18F-fluciclovine PET/CT to distinguish radiation necrosis from tumor progression for brain metastases treated with radiosurgery: results of a prospective pilot study.

PURPOSE: Distinguishing radiation necrosis from tumor progression among patients with brain metastases previously treated with stereotactic radiosurgery represents a common diagnostic challenge. We performed a prospective pilot study to determine whether PET/CT with 18F-fluciclovine, a widely available amino acid PET radiotracer, repurposed intracranially, can accurately diagnose equivocal lesions. METHODS: Adults with brain metastases previously treated with radiosurgery presenting with a follow-up tumor-protocol MRI brain equivocal for radiation necrosis versus tumor progression underwent an 18F-fluciclovine PET/CT of the brain within 30 days. The reference standard for final diagnosis consisted of clinical follow-up until multidisciplinary consensus or tissue confirmation. RESULTS: Of 16 patients imaged from 7/2019 to 11/2020, 15 subjects were evaluable with 20 lesions (radiation necrosis, n = 16; tumor progression, n = 4). Higher SUVmax statistically significantly predicted tumor progression (AUC = 0.875; p = 0.011). Lesion SUVmean (AUC = 0.875; p = 0.018), SUVpeak (AUC = 0.813; p = 0.007), and SUVpeak-to-normal-brain (AUC = 0.859; p = 0.002) also predicted tumor progression, whereas SUVmax-to-normal-brain (p = 0.1) and SUVmean-to-normal-brain (p = 0.5) did not. Qualitative visual scores were significant predictors for readers 1 (AUC = 0.750; p < 0.001) and 3 (AUC = 0.781; p = 0.045), but not for reader 2 (p = 0.3). Visual interpretations were significant predictors for reader 1 (AUC = 0.898; p = 0.012) but not for reader 2 (p = 0.3) or 3 (p = 0.2). CONCLUSIONS: In this prospective pilot study of patients with brain metastases previously treated with radiosurgery presenting with a contemporary MRI brain with a lesion equivocal for radiation necrosis versus tumor progression, 18F-fluciclovine PET/CT repurposed intracranially demonstrated encouraging diagnostic accuracy, supporting the pursuit of larger clinical trials which will be necessary to establish diagnostic criteria and performance.

Evaluation of Polymeric Nanomedicines Targeted to PSMA: Effect of Ligand on Targeting Efficiency.

Targeted nanomedicines offer a strategy for greatly enhancing accumulation of a therapeutic within a specific tissue in animals. In this study, we report on the comparative targeting efficiency toward prostate-specific membrane antigen (PSMA) of a number of different ligands that are covalently attached by the same chemistry to a polymeric nanocarrier. The targeting ligands included a small molecule (glutamate urea), a peptide ligand, and a monoclonal antibody (J591). A hyperbranched polymer (HBP) was utilized as the nanocarrier and contained a fluorophore for tracking/analysis, whereas the pendant functional chain-ends provided a handle for ligand conjugation. Targeting efficiency of each ligand was assessed in vitro using flow cytometry and confocal microscopy to compare degree of binding and internalization of the HBPs by human prostate cancer (PCa) cell lines with different PSMA expression status (PC3-PIP (PSMA+) and PC3-FLU (PSMA-). The peptide ligand was further investigated in vivo, in which BALB/c nude mice bearing subcutaneous PC3-PIP and PC3-FLU PCa tumors were injected intravenously with the HBP-peptide conjugate and assessed by fluorescence imaging. Enhanced accumulation in the tumor tissue of PC3-PIP compared to PC3-FLU highlighted the applicability of this system as a future imaging and therapeutic delivery vehicle.

Hybrid PET/MR imaging: physics and technical considerations.

