Recurrent Germline DLST Mutations in Individuals with Multiple Pheochromocytomas and Paragangliomas.

Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and 13C5-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (∼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.

Different Effects of RNAi-Mediated Downregulation or Chemical Inhibition of NAMPT in an Isogenic IDH Mutant and Wild-Type Glioma Cell Model.

The IDH1R132H mutation in glioma results in the neoenzymatic function of IDH1, leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG), alterations in energy metabolism and changes in the cellular redox household. Although shifts in the redox ratio NADPH/NADP+ were described, the consequences for the NAD+ synthesis pathways and potential therapeutic interventions were largely unexplored. Here, we describe the effects of heterozygous IDH1R132H on the redox system in a CRISPR/Cas edited glioblastoma model and compare them with IDH1 wild-type (IDH1wt) cells. Besides an increase in 2-HG and decrease in NADPH, we observed an increase in NAD+ in IDH1R132H glioblastoma cells. RT-qPCR analysis revealed the upregulation of the expression of the NAD+ synthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Knockdown of NAMPT resulted in significantly reduced viability in IDH1R132H glioblastoma cells. Given this dependence of IDH1R132H cells on NAMPT expression, we explored the effects of the NAMPT inhibitors FK866, GMX1778 and GNE-617. Surprisingly, these agents were equally cytotoxic to IDH1R132H and IDH1wt cells. Altogether, our results indicate that targeting the NAD+ synthesis pathway is a promising therapeutic strategy in IDH mutant gliomas; however, the agent should be carefully considered since three small-molecule inhibitors of NAMPT tested in this study were not suitable for this purpose.

Exploring Nitric Oxide (NO)-Releasing Celecoxib Derivatives as Modulators of Radioresponse in Pheochromocytoma Cells.

COX-2 can be considered as a clinically relevant molecular target for adjuvant, in particular radiosensitizing treatments. In this regard, using selective COX-2 inhibitors, e.g., in combination with radiotherapy or endoradiotherapy, represents an interesting treatment option. Based on our own findings that nitric oxide (NO)-releasing and celecoxib-derived COX-2 inhibitors (COXIBs) showed promising radiosensitizing effects in vitro, we herein present the development of a series of eight novel NO-COXIBs differing in the peripheral substitution pattern and their chemical and in vitro characterization. COX-1 and COX-2 inhibition potency was found to be comparable to the lead NO-COXIBs, and NO-releasing properties were demonstrated to be mainly influenced by the substituent in 4-position of the pyrazole (Cl vs. H). Introduction of the N-propionamide at the sulfamoyl residue as a potential prodrug strategy lowered lipophilicity markedly and abolished COX inhibition while NO-releasing properties were not markedly influenced. NO-COXIBs were tested in vitro for a combination with single-dose external X-ray irradiation as well as [177Lu]LuCl3 treatment in HIF2α-positive mouse pheochromocytoma (MPC-HIF2a) tumor spheroids. When applied directly before X-ray irradiation or 177Lu treatment, NO-COXIBs showed radioprotective effects, as did celecoxib, which was used as a control. Radiosensitizing effects were observed when applied shortly after X-ray irradiation. Overall, the NO-COXIBs were found to be more radioprotective compared with celecoxib, which does not warrant further preclinical studies with the NO-COXIBs for the treatment of pheochromocytoma. However, evaluation as radioprotective agents for healthy tissues could be considered for the NO-COXIBs developed here, especially when used directly before irradiation.

Design, synthesis, and evaluation of positron emission tomography/fluorescence dual imaging probes for targeting facilitated glucose transporter 1 (GLUT1).

