Christian Vanhove

Comparison of the Biodistribution and Tumor Targeting of Two 99mTc-Labeled Anti-EGFR Nanobodies in Mice, Using Pinhole SPECT/Micro-CT

Camelidae possess an unusual class of antibodies devoid of light chains. Nanobodies are intact antigen-binding fragments that are stable, easily generated against different targets, and fully functional. Their rapid clearance from the blood circulation favors their use as imaging agents. We compared the in vivo tumor uptake and biodistribution of 2 anti–epidermal growth factor receptor (anti-EGFR) Nanobodies, 99mTc-7C12 and 99mTc-7D12. Methods: Nanobodies were labeled via their hexahistidine tail with 99mTc-tricarbonyl (99mTc(CO)3) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFR-negative) xenografts were intravenously injected with 99mTc-7C12 and 99mTc-7D12 on separate days. Pinhole SPECT/micro-CT images were acquired at 1 h after administration to assess noninvasively the biodistribution and tumor targeting of the labeled compounds. Pinhole SPECT and micro-CT images from the same mouse were automatically fused on the basis of a mathematic rigid-body-transformation algorithm using six 57Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm3) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either 99mTc-7C12 or 99mTc-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g). Results: Binding of both tracers was receptor-specific. Image analysis showed high and similar tumor uptake values for both 99mTc-7C12 and 99mTc-7D12 (4.55 ± 0.24 %IA/cm3 and 4.62 ± 0.36 %IA/cm3, respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16 ± 0.14 for 99mTc-7C12 and 1.49 ± 0.60 for 99mTc-7D12). 99mTc-7C12 showed significantly higher kidney uptake (63.48 ± 2.36 vs. 56.25 ± 2.46 %IA/cm3) and lower liver uptake (2.55 ± 0.26 vs. 4.88 ± 0.86 %IA/cm3) than did 99mTc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-to-background ratio; however, 99mTc-7C12 showed higher tumor uptake (9.11 ± 1.12 %IA/g) than did 99mTc-7D12 (6.09 ± 0.77 %IA/g). 99mTc-7D12 demonstrated significantly higher blood activity than did 99mTc-7C12, but both showed short plasma half-lives (<10 min).Conclusion: The Nanobody fragments used here show high tumor uptake, low liver uptake, and rapid blood clearance. Nanobodies are promising probes for noninvasive radioimmunodetection of specific targets early after administration. On the basis of its favorable biodistribution, 99mTc-7C12 was selected for further studies.

Interest of the ordered subsets expectation maximization (OS-EM) algorithm in pinhole single-photon emission tomography reconstruction : A phantom study

Abstract.Pinhole single-photon emission tomography (SPET) has been proposed to improve the trade-off between sensitivity and resolution for small organs located in close proximity to the pinhole aperture. This technique is hampered by artefacts in the non-central slices. These artefacts are caused by truncation and by the fact that the pinhole SPET data collected in a circular orbit do not contain sufficient information for exact reconstruction. The ordered subsets expectation maximization (OS-EM) algorithm is a potential solution to these problems. In this study a three-dimensional OS-EM algorithm was implemented for data acquired on a single-head gamma camera equipped with a pinhole collimator (PH OS-EM). The aim of this study was to compare the PH OS-EM algorithm with the filtered back-projection algorithm of Feldkamp, Davis and Kress (FDK)and with the conventional parallel-hole geometry as a whole,using a line source phantom, Picker’s thyroid phantom and a phantom mimicking the human cervical column. Correction for the angular dependency of the sensitivity in the pinhole geometrywas based on a uniform flood acquisition. The projection data were shifted according to the measured centre of rotation. No correction was made for attenuation, scatter or distance-dependent camera resolution. The resolution measured with the line source phantom showed a significant improvement with PH OS-EM as compared with FDK, especially in the axial direction. Using Picker’s thyroid phantom, one iteration with eight subsets was sufficient to obtain images with similar noise levels in uniform regions of interest to those obtained with the FDK algorithm. With these parameters the reconstruction time was 2.5 times longer than for the FDK method. Furthermore, there was a reduction in the artefacts caused by the circular orbit SPET acquisition. The images obtained from the phantom mimicking the human cervical column indicated that the improvement in image quality with PH OS-EM is relevant for future clinical useand that the improvements obtained using the OS-EM algorithm are more significant for the pinhole geometry than for the conventional parallel-hole geometry. We conclude that PH OS-EM is a practical and promising alternative for pinhole SPET reconstruction.

