1 June 2008 , Number 3

For the third time in less than two years, Beirut has been turned into a battleground, with Hizbollah and the opposition taking over the city. The government and its allies did not engage militarily, managing to avoid civil war and becoming a regional battlefield. The Arab League is now trying to sort things out, highlighting the regional and international dimensions.

Research Event

Event participants

Fadi El-Jardali, Professor of Health Policy and Systems, American University of Beirut
Moderator: Nadim Houry, Executive Director, Arab Reform Initiative

Members Event Webinar

Event participants

Dr Munzer al-Khalil, Head, Idlib Health Directorate
Raed Al Saleh, Director, Syria Civil Defence (The White Helmets)
Alaa Rajaa Mughrabieh, Child Protection Officer, Hurras Network
Chair: Dr Lina Khatib, Director, Middle East and North Africa Programme, Chatham House

7 May 2020 , Volume 96, Number 3

Insulin-like growth factors (IGFs), specifically IGF1 and IGF2, promote glucose metabolism, with their availability regulated by IGF-binding proteins (IGFBPs). We hypothesized that IGF1 and IGF2 levels, or their bioavailability, are reduced during type 1 diabetes development. Total serum IGF1, IGF2, and IGFBP1–7 levels were measured in an age-matched, cross-sectional cohort at varying stages of progression to type 1 diabetes. IGF1 and IGF2 levels were significantly lower in autoantibody (AAb)⁺ compared with AAb^– relatives of subjects with type 1 diabetes. Most high-affinity IGFBPs were unchanged in individuals with pre–type 1 diabetes, suggesting that total IGF levels may reflect bioactivity. We also measured serum IGFs from a cohort of fasted subjects with type 1 diabetes. IGF1 levels significantly decreased with disease duration, in parallel with declining β-cell function. Additionally, plasma IGF levels were assessed in an AAb⁺ cohort monthly for a year. IGF1 and IGF2 showed longitudinal stability in single AAb⁺ subjects, but IGF1 levels decreased over time in subjects with multiple AAb and those who progressed to type 1 diabetes, particularly postdiagnosis. In sum, IGFs are dysregulated both before and after the clinical diagnosis of type 1 diabetes and may serve as novel biomarkers to improve disease prediction.

Sodium–glucose cotransport 2 inhibitors (SGLT2i) lower plasma glucose but stimulate endogenous glucose production (EGP). The current study examined the effect of dapagliflozin on EGP while clamping plasma glucose, insulin, and glucagon concentrations at their fasting level. Thirty-eight patients with type 2 diabetes received an 8-h measurement of EGP ([3-³H]-glucose) on three occasions. After a 3-h tracer equilibration, subjects received 1) dapagliflozin 10 mg (n = 26) or placebo (n = 12); 2) repeat EGP measurement with the plasma glucose concentration clamped at the fasting level; and 3) repeat EGP measurement with inhibition of insulin and glucagon secretion with somatostatin infusion and replacement of basal plasma insulin and glucagon concentrations. In study 1, the change in EGP (baseline to last hour of EGP measurement) in subjects receiving dapagliflozin was 22% greater (+0.66 ± 0.11 mg/kg/min, P < 0.05) than in subjects receiving placebo, and it was associated with a significant increase in plasma glucagon and a decrease in the plasma insulin concentration compared with placebo. Under glucose clamp conditions (study 2), the change in plasma insulin and glucagon concentrations was comparable in subjects receiving dapagliflozin and placebo, yet the difference in EGP between dapagliflozin and placebo persisted (+0.71 ± 0.13 mg/kg/min, P < 0.01). Under pancreatic clamp conditions (study 3), dapagliflozin produced an initial large decrease in EGP (8% below placebo), followed by a progressive increase in EGP that was 10.6% greater than placebo during the last hour. Collectively, these results indicate that 1) the changes in plasma insulin and glucagon concentration after SGLT2i administration are secondary to the decrease in plasma glucose concentration, and 2) the dapagliflozin-induced increase in EGP cannot be explained by the increase in plasma glucagon or decrease in plasma insulin or glucose concentrations.