Increased energy metabolism followed by enhanced glucose consumption is a hallmark of cancer. Most cancer cells show overexpression of facilitated hexose transporter GLUT1, including breast cancer. GLUT1 is the main transporter for 2-deoxy-2-[18F]fluoro-d-glucose (2-[18F]FDG), the gold standard of positron emission tomography (PET) imaging in oncology. The present study's goal was to develop novel glucose-based dual imaging probes for their use in tandem PET and fluorescence (Fl) imaging. A glucosamine scaffold tagged with a fluorophore and an 18F-label should confer selectivity to GLUT1. Out of five different compounds, 2-deoxy-2-((7-sulfonylfluoro-2,1,3-benzoxadiazol-4-yl)amino)-d-glucose (2-FBDG) possessed favorable fluorescent properties and a similar potency as 2-deoxy-2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)-d-glucose (2-NBDG) in competing for GLUT1 transport against 2-[18F]FDG in breast cancer cells. Radiolabeling with 18F was achieved through the synthesis of prosthetic group 7-fluoro-2,1,3-benzoxadiazole-4-sulfonyl [18F]fluoride ([18F]FBDF) followed by the reaction with glucosamine. The radiotracer was finally analyzed in vivo in a breast cancer xenograft model and compared to 2-[18F]FDG. Despite favourable in vitro fluorescence imaging properties, 2-[18F]FBDG was found to lack metabolic stability in vivo, resulting in radiodefluorination. Glucose-based 2-[18F]FBDG represents a novel dual-probe for GLUT1 imaging using FI and PET with the potential for further structural optimization for improved metabolic stability in vivo.

Metabolomics, machine learning and immunohistochemistry to predict succinate dehydrogenase mutational status in phaeochromocytomas and paragangliomas.

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours with a hereditary background in over one-third of patients. Mutations in succinate dehydrogenase (SDH) genes increase the risk for PPGLs and several other tumours. Mutations in subunit B (SDHB) in particular are a risk factor for metastatic disease, further highlighting the importance of identifying SDHx mutations for patient management. Genetic variants of unknown significance, where implications for the patient and family members are unclear, are a problem for interpretation. For such cases, reliable methods for evaluating protein functionality are required. Immunohistochemistry for SDHB (SDHB-IHC) is the method of choice but does not assess functionality at the enzymatic level. Liquid chromatography-mass spectrometry-based measurements of metabolite precursors and products of enzymatic reactions provide an alternative method. Here, we compare SDHB-IHC with metabolite profiling in 189 tumours from 187 PPGL patients. Besides evaluating succinate:fumarate ratios (SFRs), machine learning algorithms were developed to establish predictive models for interpreting metabolite data. Metabolite profiling showed higher diagnostic specificity compared to SDHB-IHC (99.2% versus 92.5%, p = 0.021), whereas sensitivity was comparable. Application of machine learning algorithms to metabolite profiles improved predictive ability over that of the SFR, in particular for hard-to-interpret cases of head and neck paragangliomas (AUC 0.9821 versus 0.9613, p = 0.044). Importantly, the combination of metabolite profiling with SDHB-IHC has complementary utility, as SDHB-IHC correctly classified all but one of the false negatives from metabolite profiling strategies, while metabolite profiling correctly classified all but one of the false negatives/positives from SDHB-IHC. From 186 tumours with confirmed status of SDHx variant pathogenicity, the combination of the two methods resulted in 185 correct predictions, highlighting the benefits of both strategies for patient management. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Germline SDHB-inactivating mutation in gastric spindle cell sarcoma.

Gastrointestinal stromal tumors (GISTs) are the most frequent mesenchymal tumors of the gastrointestinal tract. Inactivating mutations or epigenetic deregulation of succinate dehydrogenase complex (SDH) genes are considered defining features of a subset of GIST occurring in the stomach. Based on comprehensive molecular profiling and biochemical analysis within a precision oncology program, we identified hallmarks of SDH deficiency (germline SDHB-inactivating mutation accompanied by somatic loss of heterozygosity, lack of SDHB expression, global DNA hypermethylation, and elevated succinate/fumarate ratio) in a 40-year-old woman with undifferentiated gastric spindle cell sarcoma that did not meet the diagnostic criteria for other mesenchymal tumors of the stomach, including GIST. These data reveal that the loss of SDH function can be involved in the pathogenesis of non-GIST sarcoma of the gastrointestinal tract.

Mutations in MDH2, Encoding a Krebs Cycle Enzyme, Cause Early-Onset Severe Encephalopathy.