In just over a decade, hybrid imaging with FDG PET/CT has become a standard bearer in the management of cancer patients. An exquisitely sensitive whole-body imaging modality, it combines the ability to detect subtle biologic changes with FDG PET and the anatomic information offered by CT scans. With advances in MR technology and advent of novel targeted PET radiotracers, hybrid PET/MRI is an evolutionary technique that is poised to revolutionize hybrid imaging. It offers unparalleled spatial resolution and functional multi-parametric data combined with biologic information in the non-invasive detection and characterization of diseases, without the deleterious effects of ionizing radiation. This article reviews the basic principles of FDG PET and MR imaging, discusses the salient technical developments of hybrid PET/MR systems, and provides an introduction to FDG PET/MR image acquisition.

Improving the biodistribution of PSMA-targeting tracers with a highly negatively charged linker.

BACKGROUND: Prostate specific membrane antigen (PSMA) is overexpressed in prostate cancer and in tumor vasculature. Small molecule based inhibitors of PSMA have promised to provide sensitive detection of primary and metastatic prostate tumors. Although significant progress has been made, many of the radiolabeled imaging agents exhibit non-specific background binding. Prevailing tracer designs focus on high affinity urea-based inhibitors with strategically placed hydrophobic patches that interact favorably with the substrate tunnel of PSMA. We hypothesized that a novel PSMA inhibitor design incorporating highly negatively charged linkers may minimize non-specific binding and decrease overall background. METHODS: Through iterative redesign, we generated a series of PSMA inhibitors with highly negatively charged linkers that connect to urea inhibitors and bulky radionuclide chelates. We then performed in vivo imaging and biodistribution studies with the radiolabeled tracers. RESULTS: The tracers derived from our iterative redesign have affinities for PSMA comparable to the "parent" urea ligand Cys-C(O)-Glu. Using a fluorine-18 labeled PSMA targeting tracer, we found that these highly negatively charged molecules exhibit rapid renal excretion with minimal non-specific binding. The biodistribution data at 2 hr showed 4.6%ID/g PC3-PIP tumor uptake with spleen, liver, bone, and blood background levels of 0.1%, 0.17%, 0.1%, and 0.04%, respectively. CONCLUSION: Placement of multiple negative charges in the linker region of PSMA tracers significantly reduced the non-specific background binding without significant reduction of binding affinity. This increased tumor/background contrast in positron emission tomography promises to provide more sensitive tumor detection while decreasing the overall radiation exposure to patients.

Ictal single photon emission computed tomography in epileptic auras.

OBJECTIVES: Little is known about whether ictal single photon emission computed tomography (SPECT) during an isolated aura can localize the epileptogenic zone (EZ). This study seeks to evaluate the yield of ictal SPECT injection in isolated epileptic auras. METHODS: We identified 20 patients with focal epilepsy studied during 26 isolated auras by ictal interictal subtraction SPECT coregistered to magnetic resonance imaging (SISCOM). Studies were rated by two readers who blindly scored the images for presence or absence of an area of dominant hyperperfusion and the lateralization and localization of ictal hyperperfusion; kappa statistics were calculated. Results are correlated with the localization or lateralization of the EZ, time of injection, and electroencephalography (EEG) findings during aura. RESULTS: Fourteen (53%) of 26 injections in 13 patients were rated by both readers as having an area of dominant hyperperfusion with poor interobserver agreement (k = 0.128). Nine of 26 injections in eight patients were correctly lateralized to the side of the EZ (κ = 0.46), but only one of 21 injections in one patient was correctly localized (κ = 0.146). No difference was found when comparing temporal and extratemporal cases. Studies obtained in auras with ictal EEG change were no more likely to be correctly localized than in ones without (p = 0.19). The timing of injection was not a predictor of success. SIGNIFICANCE: Ictal SPECT injection during an isolated aura has a low yield of correct localization of the EZ and cannot be relied on alone during presurgical evaluation. A repeat injection during a seizure with clinical signs and ictal EEG accompaniment is recommended.

A novel PSMA-targeting tracer with highly negatively charged linker demonstrates decreased salivary gland uptake in mice compared to [68Ga]Ga-PSMA-11.

