An anti-SARS-CoV-2 non-neutralizing antibody with Fc-effector function defines a new NTD epitope and delays neuroinvasion and death in K18-hACE2 mice

Emerging evidence in animal models indicate that both neutralizing activity and Fc- mediated effector functions of neutralizing antibodies contribute to protection against SARS-CoV-2. It is unclear if antibody effector functions alone could protect against SARS-CoV-2. Here we isolated CV3-13, a non-neutralizing antibody from a convalescent individual with potent Fc-mediated effector functions that targeted the N- terminal domain (NTD) of SARS-CoV-2 Spike. The cryo-EM structure of CV3-13 in complex with SAR-CoV-2 spike revealed that the antibody bound from a distinct angle of approach to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, an Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Thus, we demonstrate that efficient Fc-mediated effector functions can contribute to the in vivo efficacy of anti-SARS-CoV-2 monoclonal antibodies in the absence of neutralization.

Contribution of single mutations to selected SARS-CoV-2 emerging variants Spike antigenicity

Towards the end of 2020, multiple variants of concern (VOCs) and variants of interest (VOIs) have arisen from the original SARS-CoV-2 Wuhan-Hu-1 strain. Mutations in the Spike protein are highly scrutinized for their impact on transmissibility, pathogenesis and vaccine efficacy. Here, we contribute to the growing body of literature on emerging variants by evaluating the impact of single mutations on the overall antigenicity of selected variants and their binding to the ACE2 receptor. We observe a differential contribution of single mutants to the global variants phenotype related to ACE2 interaction and antigenicity. Using biolayer interferometry, we observe that enhanced ACE2 interaction is mostly modulated by a decrease in off-rate. Finally, we made the interesting observation that the Spikes from tested emerging variants bind better to ACE2 at 37{degrees}C compared to the D614G variant. Whether improved ACE2 binding at higher temperature facilitates emerging variants transmission remain to be demonstrated.

Impact of temperature on the affinity of SARS-CoV-2 Spike for ACE2

The seasonal nature in the outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. The current COVID-19 pandemic makes no exception, and temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2. The receptor binding domain (RBD) of the Spike glycoprotein binds to the angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Studying the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike to ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide, bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

A single BNT162b2 mRNA dose elicits antibodies with Fc-mediated effector functions and boost pre-existing humoral and T cell responses

The standard dosing of the Pfizer/BioNTech BNT162b2 mRNA vaccine validated in clinical trials includes two doses administered three weeks apart. While the decision by some public health authorities to space the doses because of limiting supply has raised concerns about vaccine efficacy, data indicate that a single dose is up to 90% effective starting 14 days after its administration. We analyzed humoral and T cells responses three weeks after a single dose of this mRNA vaccine. Despite the proven efficacy of the vaccine at this time point, no neutralizing activity were elicited in SARS-CoV-2 naive individuals. However, we detected strong anti-receptor binding domain (RBD) and Spike antibodies with Fc-mediated effector functions and cellular responses dominated by the CD4+ T cell component. A single dose of this mRNA vaccine to individuals previously infected by SARS-CoV-2 boosted all humoral and T cell responses measured, with strong correlations between T helper and antibody immunity. Neutralizing responses were increased in both potency and breadth, with distinctive capacity to neutralize emerging variant strains. Our results highlight the importance of vaccinating uninfected and previously-infected individuals and shed new light into the potential role of Fc-mediated effector functions and T cell responses in vaccine efficacy. They also provide support to spacing the doses of two-vaccine regimens to vaccinate a larger pool of the population in the context of vaccine scarcity against SARS-CoV-2.