MDH2 encodes mitochondrial malate dehydrogenase (MDH), which is essential for the conversion of malate to oxaloacetate as part of the proper functioning of the Krebs cycle. We report bi-allelic pathogenic mutations in MDH2 in three unrelated subjects presenting with early-onset generalized hypotonia, psychomotor delay, refractory epilepsy, and elevated lactate in the blood and cerebrospinal fluid. Functional studies in fibroblasts from affected subjects showed both an apparently complete loss of MDH2 levels and MDH2 enzymatic activity close to null. Metabolomics analyses demonstrated a significant concomitant accumulation of the MDH substrate, malate, and fumarate, its immediate precursor in the Krebs cycle, in affected subjects' fibroblasts. Lentiviral complementation with wild-type MDH2 cDNA restored MDH2 levels and mitochondrial MDH activity. Additionally, introduction of the three missense mutations from the affected subjects into Saccharomyces cerevisiae provided functional evidence to support their pathogenicity. Disruption of the Krebs cycle is a hallmark of cancer, and MDH2 has been recently identified as a novel pheochromocytoma and paraganglioma susceptibility gene. We show that loss-of-function mutations in MDH2 are also associated with severe neurological clinical presentations in children.

The cost of improving nutritional outcomes through food-assisted maternal and child health and nutrition programmes in Burundi and Guatemala.

Evidence on the cost-effectiveness of multisectoral maternal and child health and nutrition programmes is scarce. We conducted a prospective costing study of two food-assisted maternal and child health and nutrition programmes targeted to pregnant women and children during the first 1,000 days (pregnancy to 2 years). Each was paired with a cluster-randomized controlled trial to evaluate impact and compare the optimal quantity and composition of food rations (Guatemala, five treatment arms) and their optimal timing and duration (Burundi, three treatment arms). We calculated the total and per beneficiary cost, conducted cost consequence analyses, and estimated the cost savings from extending the programme for 2 years. In Guatemala, the programme model with the lowest cost per percentage point reduction in stunting provided the full-size family ration with an individual ration of corn-soy blend or micronutrient powder. Reducing family ration size lowered costs but failed to reduce stunting. In Burundi, providing food assistance for the full 1,000 days led to the lowest cost per percentage point reduction in stunting. Reducing the duration of ration eligibility reduced per beneficiary costs but was less effective. A 2-year extension could have saved 11% per beneficiary in Guatemala and 18% in Burundi. We found that investments in multisectoral nutrition programmes do not scale linearly. Programmes providing smaller rations or rations for shorter durations, although less expensive per beneficiary, may not provide the necessary dose to improve (biological) outcomes. Lastly, delivering effective programmes for longer periods can generate cost savings by dispersing start-up costs and lengthening peak operating capacity.

Targeting phosphatidylserine for radionuclide-based molecular imaging of apoptosis.

One major characteristic of programmed cell death (apoptosis) results in the increased expression of phosphatidylserine (PS) on the outer membrane of dying cells. Consequently, PS represents an excellent target for non-invasive imaging of apoptosis by single-photon emission computed tomography (SPECT) and positron emission tomography (PET). Annexin V is a 36 kDa protein which binds with high affinity to PS in the presence of Ca2+ ions. This makes radiolabeled annexins valuable apoptosis imaging agents for clinical and biomedical research applications for monitoring apoptosis in vivo. However, the use of radiolabeled annexin V for in vivo imaging of cell death has been met with a variety of challenges which have prevented its translation into the clinic. These difficulties include: complicated and time-consuming radiolabeling procedures, sub-optimal biodistribution, inadequate pharmacokinetics leading to poor tumour-to-blood contrast ratios, reliance upon Ca2+ concentrations in vivo, low tumor tissue penetration, and an incomplete understanding of what constitutes the best imaging protocol following induction of apoptosis. Therefore, new concepts and improved strategies for the development of PS-binding radiotracers are needed. Radiolabeled PS-binding peptides and various Zn(II) complexes as phosphate chemosensors offer an innovative strategy for radionuclide-based molecular imaging of apoptosis with PET and SPECT. Radiolabeled peptides and Zn(II) complexes provide several advantages over annexin V including better pharmacokinetics due to their smaller size, better availability, simpler synthesis and radiolabeling strategies as well as facilitated tissue penetration due to their smaller size and faster blood clearance profile allowing for optimized image contrast. In addition, peptides can be structurally modified to improve metabolic stability along with other pharmacokinetic and pharmacodynamic properties. The present review will summarize the current status of radiolabeled annexins, peptides and Zn(II) complexes developed as radiotracers for imaging apoptosis through targeting PS utilizing PET and SPECT imaging.

Novel methods in adrenal research: a metabolomics approach.