An algorithm for total variation regularization in high-dimensional linear problems

This paper describes an iterative algorithm for high-dimensional linear inverse problems, which is regularized by a differentiable discrete approximation of the total variation (TV) penalty. The algorithm is an interlaced iterative method based on optimization transfer with a separable quadratic surrogate for the TV penalty. The surrogate cost function is optimized using the block iterative regularized algebraic reconstruction technique (RSART). A proof of convergence is given and convergence is illustrated by numerical experiments with simulated parallel-beam computerized tomography (CT) data. The proposed method provides a block-iterative and convergent, hence efficient and reliable, algorithm to investigate the effects of TV regularization in applications such as CT.

Phase I Study of 68Ga-HER2-Nanobody for PET/CT Assessment of HER2 Expression in Breast Carcinoma

Human epidermal growth factor receptor 2 (HER2) status is one of the major tumor characteristics in breast cancer to guide therapy. Anti-HER2 treatment has clear survival advantages in HER2-positive breast carcinoma patients. Heterogeneity in HER2 expression between primary tumor and metastasis has repeatedly been described, resulting in the need to reassess HER2 status during the disease course. To avoid repeated biopsy with potential bias due to tumor heterogeneity, Nanobodies directed against HER2 have been developed as probes for molecular imaging. Nanobodies, which are derived from unique heavy-chain-only antibodies, are the smallest antigen-binding antibody fragments and have ideal characteristics for PET imaging. The primary aims were assessment of safety, biodistribution, and dosimetry. The secondary aim was to investigate tumor-targeting potential. Methods: In total, 20 women with primary or metastatic breast carcinoma (score of 2+ or 3+ on HER2 immunohistochemical assessment) were included. Anti-HER2-Nanobody was labeled with 68Ga via a NOTA derivative. Administered activities were 53–174 MBq (average, 107 MBq). PET/CT scans for dosimetry assessment were obtained at 10, 60, and 90 min after administration. Physical evaluation and blood analysis were performed for safety evaluation. Biodistribution was analyzed for 11 organs using MIM software; dosimetry was assessed using OLINDA/EXM. Tumor-targeting potential was assessed in primary and metastatic lesions. Results: No adverse reactions occurred. A fast blood clearance was observed, with only 10% of injected activity remaining in the blood at 1 h after injection. Uptake was seen mainly in the kidneys, liver, and intestines. The effective dose was 0.043 mSv/MBq, resulting in an average of 4.6 mSv per patient. The critical organ was the urinary bladder wall, with a dose of 0.406 mGy/MBq. In patients with metastatic disease, tracer accumulation well above the background level was demonstrated in most identified sites of disease. Primary lesions were more variable in tracer accumulation. Conclusion: 68Ga-HER2-Nanobody PET/CT is a safe procedure with a radiation dose comparable to other routinely used PET tracers. Its biodistribution is favorable, with the highest uptake in the kidneys, liver, and intestines but very low background levels in all other organs that typically house primary breast carcinoma or tumor metastasis. Tracer accumulation in HER2-positive metastases is high, compared with normal surrounding tissues, and warrants further assessment in a phase II trial.