A small number of early introductions seeded widespread transmission of SARS-CoV-2 in Quebec, Canada

Using genomic epidemiology, we investigated the arrival of SARS-CoV-2 to Quebec, the Canadian province most impacted by COVID-19, with >280,000 positive cases and >10,000 deaths in a population of 8.5 million as of March 1st, 2021. We report 2,921 high-quality SARS-CoV-2 genomes in the context of >12,000 publicly available genomes sampled globally over the first pandemic wave (up to June 1st, 2020). By combining phylogenetic and phylodynamic analyses with epidemiological data, we quantify the number of introduction events into Quebec, identify their origins, and characterize the spatio-temporal spread of the virus. Conservatively, we estimated at least 500 independent introduction events, the majority of which happened from spring break until two weeks after the Canadian border closed for non-essential travel. Subsequent mass repatriations did not generate large transmission lineages (>50 cases), likely due to mandatory quarantine measures in place at the time. Consistent with common spring break and snowbird destinations, most of the introductions were inferred to have originated from Europe via the Americas. Fewer than 100 viral introductions arrived during spring break, of which 5-10 led to the largest transmission lineages of the first wave (accounting for 36-58% of all sequenced infections). These successful viral transmission lineages dispersed widely across the province, consistent with founder effects and superspreading dynamics. Transmission lineage size was greatly reduced after March 11th, when a quarantine order for returning travelers was enacted. While this suggests the effectiveness of early public health measures, the biggest transmission lineages had already been ignited prior to this order. Combined, our results reinforce how, in the absence of tight travel restrictions or quarantine measures, fewer than 100 viral introductions in a week can ensure the establishment of extended transmission chains.

Cross-sectional evaluation of humoral responses against SARS-CoV-2 Spike

The SARS-CoV-2 virus is responsible for the current worldwide coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoprotein of SARS-CoV-2 mediates viral entry and is the main target for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic and public health interventions. Here we performed a cross-sectional study on 106 SARS-CoV-2-infected individuals to evaluate humoral responses against the SARS-CoV-2 Spike. The vast majority of infected individuals elicited anti-Spike antibodies within 2 weeks after the onset of symptoms. The levels of receptor-binding domain (RBD)-specific IgG persisted overtime, while the levels of anti-RBD IgM decreased after symptoms resolution. Some of the elicited antibodies cross-reacted with other human coronaviruses in a genus-restrictive manner. While most of individuals developed neutralizing antibodies within the first two weeks of infection, the level of neutralizing activity was significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection.

A dual-labeled Annexin A5 is not suited for SPECT imaging of brain cell death in experimental murine stroke.

Cell death is one of the pathophysiological hallmarks after stroke. Markers to image cell death pathways in vivo are highly desirable. We previously showed that fluorescently labeled Annexin A5 (AnxA5), which binds specifically to phosphatidylserine (PS) on dead/dying cells, can be used in experimental stroke for monitoring cell death with optical imaging. Here we investigated whether dual-labeled AnxA5 (technetium and fluorescence label) can be used for single-photon emission computed tomography (SPECT) of cell death in the same model. C57Bl6/N mice were subjected to 60-minute middle cerebral artery occlusion (MCAO) and underwent SPECT imaging at 24, 48, and 72 hours afterwards. They were injected intravenously with either PS-binding AnxA5 or the nonfunctional AnxA5 (negative control), labeled with 99mTc and Alexa Fluor 568, respectively. After SPECT imaging, brain sections were cut for autoradiography and fluorescence microscopy. Ethanol-induced cell death in the femur muscle was used as positive control. We detected dual-labeled AnxA5 in the model of ethanol-induced cell death in the femur muscle, but not after MCAO at any time point, either with SPECT or with ex vivo autoradiography or fluorescence microscopy. Dual-labeled AnxA5 appears to be unsuited for visualizing death of brain cells in this MCAO model.

XTEN-annexin A5: XTEN allows complete expression of long-circulating protein-based imaging probes as recombinant alternative to PEGylation.