Metabolic alterations have implications in a spectrum of tissue functions and disease. Aided by novel molecular biological and computational tools, our understanding of physiological and pathological processes underpinning endocrine and endocrine-related disease has significantly expanded over the last decade. Herein, we focus on novel metabolomics-related methodologies in adrenal research: in situ metabolomics by mass spectrometry imaging, steroid metabolomics by gas and liquid chromatography-mass spectrometry, energy pathway metabologenomics by liquid chromatography-mass spectrometry-based metabolomics of Krebs cycle intermediates, and cellular reprogramming to generate functional steroidogenic cells and hence to modulate the steroid metabolome. All four techniques to assess and/or modulate the metabolome in biological systems provide tremendous opportunities to manage neoplastic and non-neoplastic disease of the adrenal glands in the era of precision medicine. In this context, we discuss emerging clinical applications and/or promising metabolic-driven research towards diagnostic, prognostic, predictive and therapeutic biomarkers in tumours arising from the adrenal gland and extra-adrenal paraganglia as well as modern approaches to delineate and reprogram adrenal metabolism.

Metabolome-guided genomics to identify pathogenic variants in isocitrate dehydrogenase, fumarate hydratase, and succinate dehydrogenase genes in pheochromocytoma and paraganglioma.

PURPOSE: Metabolic aberrations have been described in neoplasms with pathogenic variants (PV) in the Krebs cycle genes encoding succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH). In turn, accumulation of oncometabolites succinate, fumarate, and 2-hydroxyglutarate can be employed to identify tumors with those PV . Additionally, such metabolic readouts may aid in genetic variant interpretation and improve diagnostics. METHODS: Using liquid chromatography-mass spectrometry, 395 pheochromocytomas and paragangliomas (PPGLs) from 391 patients were screened for metabolites to indicate Krebs cycle aberrations. Multigene panel sequencing was applied to detect driver PV in cases with indicative metabolite profiles but undetermined genetic drivers. RESULTS: Aberrant Krebs cycle metabolomes identified rare cases of PPGLs with germline PV in FH and somatic PV in IDHx and SDHx, including the first case of a somatic IDH2 PV in PPGL. Metabolomics also reliably identified PPGLs with SDHx loss-of-function (LOF) PV. Therefore we utilized tumor metabolite profiles to further classify variants of unknown significance in SDHx, thereby enabling missense variants associated with SDHx LOF to be distinguished from benign variants. CONCLUSION: We propose incorporation of metabolome data into the diagnostics algorithm in PPGLs to guide genetic testing and variant interpretation and to help identify rare cases with PV in FH and IDHx.

Role of MDH2 pathogenic variant in pheochromocytoma and paraganglioma patients.

PURPOSE: MDH2 (malate dehydrogenase 2) has recently been proposed as a novel potential pheochromocytoma/paraganglioma (PPGL) susceptibility gene, but its role in the disease has not been addressed. This study aimed to determine the prevalence of MDH2 pathogenic variants among PPGL patients and determine the associated phenotype. METHODS: Eight hundred thirty patients with PPGLs, negative for the main PPGL driver genes, were included in the study. Interpretation of variants of unknown significance (VUS) was performed using an algorithm based on 20 computational predictions, by implementing cell-based enzymatic and immunofluorescence assays, and/or by using a molecular dynamics simulation approach. RESULTS: Five variants with potential involvement in pathogenicity were identified: three missense (p.Arg104Gly, p.Val160Met and p.Ala256Thr), one in-frame deletion (p.Lys314del), and a splice-site variant (c.429+1G>T). All were germline and those with available biochemical data, corresponded to noradrenergic PPGL. CONCLUSION: This study suggests that MDH2 pathogenic variants may play a role in PPGL susceptibility and that they might be responsible for less than 1% of PPGLs in patients without pathogenic variants in other major PPGL driver genes, a prevalence similar to the one recently described for other PPGL genes. However, more epidemiological data are needed to recommend MDH2 testing in patients negative for other major PPGL genes.

Primary fibroblast co-culture stimulates growth and metabolism in Sdhb-impaired mouse pheochromocytoma MTT cells.

