed with Life Technologies’ HID-Ion AmpliSeq™ Identity Panel v2.2 using the Ion PGM™ massively parallel sequencing (MPS) system. From this panel we used 134 autosomal SNPs. Using the reference samples of three donors, we evaluated the strength of evidence with likelihood ratio (LR) calculations using the open-source quantitative EuroForMix program and compared the results with a previous study using a qualitative software (LRmix). SNP analysis is a special case of STRs, restricted to a maximum of two alleles per locus. We showed that simple two-person mixtures can be readily analysed with both LRmix and Euroformix, but the performance of three- or more person mixtures is generally inefficient with LRmix. Taking account of the “peak height” information, by substituting ‘sequence read’ coverage values from the MPS data for each SNP allele, greatly improves the discrimination between true and non-contributors. The higher the mixture proportion (Mx) of the person of interest is, the higher the LR. Simulation experiments (up to six contributors) showed that the strength of the evidence is dependent upon Mx, but relatively insensitive to the number of contributors. If a higher number of loci were multiplexed, the analysis of mixtures would be much improved, because the extra information would enable lower Mx values to be evaluated. In summary, incorporating the 'sequence read' (coverage) into the quantitative model shows a significant benefit over the qualitative approach. Calculations are quite fast (six seconds for three contributors).     

Towards in vivo nuclear microscopy: iodine-125 imaging in mice using micro-pinholes

Position-sensitive gamma-radiation detectors equipped with collimators have been used for in vivo imaging of the distribution of radiolabelled molecules in laboratory animals and humans for several decades. To date, the best image resolution achieved in a rodent is on the order of 1 mm. Here we demonstrate how a basic and compact gamma camera can be constructed for in vivo radionuclide imaging in small animals, at much higher spatial resolution. Resolution improvements were obtained by combining dense, shaped, micro-pinhole apertures with iodine-125, an isotope with low energy emissions, ease of incorporation into a wide range of molecules, and straightforward translation into the clinic via other isotopes of iodine that are suitable for nuclear medicine imaging. (125)I images of test distributions and a mouse thyroid have been obtained at a resolution of as high as 200 microm using this simple bench-top camera. Possible future applications and extension to ultra-high-resolution emission tomography are discussed.     

In vivo imaging of the human thyroid with a positron camera using 124I

A high-density avalanche chamber (HIDAC) positron camera was used for tomographic imaging of the human thyroid in vivo. Images were made 7 and 24 h after the oral administration of the positron-emitting radionuclide, sodium iodide 124I (with activities varying between 0.3 and 1 mCi), to patients scheduled for either partial thyroidectomy or radioiodine treatment. The results of thyroid imaging performed on 38 patients and their clinical relevance are discussed; as an illustration, three typical cases are presented. In Graves' disease, it was found that, whereas standard 131I and 124I scintigrams showed a diffuse goitre, positron images indicated a marked heterogeneity of the activity distribution, with "cold" areas in 8 out of the 11 cases studied. In conventional scintigrams, multinodular goitre showed a non-uniform radioiodine distribution, while positron images revealed considerable regional differences of activity uptake, with hot and cold areas in all of the 13 cases studied. As a consequence of the high spatial resolution of the camera [2.5 mm full width at half maximum (FWHM)], the functional volume of the thyroid may be estimated from 2 mm-thick transverse tomographic sections to within about 13%. This estimate may be compared with the measured volume after partial thyroidectomy, and in a follow-up scan, a further estimate can be made of the residual thyroid tissue remaining within the patient's body. In the case of radioiodine treatment in Graves' disease and multinodular goitre, the appropriate therapeutic dose of 131I can calculated according to the functional volume of the thyroid estimated from 124I tomographic images.     

The WOMAC score can be reliably used to classify patient satisfaction after total knee arthroplasty

Purpose

The primary aim of this study was to define a classification in the WOMAC score after total knee arthroplasty (TKA) according to patient satisfaction. The secondary aims were to describe patient demographics for each level of satisfaction.