UNLABELLED: The coupling of polyethylene glycol (PEG) to proteins (PEGylation) has become a standard method to prolong blood circulation of imaging probes and other proteins, liposomes, and nanoparticles. However, concerns have arisen about the safety of PEG, especially with respect to its poor biodegradability and antibody formation, including new evidence about preformed anti-PEG antibodies in a quarter of healthy blood donors. Here, we apply a new hydrophilic polypeptide XTEN to extend the blood half-life of an imaging probe. As an example, we chose annexin A5 (AnxA5), a recombinant 35-kD protein extensively used for the in vitro and in vivo detection of apoptosis, that has a blood half-life of less than 7 min in mice, limiting its accumulation in target tissues and therefore limiting its utility as an imaging reagent. METHODS: The sequence of XTEN was developed by Volker Schellenberger and colleagues by evolutionary in vitro optimization to yield PEG-like properties but provides several key advantages in comparison to PEG. The DNA of a 288-amino-acid version of XTEN with an additional N-terminal cysteine for site-directed coupling was fused to AnxA5 (XTEN-AnxA5). The fusion protein could be highly expressed in Escherichia coli and efficiently purified using XTEN conveniently as a purification tag. It was labeled with a thiol-reactive fluorescent dye and via a chelator with a radionuclide. RESULTS: SPECT/CT imaging revealed a blood half-life of about 1 h in mice, markedly longer than the 7-min blood half-life for unmodified AnxA5, which should allow improved imaging of target tissues with low perfusion. In comparison to AnxA5, XTEN-AnxA5 demonstrated a substantially higher accumulation in tumors under chemotherapy in near-infrared fluorescence imaging. CONCLUSION: The presented method allows the expression and production of high amounts of long-circulating XTEN-AnxA5 without the necessity of PEGylation, thereby simplifying the synthesis while avoiding labeling-induced inactivation of AnxA5 and potential adverse effects of PEG. It is readily applicable to other recombinant protein or peptide-based imaging probes and allows fine-tuning of the desired blood half-life, because longer XTEN variants yield longer blood half-lives.

Contribution of 68Ga-DOTATOC PET/CT to target volume delineation of skull base meningiomas treated with stereotactic radiation therapy.

PURPOSE: To investigate the potential impact of 68Ga-DOTATOC positron emission tomography (68Ga-DOTATOC-PET) in addition to magnetic resonance imaging (MRI) and computed tomography (CT) for retrospectively assessing the gross tumor volume (GTV) delineation of meningiomas of the skull base in patients treated with fractionated stereotactic radiation therapy (FSRT). METHODS AND MATERIALS: The study population consisted of 48 patients with 54 skull base meningiomas, previously treated with FSRT. After scans were coregistered, the GTVs were first delineated with MRI and CT data (GTVMRI/CT) and then by PET (GTVPET) data. The overlapping regions of both datasets resulted in the GTVcommon, which was enlarged to the GTVfinal by adding volumes defined by only one of the complementary modalities (GTVMRI/CT-added or GTVPET-added). We then evaluated the contribution of conventional imaging modalities (MRI, CT) and 68Ga-DOTATOC-PET to the GTVfinal, which was used for planning purposes. RESULTS: Forty-eight of the 54 skull base lesions in 45 patients showed increased 68Ga-DOTATOC uptake and were further analyzed. The mean GTVMRI/CT and GTVPET were approximately 21 cm3 and 25 cm3, with a common volume of approximately 15 cm3. PET contributed a mean additional GTV of approximately 1.5 cm3 to the common volume (16%±34% of the GTVcommon). Approximately 4.5 cm3 of the GTVMRI/CT was excluded from the contribution to the common volume. The resulting mean GTVfinal was significantly smaller than both the GTVMRI/CT and the GTVPET. Compared with the initial GTVMRI/CT, the addition of 68Ga-DOTATOC-PET resulted in more than 10% modification of the size of the GTVfinal in 32 (67%) meningiomas CONCLUSIONS: 68Ga-DOTATOC-PET/CT seems to improve the target volume delineation in skull base meningiomas, often leading to a reduction of GTV compared with results from conventional imaging (MRI and CT).

Brain perfusion SPECT in the mouse: normal pattern according to gender and age.