Pheochromocytomas and paragangliomas (PGLs) due to mutations of succinate dehydrogenase (SDH) B, a subunit of the SDH complex with a role in the Krebs cycle and the respiratory chain, tend to be larger at diagnosis and more prone to metastatic disease than other tumors. This presentation contrasts with the behavior of some cell line models of SDHB impairment, which show reduced growth compared to wild type. We hypothesize that reduced growth of SDHB-impaired monolayer culture models might reflect lack of support from sources within the tumor microenvironment. The present study therefore investigates how the microenvironment, modeled here by fibroblast co-culture, modulates cell metabolism, growth and invasion in an Sdhb-impaired mouse pheochromocytoma cell line. We employed two different constructs of short hairpin RNA to knockdown Sdhb and compared growth in a monolayer with and without fibroblast co-culture. Sdhb-silenced cells showed functional impairment of SDH with elevated succinate to fumarate ratio and decreased oxidative capacity. Cell growth was delayed with an increase in doubling time of 2 h or 20 h. Clonogenic cell survival and viability, on the other hand, were either unchanged or increased compared to control. In standard monolayer culture, no differences in pro-metastatic features were present. Co-culture with primary mouse fibroblast reversed the difference of proliferation between control and Sdhb knockdown but was unable to significantly influence invasiveness under these culture conditions. Metabolic studies identified that lactate secreted by fibroblasts was taken up preferentially by Sdhb-silenced cells. In summary, the present study identified a potential role for the tumor microenvironment in influencing phenotypic features of SDHB-mutated PGLs, providing a basis for the use of therapies targeted towards the tumor microenvironment.

Periprocedural anticoagulation during left atrial ablation: interrupted and uninterrupted vitamin K-antagonists or uninterrupted novel anticoagulants.

BACKGROUND: There is a lack of data on anticoagulation requirements during ablation of atrial fibrillation (AF). This study compares different oral anticoagulation (OAC) strategies to evaluate risk of bleeding and thromboembolic complications. METHODS: We conducted a single-centre study in patients undergoing left atrial ablation of AF. Three groups were defined: 1) bridging: interrupted vitamin-K-antagonists (VKA), INR ≤2, and bridging with heparin; 2) VKA: uninterrupted VKA and INR of > 2; 3) DOAC: uninterrupted direct oral anticoagulants. Bleeding complications, thromboembolic events and peri-procedural heparin doses were assessed. RESULTS: In total, 780 patients were documented. At 48 h, major complications were more common in the bridging group compared to uninterrupted VKA and DOAC groups (OR: 3.42, 95% CI: 1.29-9.10 and OR: 3.01, 95% CI: 1.19-7.61), largely driven by differences in major pericardial effusion (OR: 4.86, 95% CI: 1.56-15.99 and OR: 4.466, 95% CI, 1.52-13.67) and major vascular events (OR: 2.92, 95% CI: 0.58-14.67 and OR: 9.72, 95% CI: 1.00-94.43). Uninterrupted VKAs and DOACs resulted in similar odds of major complications (overall OR: 1.14, 95% CI: 0.44-2.92), including cerebrovascular events (OR: 1.21, 95% CI: 0.27-5.45). However, whereas only TIAs were observed in DOAC and bridging groups, strokes also occurred in the VKA group. Rates of minor complications (pericardial effusion, vascular complications, gastrointestinal hemorrhage) and major/minor groin hemorrhage were similar across groups. CONCLUSION: Our dataset illustrates that uninterrupted VKA and DOAC have a better risk-benefit profile than VKA bridging. Bridging was associated with a 4.5× increased risk of complications and should be avoided, if possible.

Targeting Phosphatidylserine with a 64Cu-Labeled Peptide for Molecular Imaging of Apoptosis.