BACKGROUND: The current generation of radiolabeled PSMA-targeting therapeutic agents is limited by prominent salivary gland binding, which results in dose-limiting xerostomia from radiation exposure. JB-1498 is a urea-based small molecule with a highly negatively charged linker targeting prostate specific membrane antigen (PSMA). Prior work on a similar tracer with the same negatively charged linker demonstrated low normal organ/soft tissue background uptake compared to [68Ga]Ga-PSMA-11. The purpose of this study was to investigate if [68Ga]Ga-JB-1498 had reduced salivary gland uptake in mice compared to [68Ga]Ga-PSMA-11. RESULTS: JB-1498 demonstrated high affinity for PSMA binding and tumor uptake in a murine tumor model. In an initial biodistribution study with low molar activity, [68Ga]Ga-JB-1498 demonstrated salivary gland uptake of 0.13 ± 0.01%ID/g. In a second biodistribution study in non-tumor-bearing mice with high molar activity, [68Ga]Ga-JB1498 demonstrated salivary gland uptake of 0.39 ± 0.24% ID/g and kidney activity of 10.12 ± 1.73% ID/g at one hour post IV injection. This salivary gland uptake is significantly less than the published uptake of [68Ga]Ga-PSMA-11. Micro-PET visually confirmed the findings of the biodistribution studies. Dynamic micro-PET imaging demonstrated gradually decreasing [68Ga]Ga-JB1498 activity in salivary glands and kidneys, compared to gradually increasing [68Ga]Ga-PSMA-11 activity in these two organs during the first hour. CONCLUSION: Biodistribution and micro-PET imaging of [68Ga]Ga-JB-1498 demonstrate significantly decreased salivary gland uptake and different pharmacokinetic behavior in kidneys and salivary glands in mice compared to [68Ga]Ga-PSMA-11. Our findings suggest that constructing a PSMA-targeting molecule with a highly negatively charged linker is a promising strategy to reduce salivary gland uptake of GCP-II/PSMA ligands in theranostic applications.

Multi-Modality Imaging in Vasculitis.

Systemic vasculitides are a rare and complex group of diseases that can affect multiple organ systems. Clinically, presentation may be vague and non-specific and as such, diagnosis and subsequent management are challenging. These entities are typically classified by the size of vessel involved, including large-vessel vasculitis (giant cell arteritis, Takayasu's arteritis, and clinically isolated aortitis), medium-vessel vasculitis (including polyarteritis nodosa and Kawasaki disease), and small-vessel vasculitis (granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis). There are also other systemic vasculitides that do not fit in to these categories, such as Behcet's disease, Cogan syndrome, and IgG4-related disease. Advances in medical imaging modalities have revolutionized the approach to diagnosis of these diseases. Specifically, color Doppler ultrasound, computed tomography and angiography, magnetic resonance imaging, positron emission tomography, or invasive catheterization as indicated have become fundamental in the work up of any patient with suspected systemic or localized vasculitis. This review presents the key diagnostic imaging modalities and their clinical utility in the evaluation of systemic vasculitis.

Novel Depiction of Collateral Flow in Chronic Thromboembolic Pulmonary Hypertension Using 4D Dynamic 82 Rb PET/CT.

ABSTRACT: CT pulmonary angiogram and ventilation-perfusion scintigraphy are the 2 primary imaging modalities for evaluating patients with CTEPH (chronic thromboembolic pulmonary hypertension). PET/CT and MRI currently have a limited role in the evaluation of acute or chronic pulmonary embolism. We present incidentally captured dynamic pulmonary perfusion images in a patient with history of CTEPH who underwent 82 Rb myocardial perfusion PET/CT for evaluation of chest pain. Analysis of the PET data revealed delayed perfusion of the affected lobes suggesting collateralization, highlighting a potentially new imaging paradigm for assessment of pulmonary perfusion.

A Perspective on the Evolving Story of PSMA Biology, PSMA-Based Imaging, and Endoradiotherapeutic Strategies.