Regional cerebral blood flow (rCBF) is a useful surrogate marker of neuronal activity and a parameter of primary interest in the diagnosis of many diseases. The increasing use of mouse models spawns the demand for in vivo measurement of rCBF in the mouse. Small animal SPECT provides excellent spatial resolution at adequate sensitivity and is therefore a promising tool for imaging the mouse brain. This study evaluates the feasibility of mouse brain perfusion SPECT and assesses the regional pattern of normal Tc-99m-HMPAO uptake and the impact of age and gender. Whole-brain kinetics was compared between Tc-99m-HMPAO and Tc-99m-ECD using rapid dynamic planar scans in 10 mice. Assessment of the regional uptake pattern was restricted to the more suitable tracer, HMPAO. Two HMPAO SPECTs were performed in 18 juvenile mice aged 7.5 ± 1.5weeks, and in the same animals at young adulthood, 19.1 ± 4.0 weeks (nanoSPECT/CTplus, general purpose mouse apertures: 1.2kcps/MBq, 0.7mm FWHM). The 3-D MRI Digital Atlas Database of an adult C57BL/6J mouse brain was used for region-of-interest (ROI) analysis. SPECT images were stereotactically normalized using SPM8 and a custom made, left-right symmetric HMPAO template in atlas space. For testing lateral asymmetry, each SPECT was left-right flipped prior to stereotactical normalization. Flipped and unflipped SPECTs were compared by paired testing. Peak brain uptake was similar for ECD and HMPAO: 1.8 ± 0.2 and 2.1 ± 0.6 %ID (p=0.357). Washout after the peak was much faster for ECD than for HMPAO: 24 ± 7min vs. 4.6 ± 1.7h (p=0.001). The general linear model for repeated measures with gender as an intersubject factor revealed an increase in relative HMPAO uptake with age in the neocortex (p=0.018) and the hippocampus (p=0.012). A decrease was detected in the midbrain (p=0.025). Lateral asymmetry, with HMPAO uptake larger in the left hemisphere, was detected primarily in the neocortex, both at juvenile age (asymmetry index AI=2.7 ± 1.7%, p=0.000) and at young adult age (AI=2.4 ± 1.7%, p=0.000). Gender had no effect on asymmetry. Voxel-wise testing confirmed the ROI-based findings. In conclusion, high-resolution HMPAO SPECT is a promising technique for measuring rCBF in preclinical research. It indicates lateral asymmetry of rCBF in the mouse brain as well as age-related changes during late maturation. ECD is not suitable as tracer for brain SPECT in the mouse because of its fast clearance from tissue indicating an interspecies difference in esterase activity between mice and humans.

Straightforward thiol-mediated protein labelling with DTPA: Synthesis of a highly active 111In-annexin A5-DTPA tracer.

BACKGROUND: Annexin A5 (anxA5) has been found useful for molecular imaging of apoptosis and other biological processes. METHODS: Here, we report an optimised two-step synthesis of annexin A5-diethylene triamine pentaacetic acid (DTPA) (anxA5-DTPA) for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with a single purification step. The use of a recombinant annexin A5 (cys-anxA5) with a single thiol group allowed regionally specific coupling, without affecting the binding domain of cys-anxA5. RESULTS: The metal complexing capacity of anxA5-DTPA was investigated by labelling with 111In3+ and Eu3+. Binding of modified anxA5-DTPA to apoptotic cells was tested in competition experiments with a fluorescent anxA5 derivative (anxA5-FITC) using flow cytometry and compared with that of wildtype anxA5 or non-binding anxA5-DTPA (M1234-anxA5-DTPA). The binding affinity to apoptotic cells of the anxA5-DTPA conjugate does not differ from that of wildtype anxA5. CONCLUSIONS: This two-step synthesis of annexin A5-DTPA resulted in biologically active anxA5-DTPA, which can be labelled with radionuclides for use in SPECT and PET imaging.

The surface molecular functionality of decellularized extracellular matrices.

Decellularization of tissues and organs is a successful platform technology for creating scaffolding materials for tissue engineering and regenerative medicine. It has been suggested that the success of these materials upon implantation is due to the molecular signals provided by the remaining scaffold extracellular matrix (ECM) components presented to probing cells in vivo as they repopulate the surface. For this study, decellularized matrices were created from esophagus, bladder, and small intestine harvested from adult male Fischer 344 rats. The three decellularized matrices (each originating from source tissues which included an epithelial lining on their luminal surfaces) were immunostained for collagen IV and laminin to determine basement membrane retention. Scanning electron micrographs of the surfaces were used to provide insight into the surface topography of each of the decellularized tissues. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to generate high-resolution mass spectra for the surfaces of each scaffold. This surface-sensitive technique allows for detailed molecular analysis of the outermost 1-2 nm of a material and has been applied previously to thin protein films and secreted ECM proteins on poly(N-isopropyl acrylamide) (polyNIPAAM) surfaces. To extract trends from within the complex ToF-SIMS dataset, a multivariate analysis technique, principal component analysis (PCA), was employed. Using this method, a molecular fingerprint of each surface was created and separation was seen in the PCA scores between the decellularized esophagus and the decellularized small intestine samples. The PCA scores for the decellularized bladder sample fell between the previous two decellularized samples. Protein films of common extracellular matrix constituents (collagen IV, collagen I, laminin, and Matrigel) were also investigated. The PCA results from these protein films were used to develop qualitative hypotheses for the relationship of the key fragments identified from the PCA of the decellularized ECMs.