Localization, mechanism and reduction of renal retention of technetium-99m labeled epidermal growth factor receptor-specific nanobody in mice

BACKGROUND Nanobodies are single-domain antigen binding fragments derived from functional heavy-chain antibodies elicited in Camelidae. They are powerful probes for radioimmunoimaging, but their renal uptake is relatively high. In this study we have evaluated the role of megalin on the renal uptake of anti-EGFR (99m)Tc-7C12 nanobody and the potency of gelofusine and/or lysine to reduce renal uptake of (99m)Tc-7C12. METHODS First we compared the renal uptake of (99m)Tc-7C12 in megalin-deficient and megalin-wild-type mice using pinhole SPECT/microCT and ex vivo analysis. The effect of gelofusine and lysine administration on renal accumulation of (99m)Tc-7C12 was analyzed in CD-1 mice divided into lysine preload at 30 min before tracer injection (LysPreload), LysPreload + gelofusine coadministration (LysPreload + GeloCoad), lysine coadministration (LysCoad), gelofusine coadministration (GeloCoad) and LysCoad + GeloCoad. The combined effect of gelofusine and lysine on tumor uptake was tested in mice xenografts. RESULTS Renal uptake of (99m)Tc-7C12 was 44.22 ± 3.46% lower in megalin-deficient compared with megalin-wild-type mice. In CD-1 mice, lysine preload had no effect on the renal retention whereas coinjection of lysine or gelofusine with the tracer resulted in 25.12 ± 2.99 and 36.22 ± 3.07% reduction, respectively. The combined effect of gelofusine and lysine was the most effective, namely a reduction of renal retention of 45.24 ± 2.09%. Gelofusine and lysine coadministration improved tumor uptake. CONCLUSION Megalin contributes to the renal accumulation of (99m)Tc-7C12. Gelofusine and lysine coinjection with the tracer reduces the renal uptake while tumor uptake is improved. Although this methodology allows for optimization of imaging protocol using nanobodies, further improvements are needed before using these molecules for radionuclide therapy.

In vivo disassembly of IV administered siRNA matrix nanoparticles at the renal filtration barrier

Intravenous administration of siRNA nanocarriers may provide unique therapeutic opportunities for tissue-specific gene silencing. Although often engineered to overcome the numerous barriers that exist in the systemic circulation, many nanocarriers fail in extending the circulation time of the siRNA. A more detailed assessment of the different clearance mechanisms that are in play after intravenous injection could therefore be of value to improve siRNA nanocarrier design. In this report, the biodistribution in mice of siRNA loaded dextran nanogels was investigated in detail. Both single photon emission computed tomography (SPECT) imaging and fluorescence single particle tracking (fSPT) indicate that the particles are rapidly cleared from the circulation. PEGylation of the nanogels was not able to increase the half-life in the bloodstream. Carrier disassembly in the systemic circulation and phagocytic clearance are known to facilitate the elimination of siRNA nanoparticles. Additionally, it is demonstrated for dextran nanogels that also the kidneys play an important role in their elimination from the bloodstream. SPECT imaging revealed an accumulation of the siRNA loaded dextran nanogels in the kidneys shortly after intravenous injection and a significantly delayed transition of siRNA from kidney to bladder, as opposed to the injection of free siRNA. These data indicate that components of the glomerular filtration barrier may contribute to the dissociation of siRNA from its carrier, as was recently suggested for cationic cyclodextrin siRNA polyplexes. This clearance mechanism should therefore be taken into account when designing siRNA nanocarriers for intravenous administration.

Small animal imaging with multi-pinhole SPECT

With Single Photon Emission Computed Tomography (SPECT), images of minute concentrations of tracer molecules can be acquired, allowing in vivo molecular imaging. For human imaging, the SPECT system has a modest spatial resolution of 5-15 mm, a large field of view and a high sensitivity. Using multi-pinhole SPECT, one can trade in field of view for resolution with preserved sensitivity, which enables the implementation of a small animal SPECT system with an improved resolution, currently ranging from 0.3 to 2 mm, in a much smaller field of view. The unconventional collimation and the more stringent resolution requirements pose problems that are not present in clinical SPECT imaging. This paper discusses how these problems can be solved to implement micro-SPECT imaging on a rotating gamma camera.