Rationale: Lymphatic tracers can help visualize the lymphatic drainage patterns and sentinel nodes of individual prostate cancer patients. To determine the role of nuclear medicine, in particular the positional guidance of a SPECT/CT-based 3D imaging roadmap, in this process we studied to which extend fluorescence-guidance underestimated the number of target lesions. Methods: SPECT/CT imaging was performed after intraprostatic tracer administration of either ICG-^99mTc-nanocolloid (hybrid tracer group) or ^99mTc-nanocolloid to create a roadmap that depicted all sentinel nodes (SNs). Patients who received ^99mTc-nanocolloid were injected with "free" ICG immediately prior to surgery ("free" ICG group). Before unblinding, fluorescence-guidance was used for intraoperative SN identification. This was followed by extended pelvic lymph node dissection (ePLND). Following unblinding of the SPECT/CT images, the number of missed SN’s were recorded and their resection was pursued when the anatomy allowed. Results: Preoperative SPECT/CT revealed no differences in the SN identification rate between ICG-^99mTc-nanocolloid and ^99mTc-nanocolloid. However, fluorescence-guidance only allowed intraoperative removal of all SNs in 40% of patients in the hybrid tracer group and in 20% of patients in the "free" ICG group. Overall, 75.9% of the intraoperatively resected SNs in the hybrid tracer group and 51.8% of the SNs in the "free" ICG group were removed solely under fluorescence-guidance. During ePLND 22 additional SNs were resected (7 in the hybrid tracer group and 15 in the "free" ICG group). After unblinding 18 remaining SNs were identified (6 in the hybrid group and 12 in the "free" ICG group). In the "free" ICG group, ex vivo evaluation of the excised specimens revealed that 14 SNs removed under ePLND or after unblinding contained radioactivity but no fluorescence. Conclusion: The preoperative imaging roadmap provided by SPECT/CT enhanced the detection of prostate SNs in more ectopic locations in 17 of the 25 patients and the hybrid tracer ICG-^99mTc-nanocolloid was shown to outperform "free" ICG. Overall, fluorescence-guided pelvic nodal surgery underestimated the number of SNs in 60-80% of patients.

Calculation of radiation dosimetry in targeted nuclear medicine therapies is traditionally resource-intensive requiring multiple post-therapy SPECT acquisitions. An alternative approach is to take advantage of existing pharmacokinetic data from these smaller cohorts to enable dose computation from a single post-treatment scan in a manner that may be applied to a much broader patient population. Methods: In this work, a technical description for simplified dose estimation is presented and applied to assessment of ¹⁷⁷Lu-PSMA-617 therapy (Prostate-Specific Membrane Antigen) for metastatic prostate cancer. By normalizing existing time-activity curves to a single measurement time, it is possible to calculate a mean and range of time-integrated activity values which relate to radiation absorbed dose. To assist with accurate pharmacokinetic modelling of the training cohort, a method for contour-guided image registration was developed. Results: Tissue-specific dose conversion factors for common post-treatment imaging times are reported along with a characterization of added uncertainty in comparison to a traditional serial imaging protocol. Single time point dose factors for tumor were determined to be 11.0, 12.1, 13.6, and 15.2 Gy per MBq/mL at image times of 24, 48, 72, and 96 hours, respectively. For normal tissues, parotid gland factors were 6.7, 9.4, 13.3, and 19.3 Gy per MBq/mL and kidneys were 7.1, 10.3, 15.0, and 22.0 Gy per MBq/mL at those times. Tumor dose estimates were most accurate using delayed scanning at times beyond 72 hours. Dose to healthy tissues is best characterized by scanning patients in the first two days of treatment owing to the larger degree of tracer clearance in this early phase. Conclusion: The work demonstrates a means for efficient dose estimation in ¹⁷⁷Lu-PSMA-617 therapy. By providing methods to simplify and potentially automate radiation dosimetry we hope to accelerate the understanding of radiobiology and development of dose-response models in this unique therapeutic context.