Molecular imaging of programmed cell death (apoptosis) in vivo is an innovative strategy for early assessment of treatment response and treatment efficacy in cancer patients. Externalization of phosphatidylserine (PS) to the cell membrane surface of dying cells makes this phospholipid an attractive molecular target for the development of apoptosis imaging probes. In this study, we have radiolabeled PS-binding 14-mer peptide FNFRLKAGAKIRFG (PSBP-6) with positron-emitter copper-64 (64Cu) for PET imaging of apoptosis. Peptide PSBP-6 was conjugated with radiometal chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through an aminovaleric acid (Ava) linker for subsequent radiolabeling with 64Cu to prepare radiotracer 64Cu-NOTA-Ava-PSBP-6. PS-binding potencies of PSBP-6, NOTA-Ava-PSBP-6, and natCu-NOTA-Ava-PSBP-6 were determined in a competitive radiometric PS-binding assay. Radiotracer 64Cu-NOTA-Ava-PSBP-6 was studied in camptothecin-induced apoptotic EL4 mouse lymphoma cells and in a murine EL4 tumor model of apoptosis using dynamic PET imaging. Peptide PSBP-6 was also conjugated via an Ava linker with fluorescein isothiocyanate (FITC). FITC-Ava-PSBP-6 was evaluated in flow cytometry and fluorescence confocal microscopy experiments. Radiopeptide 64Cu-NOTA-Ava-PSBP-6 was synthesized in high radiochemical yields of >95%. The IC50 values for PS-binding potency of PSBP-6, NOTA-Ava-PSBP-6, and natCu-NOTA-PSBP-6 were 600 µM, 30 µM, and 23 µM, respectively. A competitive radiometric cell binding assay confirmed binding of 64Cu-NOTA-Ava-PSBP-6 to camptothecin-induced apoptotic EL4 cells in a Ca2+-independent manner. PET imaging studies demonstrated significantly higher uptake of 64Cu-NOTA-Ava-PSBP-6 in apoptotic EL4 tumors (SUV5min 0.95 ± 0.04) compared to control tumors (SUV5min 0.74 ± 0.03). Flow cytometry studies showed significantly higher binding of FITC-Ava-PSBP-6 to EL4 cells treated with camptothecin compared to untreated cells. Fluorescence microscopy studies revealed that FITC-Ava-PSBP-6 was binding to cell membranes of early apoptotic cells, but was internalized in late apoptotic and necrotic cells. The present study showed that radiotracer 64Cu-NOTA-Ava-PSBP-6 holds promise as a first peptide-based PET imaging agent for molecular imaging of apoptosis. However, additional "fine-tuning" of 64Cu-NOTA-Ava-PSBP-6 is required to enhance PS-binding potency and in vivo stability to improve tumor uptake and retention.

Metabolically Stabilized (68)Ga-NOTA-Bombesin for PET Imaging of Prostate Cancer and Influence of Protease Inhibitor Phosphoramidon.

Peptide receptor-based targeted molecular imaging and therapy of cancer is on the current forefront of nuclear medicine preclinical research and clinical practice. The frequent overexpression of gastrin-releasing peptide (GRP) receptors in prostate cancer stimulated the development of radiolabeled bombesin derivatives as high affinity peptide ligands for selective targeting of the GRP receptor. In this study, we have evaluated a novel (68)Ga-labeled bombesin derivative for PET imaging of prostate cancer in vivo. In addition, we were interested in testing the recently proposed "serve-and-protect" strategy to improve metabolic stability of radiolabeled peptides in vivo and to enhance tumor uptake. GRP receptor targeting peptides NOTA-BBN2 and (nat)Ga-NOTA-BBN2 demonstrated a characteristic antagonistic profile and high binding affinity toward the GRP receptor in PC3 cells (IC50 4.6-8.2 nM). Radiolabeled peptide (68)Ga-NOTA-BBN2 was obtained from NOTA-BBN2 in radiochemical yields greater than 62% (decay-corrected). Total synthesis time was 35 min, including purification using solid-phase extraction. (68)Ga-NOTA-BBN2 exhibited favorable resistance against metabolic degradation by peptidases in vivo within the investigated time frame of 60 min. Interestingly, metabolic stability was not further enhanced in the presence of protease inhibitor phosphoramidon. Dynamic PET studies showed high tumor uptake in both PC3- and LNCaP-bearing BALB/c nude mice (SUV5min > 0.6; SUV60min > 0.5). Radiotracer (68)Ga-NOTA-BBN2 represents a novel radiometal-based bombesin derivative suitable for GRP receptor targeting in PC3 and LNCaP mouse xenografts. Further increase of metabolic stability in vivo and enhanced tumor uptake were not observed upon administration of protease inhibitor phosphoramidon. This led to the conclusion that the recently proposed "serve-and-protect" strategy may not be valid for peptides exhibiting favorable intrinsic metabolic stability in vivo.

Targeting lysyl oxidase for molecular imaging in breast cancer.