Potential impact of (68)Ga-DOTATOC PET/CT on stereotactic radiotherapy planning of meningiomas.

PURPOSE: Since meningiomas show a high expression of somatostatin receptor subtype 2, PET with (68)Ga-DOTATOC was proposed as an additional imaging modality beside CT and MRI for planning radiotherapy. We investigated the input of (68)Ga-DOTATOC-PET/CT on the definition of the "gross tumour volume" (GTV) in meningiomas, in order to assess the potential value of this method. METHODS: Prior to radiotherapy, 42 patients with meningiomas (26 f, 16 m, mean age 55) underwent MRI and (68)Ga-DOTATOC-PET/CT examinations. HISTORY: operated n = 24, radiotherapy n = 1, operation and radiotherapy n = 8, no treatment n = 9. PET/CT and MRI data were co-registered using a BrainLAB workstation. For comparison, the GTV was defined first under consideration of CT and MRI data, then using PET data. RESULTS: 3/42 patients were excluded from the analysis (two with negative PET results, one with an extensive tumour, not precisely delineable by MRI or PET/CT). The average GTV(CT/MRI) was 22(+/-19)cm(3); GTV(PET) was 23(+/-20)cm(3). Additional GTV, obtained as a result of PET was 9(+/-10)cm(3) and was observed in patients with osseous infiltration. In some pre-treated patients there were intratumoural areas (as identified in CT/MRI) without SR-expression (7(+/-11)cm(3)). Common GTV as obtained by both CT/MRI and PET was 15(+/-14)cm(3). The mean bi-directional difference between the GTV(CT/MRI) and GTV(PET) accounted to 16(+/-15)cm(3) (93%, p < 0.001). In a subgroup of seven patients with multiple meningiomas, PET showed a total of 19 lesions; nine of them were not recognizable by CT or MRI. CONCLUSION: (68)Ga-DOTATOC-PET enables delineation of SR-positive meningiomas and delivers additional information to both CT and MRI regarding the planning of stereotactic radiotherapy. The acquisition on a PET/CT scanner helps to estimate the relation of PET findings to anatomical structures and is especially useful for detection of osseous infiltration. (68)Ga-DOTATOC-PET also allows detection of additional lesions in patients with multiple meningiomas.

Modification of aminosilanized superparamagnetic nanoparticles: feasibility of multimodal detection using 3T MRI, small animal PET, and fluorescence imaging.

PURPOSE: The aim of our study was to modify an aminosilane-coated superparamagnetic nanoparticle for cell labeling and subsequent multimodal imaging using magnetic resonance imaging (MRI), positron emission tomography (PET), and fluorescent imaging in vivo. PROCEDURES: We covalently bound the transfection agent HIV-1 tat, the fluorescent dye fluorescein isothiocyanate, and the positron-emitting radionuclide gallium-68 to the particle and injected them intravenously into Wistar rats, followed by animal PET and MRI at 3.0 T. As a proof of principle hepatogenic HuH7 cells were labeled with the particles and observed for cell toxicity as well as detectability by MRI and biodistribution in vivo. RESULTS: PET imaging and MRI revealed increasing hepatic and splenic accumulation of the particles over 24 h. Adjacent in vitro studies in hepatogenic HuH7 cells showed a rapid intracellular accumulation of the particles with high labeling efficiency and without any signs of toxicity. In vivo dissemination of the labeled cells could be followed by dynamic biodistribution studies. CONCLUSIONS: We conclude that our modified superparamagnetic nanoparticles are stable under in vitro and in vivo conditions and are therefore applicable for efficient cell labeling and subsequent multimodal molecular imaging. Moreover, their multiple free amino groups suggest the possibility for further modifications and might provide interesting opportunities for various research fields.