Introduction: A new pattern of response, so-called hyper-progressive disease (HPD), is emerging during treatment with immune checkpoint inhibitors (ICI). Our aim was to investigate the prevalence of such phenomenon and to assess its association with clinical variables and metabolic parameters by ¹⁸F-fludeoxyglucose positron emission tomography/computed tomography (¹⁸F-FDG PET/CT). Methods: Data from 50 patients (34 male, 16 female, median age 73) with non-small cell lung carcinoma (NSCLC) and treated with ICI were prospectively collected. All patients underwent contrast-enhanced CT, ¹⁸F-FDG PET/CT, and complete peripheral blood sample at baseline before ICI. HPD was defined according to clinical and radiologic criteria. Because of the rapid disease progression or worsening of clinic conditions, radiologic response assessment was available for 46 patients. OS were analyzed using the Kaplan–Meier method and the log-rank test. A Cox proportional hazards regression analysis was used to evaluate factors independently associated with OS. Median follow-up was 12.4 months (9.7-15.2 months). Results: We identified the following response categories: 10 cases as complete/partial response (CR/PR), 17 cases with stable disease (SD), 5 patients with progressive disease (PD), and 14 with HPD. Among metabolic parameters we observed a statistically significant association between HPD status and tumor burden, expressed by both MTV (756.1ml for HPD vs 475.6ml for non-HPD, P = 0.011) and TLG (287.3 for HPD vs 62.1 for non-HPD, P = 0.042). Among clinical variables, 12/14 patients (85.7%) within the HPD group compared with 8/32 patients (25%) in the non-HDP group had more than two metastatic sites (p<0.001). In addition, the derived neutrophil-to-lymphocyte ratio (dNLR) and platelet counts was significantly associated with HPD status (P = 0.038, P = 0.025, respectively). Survival analysis showed a median OS of 4 months for HPD group compared with 15 months within non-HPD patients (P = 0.003). Likewise, median OS was significantly different when we considered all the response categories: CR/PR, SD, PD, and HPD (P = 0.001). Finally, Multivariate analysis identified MTV and dNLR as independent predictors for OS. Conclusion: Our results suggest that the use of ICI might represent a concern in patients with high metabolic tumor burden and inflammatory indexes at baseline. However Additional studies are needed.

Rationale: The treatment of choice for insulinomas and focal lesions in congenital hyperinsulinism (CHI) is surgery. However, intra-operative detection can be challenging. This could be overcome with intra-operative fluorescence imaging, which provides real-time lesion detection with a high spatial resolution. Here, a novel method for targeted near-infrared (NIR) fluorescence imaging of glucagon-like peptide 1 receptor (GLP-1R) positive lesions, using the GLP-1 agonist exendin-4, labeled with IRDye800CW, was examined in vitro and in vivo. Methods: A competitive binding assay was performed using Chinese hamster lung (CHL) cells transfected with the GLP-1R. Tracer biodistribution was determined in BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts. In vivo NIR fluorescence imaging of CHL-GLP-1R xenografts was performed. Localization of the tracer in the pancreatic islets of BALB/c nude mice was examined using fluorescence microscopy. Laparoscopic imaging was performed to detect the fluorescent signal of the tracer in the pancreas of mini pigs. Results: Exendin-4-IRDye800CW binds the GLP-1R with an IC50 value of 3.96 nM. The tracer accumulates in CHL-GLP-1R xenografts. Subcutaneous CHL-GLP-1R xenografts were visualized using in vivo NIR fluorescence imaging. The tracer accumulates specifically in the pancreatic islets of mice and a clear fluorescent signal was detected in the pancreas of mini pigs. Conclusion: These date provide the first in vivo evidence of the feasibility of targeted fluorescence imaging of GLP-1R positive lesions. Intra-operative lesion delineation using exendin-4-IRDye800CW could benefit open as well as laparoscopic surgical procedures for removal of insulinomas and focal lesions in CHI.