Evaluation of the radiation dose in micro-CT with optimization of the scan protocol

INTRODUCTION Micro-CT provides non-invasive anatomic evaluation of small animals. Serial micro-CT measurements are, however, hampered by the severity of ionizing radiation doses cumulating over the total period of follow-up. The dose levels may be sufficient to influence experimental outcomes such as animal survival or tumor growth. AIM This study was designed to evaluate the radiation dose of micro-CT and to optimize the scanning protocol for longitudinal micro-CT scans. METHODS AND MATERIALS Normal C57Bl/6 mice were euthanized. Radiation exposure was measured using individually calibrated lithium fluoride thermoluminescent dosimeters (TLDs). Thirteen TLDs were placed in the mice at the thyroid, lungs, liver, stomach, colon, bladder and near the spleen. Micro-CT (SkyScan 1178) was performed using two digital X-ray cameras which scanned over 180 degrees at a resolution of 83 microm, a rotation step of 1.08 degrees , 50 kV, 615 microA and 121 s image acquisition time. The TLDs were removed after each scan. CTDI(100) was measured with a 100 mm ionization chamber, centrally positioned in a 2.7 cm diameter water phantom, and rotation steps were increased to reduce both scan time and radiation dose. RESULTS Internal TLD analysis demonstrated median organ dose of 5.5 +/- 0.6 mGy per mA s, confirmed by CTDI(100) with result of 6.6 mGy per mA s. A rotation step of 2.16 resulted in qualitatively accurate images. At a resolution of 83 microm the scan time is reduced to 63 s with an estimated dose of 2.9 mGy per mA s. At 166 microm resolution, the scan time is limited to 27 s, with a concordant dose of 1.2 mGy per mA s. CONCLUSIONS The radiation dose of a standard micro-CT scan is relatively high and could influence the experimental outcome. We believe that the presented adaptation of the scan protocol allows for accurate imaging without the risk of interfering with the experimental outcome of the study.

Longitudinal Assessment of Parotid Function in Patients Receiving Tomotherapy for Head-and-Neck Cancer