INTRODUCTION: Lysyl oxidase (LOX; ExPASy ENZYME entry: EC 1.4.3.13) and members of the LOX-like family, LOXL1-LOXL4, are copper-dependent enzymes that can modify proteins of the extracellular matrix. Expression of LOX is elevated in many human cancers, including breast cancer. LOX expression correlates with the level of tissue hypoxia, and it is known to play a critical role in breast cancer metastasis. The goal of the present study was to target LOX with (1) molecular probe fluorescent labeling to visualize LOX in vitro and (2) a radiolabeled peptide to target LOX in vivo in three different preclinical models of breast cancer. METHODS: Gene expression of all five members of the LOX family was analyzed at the transcript level via microarray analysis using tissue biopsy samples from 176 patients with breast cancer. An oligopeptide sequence (GGGDPKGGGGG) was selected as a substrate-based, LOX-targeting structure. The peptide was labeled with fluorescein isothiocyanate (FITC) for confocal microscopy experiments with the murine breast cancer cell line EMT-6. In vivo molecular imaging experiments were performed using a C-terminal amidated peptide, GGGDPKGGGGG, labeled with a short-lived positron emitter, fluorine-18 ((18)F), for positron emission tomography (PET) in three different breast cancer models: EMT6, MCF-7 and MDA-MB-231. The PET experiments were carried out in the presence or absence of ß-aminopropionitrile (BAPN), an irreversible inhibitor of LOX. RESULTS: Immunostaining experiments using a LOX-specific antibody on EMT-6 cells cultured under hypoxic conditions confirmed the elevation of LOX expression in these cells. An FITC-labeled oligopeptide, FITC-Ava-GGGDPKGGGGG-NH2, was found to be localized in different cellular compartments under these conditions. After injection of [(18)F]fluorobenzoate-GGGDPKGGGGG-NH2, radioactivity uptake was visible in all three breast cancer models in vivo. Tumor uptake was reduced by predosing the animals with 2 mg of BAPN 4 h or 24 h before injection of the radiotracer. CONCLUSIONS: The present data support further investigation into the development of LOX-binding radiolabeled peptides as molecular probes for molecular imaging of LOX expression in cancer.

Rerouting the metabolic pathway of (18)F-labeled peptides: the influence of prosthetic groups.

Current translational cancer research is directed to the development of high affinity peptide ligands for targeting neuropeptide receptors overexpressed in different types of cancer. Besides their desired high binding affinity to the receptor, the suitability of radiolabeled peptides as targeting vectors for molecular imaging and therapy depends on additional aspects such as high tumor-to-background ratio, favorable clearance pattern from nontarget tissue, and sufficient metabolic stability in vivo. This study reports how a switch from the prosthetic group, N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB), to 2-deoxy-2-[(18)F]fluoro-d-glucose ([(18)F]FDG) effects the metabolic pathway of an (18)F-labeled bombesin derivative, QWAV-Sar-H-FA01010-Tle-NH2. (18)F-Labeled bombesin derivatives represent potent peptide ligands for selective targeting of gastrin-releasing peptide (GRP) receptor-expressing prostate cancer. Radiosynthesis of (18)F-labeled bombesin analogues [(18)F]FBz-Ava-BBN2 and [(18)F]FDG-AOAc-BBN2 was achieved in good radiochemical yields of ~50% at a specific activity exceeding 40 GBq/µmol. Both nonradioactive compounds FBz-Ava-BBN2 and FDG-AOAc-BBN2 inhibited binding of [(125)I]Tyr(4)-bombesin(1-14) in PC3 cells with IC50 values of 9 and 16 nM, respectively, indicating high inhibitory potency. Influence of each prosthetic group was further investigated in PC3 mouse xenografts using dynamic small animal PET imaging. In comparison to [(18)F]FBz-Ava-BBN2, total tumor uptake levels were doubled after injection of [(18)F]FDG-AOAc-BBN2 while renal elimination was increased. Blood clearance and in vivo metabolic stability were similar for both compounds. The switch from [(18)F]SFB to [(18)F]FDG as the prosthetic group led to a significant reduction in lipophilicity which resulted in more favorable renal clearance and increased tumor uptake. The presented single step radiolabeling-glycosylation approach represents an innovative strategy for site-directed peptide labeling with the short-lived positron emitter (18)F while providing a favorable pharmacokinetic profile of (18)F-labeled peptides.