In this review, we cover the evolution of knowledge on the biology of prostate-specific membrane antigen (PSMA) and its translation to therapy. The usual key to discovery is a realistic model for experimentation and for testing a hypothesis. A realistic model is especially needed in the case of the human prostate, which differs significantly from the prostate of species often used as research models. We will emphasize the genetic characterization of PSMA, the nature of the PSMA protein, and its role as a carboxypeptidase, with differing important substrates and products in different tissues. We give special prominence to the importance of PSMA as a target for imaging and therapy in prostate cancer and its underdeveloped role for imaging and targeting the neovasculature of tumors other than prostate cancer. Lastly, we bring attention to its importance in other nonprostatic tissues.

Should Low Molecular Weight PSMA Targeted Ligands Get Bigger and Use Albumin Ligands for PSMA Targeting?

Prostate Specific Membrane Antigen (PSMA) is strongly expressed in prostate cancer. Recently a number of low-molecular-weight inhibitors have demonstrated excellent PSMA targeting activity for both imaging as well as Lutecium-177 radiotherapy in human trials. The paper by Choy et al raises the question of whether we can further increase the effectiveness of PSMA targeted therapy by adding an albumin-binding entity to low-molecular-weight agents.

X-ray structure of a maquette scaffold.

Maquettes are de novo designed mimicries of nature used to test the construction and engineering criteria of oxidoreductases. One type of scaffold used in maquette construction is a four-alpha-helical bundle. The sequence of the four-alpha-helix bundle maquettes follows a heptad repeat pattern typical of left-handed coiled-coils. Initial designs were molten globular due partly to the minimalist approach taken by the designers. Subsequent iterative redesign generated several structured scaffolds with similar heme binding properties. Variant [I(6)F(13)](2), a structured scaffold, was partially resolved with NMR spectroscopy and found to have a set of mobile inter-helical packing interfaces. Here, the X-ray structure of a similar peptide ([I(6)F(13)M(31)](2) i.e. ([CGGG EIWKL HEEFLKK FEELLKL HEERLKKM](2))(2) which we call L31M), has been solved using MAD phasing and refined to 2.8A resolution. The structure shows that the maquette scaffold is an anti-parallel four-helix bundle with "up-up-down-down" topology. No pre-formed heme-binding pocket exists in the protein scaffold. We report unexpected inter-helical crossing angles, residue positions and translations between the helices. The crossing angles between the parallel helices are -5 degrees rather than the expected +20 degrees for typical left-handed coiled-coils. Deviation of the scaffold from the design is likely due to the distribution and size of hydrophobic residues. The structure of L31M points out that four identical helices may interact differently in a bundle and heptad repeats with an alternating [HPPHHPP]/[HPPHHPH] (H: hydrophobic, P: polar) pattern are not a sufficient design criterion to generate left-hand coiled-coils.

Development of targeted near-infrared imaging agents for prostate cancer.

Prostate cancer is the most common noncutaneous malignancy affecting men in North America. Radical prostatectomy remains a definitive treatment for prostate cancer. However, prostate surgeries are still performed "blindly" with the extent of tumor infiltration past the margins of the surgery only being determined postoperatively. An imaging modality that can be used during surgery is needed to help define the tumor margins. With its abundant expression in prostate cancer, prostate-specific membrane antigen (PSMA) is an ideal target for detection of prostate cancer. The purpose of this study was to develop PSMA-targeted near-infrared (NIR) optical imaging probes for intraoperative visualization of prostate cancer. We synthesized a high-affinity PSMA ligand (PSMA-1) with low molecular weight and further labeled it with commercially available NIR dyes IRDy800 and Cy5.5. PSMA-1 and PSMA-1-NIR conjugates had binding affinities better than the parent ligand Cys-CO-Glu. Selective binding was measured for each of the probes in both in vitro and in vivo studies using competitive binding and uptake studies. Interestingly, the results indicated that the pharmacokinetics of the probes was dependent of the fluorophore conjugated to the PSMA-1 ligand and varied widely. These data suggest that PSMA-targeted probes have the potential to be further developed as contrast agents for clinical intraoperative fluorescence-guided surgery.