Background and Purpose:Conventional radiotherapy is associated with high doses to the salivary glands which causes xerostomia and adverse effects on quality of life. The study aims to investigate the potential of helical tomotherapy (Hi-Art Tomotherapy®) to preserve parotid function in head-and-neck cancer patients.Patients and Methods:Seven consecutive patients treated with helical tomotherapy at the UZ Brussel, Belgium, were included. During planning, priority was attributed to planning target volume (PTV) coverage: ≥ 95% of the dose must be delivered to ≥ 95% of the PTV. Elective nodal regions received 54 Gy (1.8 Gy/fraction). A dose of 70.5 Gy (2.35 Gy/fraction) was prescribed to the primary tumor and pathologic lymph nodes = simultaneous integrated boost scheme. If possible, the mean parotid dose was kept below 26 Gy. Salivary gland function was assessed by technetium scintigraphy.Results:There was a significant dose-response relationship between mean parotid dose and functional recuperation. If the mean dose was kept < 31 Gy, a recuperation of 75% can be expected at 12 months. The authors equally observed a significant correlation between salivary excretion (SE) and the percentage of parotid gland receiving a dose < 26 Gy (V26%). In order to preserve 75% of SE, 46% of the parotid volume should receive a dose < 26 Gy.Conclusion:With the use of helical tomography the parotid gland function can largely be preserved since the mean dose to the entire gland as well as glandular volume receiving > 26 Gy can be reduced.Hintergrund und Ziel:Die konventionelle Strahlentherapie zur Behandlung von Kopf-Hals-Tumoren steht häufig im Zusammenhang mit hohen Dosisbelastungen der Speicheldrüsen. Dies verursacht Xerostomie, welche eine Beeinträchtigung der Lebensqualität zur Folge hat. Das Ziel dieser Studie ist die Untersuchung eines möglichen Vorteils helikaler Tomotherapie für die Funktionserhaltung der Ohrspeicheldrüsen.Patienten und Methodik:Eingeschlossen wurden sieben aufeinanderfolgende Patienten mit einer Nachbeobachtungszeit von 12 Monaten, die am Universitätsklinikum Brüssel (UZ Brussel), Belgien, eine helikale Tomotherapie (Hi-Art Tomotherapy®) erhielten. Bei der Planung wurde der Abdeckung des Planungszielvolumens (PTV) höchste Priorität zuerkannt: ≥ 95% des PTV mussten ≥ 95% der Dosis erhalten. Elektive Lymphabstromgebiete erhielten 54 Gy (1,8 Gy/Fraktion). Die Zielvolumendosis im Primärtumor und in pathologischen Lymphknoten betrug 70,5 Gy (2,35 Gy/Fraktion) = simultaner integrierter Boost. Nach Möglichkeit wurde die mittlere Dosis der Parotiden auf 26 Gy beschränkt. Die Funktion der Speicheldrüsen wurde durch Technetium-Szintigraphie ermittelt.Ergebnisse:Es fand sich eine signifikante Dosis-Wirkungs-Beziehung zwischen der mittleren Dosis in der Parotis und der Wiederherstellung ihrer Funktion. Bei einer mittleren Dosis < 31 Gy kann mit einer 75%igen Wiederherstellung innerhalb von 12 Monaten gerechnet werden. Die Autoren beobachteten eine signifikante Korrelation zwischen Speichelfluss (SF) und prozentualem Anteil der Parotis, der eine Dosis < 26 Gy erhielt (V26%). Um 75% des SF zu erhalten, sollten 46% des Parotisvolumens eine Dosis < 26 Gy erhalten.Schlussfolgerung:Die Möglichkeit, mittels helikaler Tomotherapie die Funktion der Speicheldrüsen zu erhalten, hängt nicht nur von der applizierten mittleren Dosis, sondern auch vom prozentualen Anteil des Volumens ab, das < 26 Gy erhält.

Improved quantification in single-pinhole and multiple-pinhole SPECT using micro-CT information

PurposeThe purpose of this study was to demonstrate the feasibility of accurate quantification in pinhole SPECT using micro-CT information.MethodsPinhole SPECT scans were performed using a clinical dual-head gamma camera. Each pinhole SPECT scan was followed by a micro-CT acquisition. Functional and anatomical images were coregistered using six point sources visible with both modalities. Pinhole SPECT images were reconstructed iteratively. Attenuation correction was based on micro-CT information. Scatter correction was based on dual and triple-energy window methods. Phantom and animal experiments were performed. A phantom containing nine vials was filled with different concentrations of 99mTc. Three vials were also filled with CT contrast agent to increase attenuation. Activity concentrations measured on the pinhole SPECT images were compared with activity concentrations measured by the dose calibrator. In addition, 11 mice were injected with 99mTc-labelled Nanobodies. After acquiring functional and anatomical images, the animals were killed and the liver activity was measured using a gamma-counter. Activity concentrations measured on the reconstructed images were compared with activity concentrations measured with the gamma counter.ResultsThe phantom experiments demonstrated an average error of −27.3 ± 15.9% between the activity concentrations measured on the uncorrected pinhole SPECT images and in the dose calibrator. This error decreased significantly to −0.1 ± 7.3% when corrections were applied for nonuniform attenuation and scatter. The animal experiment revealed an average error of −18.4 ± 11.9% between the activity concentrations measured on the uncorrected pinhole SPECT images and measured with the gamma counter. This error decreased to −7.9 ± 10.4% when attenuation and scatter correction was applied.ConclusionAttenuation correction obtained from micro-CT data in combination with scatter correction allows accurate quantification in pinhole SPECT.