Surface characterization of extracellular matrix scaffolds.

Extracellular matrix (ECM) scaffolds prepared from different tissue sources or using different methods have been demonstrated to have distinctive effects upon cell adhesion patterns and the ability to support and maintain differentiated phenotypes. It is unknown whether the molecular composition or the ultrastructure of the ECM plays a greater role in determining the phenotype of the cells with which it comes into contact. However, when implanted, the topology and ligand landscape of the material will determine the host molecules that bind and the type and behavior of cells that mediate the host response. Therefore, a comprehensive understanding of surface characteristics is essential in the design of scaffolds for specific clinical applications. The surface characteristics of ECM scaffolds derived from porcine urinary bladder, small intestine, and liver as well as the effects of two commonly used methods of chemical cross-linking upon UBM were investigated. Electron microscopy and time of flight secondary ion mass spectroscopy were used to examine the surface characteristics of the scaffolds. The results show that ECM scaffolds have unique morphologic and structural properties which are dependant on the organ or tissue from which the scaffold is harvested. Furthermore, the results show that the surface characteristics of an ECM scaffold are changed through chemical cross-linking.

Ultra-high vacuum surface analysis study of rhodopsin incorporation into supported lipid bilayers.

Planar supported lipid bilayers that are stable under ambient atmospheric and ultra-high-vacuum conditions were prepared by cross-linking polymerization of bis-sorbylphosphatidylcholine (bis-SorbPC). X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were employed to investigate bilayers that were cross-linked using either redox-initiated radical polymerization or ultraviolet photopolymerization. The redox method yields a more structurally intact bilayer; however, the UV method is more compatible with incorporation of transmembrane proteins. UV polymerization was therefore used to prepare cross-linked bilayers with incorporated bovine rhodopsin, a light-activated, G-protein-coupled receptor (GPCR). A previous study (Subramaniam, V.; Alves, I. D.; Salgado, G. F. J.; Lau, P. W.; Wysocki, R. J.; Salamon, Z.; Tollin, G.; Hruby, V. J.; Brown, M. F.; Saavedra, S. S. J. Am. Chem. Soc. 2005, 127, 5320-5321) showed that rhodopsin retains photoactivity after incorporation into UV-polymerized bis-SorbPC, but did not address how the protein is associated with the bilayer. In this study, we show that rhodopsin is retained in supported bilayers of poly(bis-SorbPC) under ultra-high-vacuum conditions, on the basis of the increase in the XPS nitrogen concentration and the presence of characteristic amino acid peaks in the ToF-SIMS data. Angle-resolved XPS data show that the protein is inserted into the bilayer, rather than adsorbed on the bilayer surface. This is the first study to demonstrate the use of ultra-high-vacuum techniques for structural studies of supported proteolipid bilayers.

An orthotopic model of pancreatic somatostatin receptor (SSTR)-positive tumors allows bimodal imaging studies using 3T MRI and animal PET-based molecular imaging of SSTR expression.

