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Introduction: Measurement of repeatability and reproducibility (R&R) is necessary to realize the full potential of positron emission tomography (PET). Several studies have evaluated the reproducibility of PET using 18F-FDG, the most common PET tracer used in oncology, but similar studies using other PET tracers are scarce. Even fewer assess agreement and R&R with statistical methods designed explicitly for the task. 18F-(2S, 4R)-4-fluoro-glutamine (18F-Gln) is a PET tracer designed for imaging glutamine uptake and metabolism. This study illustrates high reproducibility and repeatability with 18F-Gln for in vivo research. Methods: Twenty mice bearing colorectal cancer cell line xenografts were injected with ~9 MBq of 18F-Gln and imaged in an Inveon microPET. Three individuals analyzed the tumor uptake of 18F-Gln using the same set of images, the same image analysis software, and the same analysis method. Scans were randomly re-ordered for a second repeatability measurement 6 months later. Statistical analyses were performed using the methods of Bland and Altman (B&A), Gauge Reproducibility and Repeatability (Gauge R&R), and Lin's Concordance Correlation Coefficient. A comprehensive equivalency test, designed to reject a null hypothesis of non-equivalence, was also conducted. Results: In a two-way random effects Gauge R&R model, variance among mice and their measurement variance were 0.5717 and 0.024. Reproducibility and repeatability accounted for 31% and 69% of the total measurement error, respectively. B&A repeatability coefficients for analysts 1, 2, and 3 were 0.16, 0.35, and 0.49. One-half B&A agreement limits between analysts 1 and 2, 1 and 3, and 2 and 3 were 0.27, 0.47, and 0.47, respectively. The mean square deviation and total deviation index were lowest for analysts 1 and 2, while coverage probabilities and coefficients of the individual agreement were highest. Finally, the definitive agreement inference hypothesis test for equivalency demonstrated that all three confidence intervals for the average difference of means from repeated measures lie within our a priori limits of equivalence (i.e. ± 0.5%ID/g). Conclusions: Our data indicate high individual analyst and laboratory-level reproducibility and repeatability. The assessment of R&R using the appropriate methods is critical and should be adopted by the broader imaging community. [ABSTRACT FROM AUTHOR]

Purpose: Metabolic reprogramming plays an important role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Currently, positron emission tomography (PET) reporters are not used clinically to visualize altered glutamine metabolism in ccRCC, which greatly hinders detection, staging, and real-time therapeutic assessment. We sought to determine if (2S,4R)-4-[18F]fluoroglutamine ([18F]FGln) could be used to interrogate altered glutamine metabolism in ccRCC lesions in the lung. Procedures: We generated a novel ccRCC lung lesion model using the ccRCC cell line UMRC3 stably transfected with GFP and luciferase constructs. This cell line was used for characterization of [18F]FGln uptake and retention by transport analysis in cell culture and by PET/MRI (magnetic resonance imaging) in animal models. Tumor growth in animal models was monitored using bioluminescence (BLI) and MRI. After necropsy, UMRC3 tumor growth in lung tissue was verified by fluorescence imaging and histology. Results: In UMRC3 cells, [18F]FGln cell uptake was twofold higher than cell uptake in normal kidney HEK293 cells. Tracer cell uptake was reduced by 60–90% in the presence of excess glutamine in the media and by 20–50% upon treatment with V-9302, an inhibitor of the major glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2). Furthermore, in UMRC3 cells, [18F]FGln cell uptake was reduced by siRNA knockdown of ASCT2 to levels obtained by the addition of excess exogenous glutamine. Conversely, [18F]FGln cellular uptake was increased in the presence of the glutaminase inhibitor CB-839. Using simultaneous PET/MRI for visualization, retention of [18F]FGln in vivo in ccRCC lung tumors was 1.5-fold greater than normal lung tissue and twofold greater than muscle. In ccRCC lung tumors, [18F]FGln retention did not change significantly upon treatment with CB-839. Conclusions: We report one of the first direct orthotopic mouse models of ccRCC lung lesions. Using PET/MR imaging, lung tumors were easily discerned from normal tissue. Higher uptake of [18F]FGln was observed in a ccRCC cell line and lung lesions compared to HEK293 cells and normal lung tissue, respectively. [18F]FGln cell uptake was modulated by exogenous glutamine, V-9302, siRNA knockdown of ASCT2, and CB-839. Interestingly, in a pilot therapeutic study with CB-839, we observed no difference in treated tumors relative to untreated controls. This was in contrast with cellular studies, where CB-839 increased glutamine uptake. [ABSTRACT FROM AUTHOR]

d-Amino acids such as d-alanine are substrates for bacterial peptidoglycan biosynthesis and are selectively taken up by bacteria and not mammalian cells. Consequently, d-amino acid metabolism is an attractive target for antibiotic discovery and the development of bacteria-specific imaging agents. d-Fluoroalanine and the deuterium-labeled analogue fludalanine (MK641) were originally explored as antibiotics by Merck but failed in clinical trials due to unaccepted toxicity. Herein, we synthesized a fluorine-18 labeled d-fluoroalanine, d-3-[ 18 F]fluoroalanine (d-[ 18 F]FAla), and its deuterated analogue, d-3-[ 18 F]fluoroalanine- d 3 (d-[ 18 F]FAla- d 3 ), and evaluated their capability to image bacterial infection. Both d-[ 18 F]FAla and d-[ 18 F]FAla- d 3 can accumulate up to 0.64-0.78% ID/cc in the infectious area at 15 min postinjection. Despite the reduction of in vivo defluorination not being observed for deuterated 18 F-labeled d-fluoroalanine, these radiolabeled d-alanine analogues were able to differentiate bacterial infection from sterile inflammation in a soft-tissue model of S. aureus infection.

InhA, the Mycobacterium tuberculosis enoyl-ACP reductase, is a target for the tuberculosis (TB) drug isoniazid (INH). InhA inhibitors that do not require KatG activation avoid the most common mechanism of INH resistance, and there are continuing efforts to fully elucidate the enzyme mechanism to drive inhibitor discovery. InhA is a member of the short-chain dehydrogenase/reductase superfamily characterized by a conserved active site Tyr, Y158 in InhA. To explore the role of Y158 in the InhA mechanism, this residue has been replaced by fluoroTyr residues that increase the acidity of Y158 up to ∼3200-fold. Replacement of Y158 with 3-fluoroTyr (3-FY) and 3,5-difluoroTyr (3,5-F 2 Y) has no effect on k cat app / K M app nor on the binding of inhibitors to the open form of the enzyme ( K i app ), whereas both k cat app / K M app and K i app are altered by seven-fold for the 2,3,5-trifluoroTyr variant (2,3,5-F 3 Y158 InhA). 19 F NMR spectroscopy suggests that 2,3,5-F 3 Y158 is ionized at neutral pH indicating that neither the acidity nor ionization state of residue 158 has a major impact on catalysis or on the binding of substrate-like inhibitors. In contrast, K i * app is decreased 6- and 35-fold for the binding of the slow-onset inhibitor PT504 to 3,5-F 2 Y158 and 2,3,5-F 3 Y158 InhA, respectively, indicating that Y158 stabilizes the closed form of the enzyme adopted by EI*. The residence time of PT504 is reduced ∼four-fold for 2,3,5-F 3 Y158 InhA compared to wild-type, and thus, the hydrogen bonding interaction of the inhibitor with Y158 is an important factor in the design of InhA inhibitors with increased residence times on the enzyme.