Methods

A retrospective cohort consisting of 2589 patients undergoing a primary TKA were identified from an established arthroplasty database. Patient demographics, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and short form (SF) 12 scores were collected pre-operatively and 1 year post-operatively. In addition, patient satisfaction was assessed at 1 year with four responses: very satisfied, satisfied, dissatisfied or very dissatisfied. Receiver operating characteristic (ROC) curves were used to identify values in the components and total WOMAC scores that were predictive of each level of satisfaction, which were used to define the categories of excellent, good, fair and poor.

Results

At 1 year, there were 1740 (67.5%) very satisfied, 572 (22.2%) satisfied, 190 (7.4%) dissatisfied and 76 (2.9%) very dissatisfied patients. ROC curve analysis identified excellent, good, fair and poor categories for the pain (>?78, 59–78, 44–58, <?44), function (>?72, 54–72, 41–53, <?41), stiffness (>?69, 56–69, 43–55, <?43) and total (>?75, 56–75, 43–55, <?43) WOMAC scores, respectively. Patients with lung disease, diabetes, gastric ulcer, kidney disease, liver disease, depression, back pain, with worse pre-operative functional scores (WOMAC and SF-12) and those with less of an improvement in the scores, had a significantly lower level of satisfaction.

Conclusion

This study has defined a post-operative classification of excellent, good, fair and poor for the components and total WOMAC scores after TKA. The predictors of level of satisfaction should be recognised in clinical practice and patients at risk of a lower level of satisfaction should be made aware in the pre-operative consent process.

Level of evidence

III.

     

MRI can be used to assess advanced T-stage colon carcinoma as well as rectal carcinoma

Purpose

To evaluate the feasibility of assessing advanced T-stage (T3–T4) colorectal carcinomas by correlating MRI with histopathological findings.

Materials and methods

The study population comprised 31 patients with 32 lesions (22 colon and 10 rectal carcinomas). The relationship between the tumor and bowel layers on T2- and contrast-enhanced T1-weighted images (T2WI, CE-T1WI), bowel wall deformity, and the linear architecture of subserosal fat on T2WI scans was independently assessed by two radiologists. Diagnostic ability and interobserver agreement were evaluated using Fisher’s exact test and kappa statistics, respectively.

Results

The sensitivity/specificity for disrupting the outer layer on T2WI scans for the differentiation between Tis–T2 and T3–T4 colorectal carcinoma was 100/75 % (p < 0.05) for both observers; on CE-T1WI, it was 88.0/50 % (p = 0.13) for one and 96.0/50 % (p = 0.11) for the other. The sensitivity/specificity for recognizing the reticulated linear architecture to distinguish T3 from T4 colon carcinoma was 83.3/84.6 % (p < 0.05) for one reader and 100/92.3 % (p < 0.05) for the other reader.

Conclusion

Disruption of the outer low-intensity layer on T2WI scans was the most important finding for the diagnosis of T3–T4 colorectal carcinoma. The reticulated linear architecture of the fat tissue was suggestive of T4 colon carcinoma.

     

Current status and future perspectives of in vivo small animal imaging using radiolabeled nanoparticles

Small animal molecular imaging is a rapidly expanding efficient tool to study biological processes non-invasively. The use of radiolabeled tracers provides non-destructive, imaging information, allowing time related phenomena to be repeatedly studied in a single animal. In the last decade there has been an enormous progress in related technologies and a number of dedicated imaging systems overcome the limitations that the size of small animal possesses. On the other hand, nanoparticles (NPs) gain increased interest, due to their unique properties, which make them perfect candidates for biological applications. Over the past 5 years the two fields seem to cross more and more often; radiolabeled NPs have been assessed in numerous pre-clinical studies that range from oncology, till HIV treatment. In this article the current status in the tools, applications and trends of radiolabeled NPs reviewed.