Somatostatin receptor (SSTR) scintigraphy is currently used as one standard imaging modality in neuroendocrine tumors (NETs). However, future optimization of NET imaging may be achieved with positron emission tomography based methods utilizing more sensitive and specific tracers in combination with computed tomography or magnetic resonance imaging. Here we established an orthotopic mouse model that reflects relevant aspects of human pancreatic NETs such as SSTR expression, dense vascularization and metastatic disease. This model was then utilized to test the feasibility of combined magnetic resonance imaging and animal positron emission tomography. Orthotopic implantation of amphicrine, SSTR-positive pancreatic AR42J cells resulted in rapidly growing tumors, with concomitant metastatic spread into abdominal lymph nodes and peritoneal cavity. Primary tumors as well as their metastases expressed the neuroendocrine markers chromogranin A and synaptophysin. For imaging experiments, the SSTR ligands (68)Ga-DOTATOC or (68)Ga-DOTANOC were injected intravenously, and animals were subsequently examined in an animal positron emission tomography scanner and a clinical 3T (tesla) magnetic resonance imager. All animals showed radionuclide accumulation in the primary tumor. Definite anatomical correlation was achieved using digital image fusion of the positron emission tomography and magnetic resonance imaging data. (68)Ga-DOTANOC strongly accumulated in the tumor tissue (mean 6.6-fold vs. control tissues) when compared to (68)Ga-DOTATOC, which showed a higher renal clearance. In good agreement with the biodistribution data, the kidney-to-tumor ratio was higher for (68)Ga-DOTATOC (2.43-fold vs. 1.75-fold). Consequently, (68)Ga-DOTANOC achieved better signal enhancement in the primary tumor and allowed for detection of metastatic lesions. In summary, we established a novel orthotopic pancreatic SSTR-positive tumor model and used this model to provide proof of principle for the diagnostic combination of SSTR-based molecular imaging and magnetic resonance imaging. Specifically, the animal model allowed the comparative evaluation of (68)Ga-DOTANOC and (68)Ga-DOTATOC, with (68)Ga-DOTANOC providing better tumor-specific accumulation and renal activity. We conclude that this animal model will be of innovative value for further investigation in the imaging of NETs.

Diagnostic value of 123I-IMT SPECT in the follow-up of head and neck cancer.

BACKGROUND: Nuclear medicine imaging is increasingly used in the evaluation of tumors of the head and neck. In the current study, we assess the value of single-photon emission tomography (SPECT) using the amino acid tracer L-3-[123I]iodine-alpha-methyl-tyrosine (IMT) for the detection of recurrent head and neck cancer. PATIENTS AND METHODS: 45 consecutive patients with suspected recurrence of previously treated head and neck cancer were examined by IMT-SPECT using a dual head system with integrated low-dose computed tomography (CT). The accuracy of the IMT-SPECT was evaluated by correlating the findings with results of histology or clinical and CT/MRI (magnetic resonance imaging) follow-up examinations. RESULTS: The sensitivity, specificity and accuracy of IMT-SPECT in the detection of recurrent/persistent tumors were 83, 89 and 84.5%, respectively. The positive and negative predictive value amounted to 96.5 and 60%, respectively. CONCLUSION: IMT-SPECT using integrated low-dose CT appears to be a helpful complementary imaging tool for the detection of local recurrences and lymph node metastases of head and neck cancer and their differentiation from treatment-induced changes. The advantage of the method is the high positive predictive value in the diagnosis of relapsed tumors. However, a negative IMT-SPECT result does not exclude a recurrence.

Relevance of image fusion with MRI for the interpretation of I-123 iodo-methyl-tyrosine scans in patients with suspected recurrent or residual brain tumor.

PURPOSE: The aim of the study was to investigate the impact of MR/SPECT image fusion on the interpretation of I-123 iodo-methyl-tyrosine (IMT) SPECT examinations in patients with pretreated brain tumors. MATERIAL AND METHODS: In this retrospective study, 45 consecutive patients with suspected recurrent/residual gliomas (n = 41) or cerebral metastases (n = 4) were included. SPECT studies were performed using a triple-head gamma-camera system 10 minutes after injection of 300 to 370 MBq (8.1-10 mCi) I-123 IMT. Concerning MR, T1-, T2-, and FLAIR-weighted sequences as well as contrast-enhanced T1-weighted sequences were acquired by 1.5-T or 3.0-T scanners. For image fusion, the MPI-tool software package was used. SPECT and MR/SPECT fusion images were anonymized and then independently evaluated by 3 observers aware of the clinical data. Tumor localization and extent were evaluated and correlated with histopathology or clinical follow up, including MR imaging. RESULTS: In 10 of 45 (22%) patients, image fusion had a significant impact on the interpretation of scans: 5 suspected SPECT findings were correctly classified as physiological or therapy-related; in another 5 patients, image fusion added relevant clinical information on tumor extent (infiltration of the contralateral hemisphere n = 3, infiltration of the brain stem n = 2). CONCLUSIONS: According to our data, image fusion is crucial for the interpretation of positive I-123 IMT SPECT findings.