Robotic real-time near infrared targeted fluorescence imaging in a murine model of prostate cancer: a feasibility study.

OBJECTIVE: To evaluate the detection of near-infrared fluorescence from prostate tumors stained with a prostate-specific membrane antigen (PSMA)-targeted tracer developed in our institution with a novel robotic imaging system. METHODS: Prostate cancer cell lines PC3-pip (PSMA positive) and PC3-flu (PSMA negative) were implanted subcutaneously into 6 immunodeficient mice. When tumors reached 5 mm, a PSMA-targeted fluorescent conjugate was injected intravenously. The first 3 mice underwent near-infrared imaging immediately and hourly up to 4 hours after injection to determine the time necessary to obtain peak fluorescence and were killed. The last 3 mice were imaged once preoperatively and were euthanized 120 minutes later. Excision of the tumors was performed by using a novel robotic imaging system to detect near-infrared fluorescence in real time. Specimens were submitted for pathology. RESULTS: In the first 3 mice, we found 120 minutes as the time needed to observe peak fluorescence from the PSMA-positive tumors. We identified discrete near-infrared fluorescence from 2 of 3 PSMA-positive tumors with the robotic imaging system. Surgical margins were negative for all excised specimens except for one PSMA-negative tumor. CONCLUSIONS: Real-time near-infrared fluorescence imaging of prostate cancer is feasible with a novel robotic imaging system. Further research is needed to optimize the signal intensity detectable from prostate cancer with our tracer. Toxicologic studies are needed before its clinical use.

Functional imaging of cancer with emphasis on molecular techniques.

A multitude of noninvasive, quantitative, functional imaging techniques are currently in use to study tumor physiology, to probe tumor molecular processes, and to study tumor molecules and metabolites in vitro and in vivo using computed tomography (CT), magnetic resonance imaging (MRI), ultrasonography (US), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and optical imaging (OI). Such techniques can be used in conjunction with structural imaging techniques to detect, diagnose, characterize, or monitor tumors before and after therapeutic intervention. These can also be used to study tumor gene expression, to track cells and therapeutic drugs, to optimize individualized treatment planning for patients with tumors, and to foster new oncologic drug development. In this article, we review the rich variety of functional imaging techniques that are available for these purposes, which are becoming increasingly important for optimal individualized patient treatment in this day and age of "personalized medicine."

The HP-1 maquette: from an apoprotein structure to a structured hemoprotein designed to promote redox-coupled proton exchange.

Synthetic heme-binding four-alpha-helix bundles show promise as working model systems, maquettes, for understanding heme cofactor-protein assembly and function in oxidoreductases. Despite successful inclusion of several key functional elements of natural proteins into a family of heme protein maquettes, the lack of 3D structures, due principally to conformational heterogeneity, has prevented them from achieving their full potential. We report here the design and synthesis of HP-1, a disulfide-bridged two-alpha-helix peptide that self-assembles to form an antiparallel twofold symmetric diheme four-alpha-helix bundle protein with a stable conformation on the NMR time-scale. The HP-1 design strategy began with the x-ray crystal structure of the apomaquette L31M, an apomaquette derived from the structurally heterogeneous tetraheme-binding H10H24 prototype. L31M was functionally redesigned to accommodate two hemes ligated to histidines and to retain the strong coupling of heme oxidation-reduction to glutamate acid-base transitions and proton exchange that was characterized in molten globule predecessors. Heme insertion was modeled with angular constraints statistically derived from natural proteins, and the pattern of hydrophobic and hydrophilic residues on each helix was then altered to account for this large structural reorganization. The transition to structured holomaquette involved the alteration of 6 of 31 residues in each of the four identical helices and, unlike our earlier efforts, required no design intermediates. Oxidation-reduction of both hemes displays an unusually low midpoint potential (-248 mV vs. normal hydrogen electrode at pH 9.0), which is strongly coupled to proton binding, as designed.