[S-Methyl-¹¹C](±)-7-methoxy-3-(4-(4-(methylthio)phenyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol (¹¹C-NR2B-SMe) and its enantiomers were synthesized as candidates for imaging the NR2B subunit within the N-methyl-D-aspartate receptor with positron emission tomography (PET). Methods: Brains were scanned with PET for 90 min after intravenous injection of one of the candidate radioligands into rats. To detect any NR2B specific binding of radioligand in brain, various pre-blocking or displacing agents were evaluated for their impact on the PET brain imaging data. Radiometabolites from brain and other tissues were measured ex vivo and in vitro. Results: Each radioligand gave high early whole brain uptake of radioactivity, followed by a brief fast decline and then a slow final decline. ¹¹C-(S)-NR2B-SMe was studied extensively. Ex vivo measurements showed that radioactivity in rat brain at 30 min after radioligand injection was virtually unchanged radioligand. Only less lipophilic radiometabolites appeared in plasma. High-affinity NR2B ligands, Ro-25-6981, ifenprodil, and CO10124, showed increasing preblock of whole brain radioactivity retention with increasing dose (0.01 to 1.25 mg/kg, i.v.). Five 1 antagonists (FTC146, BD1407, F3, F4, and NE100) and four 1 agonists ((+)-pentazocine, (±)-PPCC, PRE-084, (+)-SKF10047) were ineffective preblocking agents, except FTC146 and F4 at high dose. Two potent 1 receptor agonists, TC1 and SA4503, showed dose-dependent preblocking effects in the presence or absence of pharmacological 1 receptor blockade with FTC146. Conclusion: ¹¹C-(S)-NR2B-SMe has adequate NR2B-specific PET signal in rat brain to warrant further evaluation in higher species. TC1 and SA4503 likely have off-target binding to NR2B in vivo.

Methods that provide rapid access to radiolabeled antibodies are vital in the development of diagnostic and radiotherapeutic agents for positron emission tomography (PET) or radioimmunotherapy. The human hepatocyte growth factor receptor (c-MET) signaling pathway is dysregulated in a number of malignancies including gastric cancer, and is an important biomarker in drug discovery. Here, we used a photoradiochemical approach to produce ⁸⁹Zr-radiolabeled onartuzumab (a monovalent, anti-human c-MET antibody), starting directly from the fully formulated drug (MetMAb). Methods: Simultaneous ⁸⁹Zr-radiolabeling and protein conjugation was performed in one-pot reactions containing ⁸⁹Zr-oxalate, the photoactive chelate DFO-aryl azide (DFO-ArN3) and MetMAb to give ⁸⁹ZrDFO-azepin-onartuzumab. As a control, ⁸⁹ZrDFO-Bn-NCS-onartuzumab was prepared via a conventional two-step process using pre-purified onartuzumab and DFO-Bn-NCS. Radiotracers were purified by using size-exclusion methods and evaluated by radiochromatography. Radiochemical stability was studied in human serum and immunoreactivity was determined by cellular binding assays using MKN-45 gastric carcinoma cells. PET imaging at multiple time points (0–72 h) was performed in female athymic nude mice bearing subcutaneous MKN-45 xenografts. Biodistribution experiments were performed after the final image. Tumor specificity of ⁸⁹ZrDFO-azepin-onartuzumab was assessed by competitive inhibition (blocking) studies. Results: Initial photoradiosynthesis experiments produced ⁸⁹ZrDFO-azepin-onartuzumab in <15 min. with an isolated decay-corrected radiochemical yield (RCY) of 24.8%, a radiochemical purity (RCP) ~90% and a molar activity (Am) of ~1.5 MBq nmol^-1. Reaction optimization improved the radiochemical conversion (RCC) of ⁸⁹ZrDFO-azepin-onartuzumab to 56.9±4.1% (n = 3), with isolated RCYs of 41.2±10.6% (n = 3), and RCPs >90%. Conventional methods produced ⁸⁹ZrDFO-Bn-NCS-onartuzumab with isolated RCY >97%, RCP >97% and Am ~14.0 MBq nmol^-1. Both radiotracers were immunoreactive and stable in human serum. PET imaging and biodistribution studies showed high tumor uptake for both radiotracers. By 72 h, tumor and liver uptake reached 15.37±5.21 %ID g^-1, 6.56±4.03 %ID g^-1, respectively for ⁸⁹ZrDFO-azepin-onartuzumab (n = 4), and 21.38±11.57 %ID g^-1 and 18.84±6.03 %ID g^-1 for ⁸⁹ZrDFO-Bn-NCS-onartuzumab (n = 4). Blocking experiments gave a statistically significant reduction in tumor uptake (6.34±0.47 %ID g^-1) of ⁸⁹ZrDFO-azepin-onartuzumab (n = 4). Conclusion: Experiments demonstrate that photoradiosynthesis is a viable alternative approach for producing ⁸⁹Zr-radiolabeled antibodies direct in protein formulation buffer which reduces protein aggregation and liver uptake.

Objective: To develop a novel approach to generate individual maps of white matter (WM) innate immune cell activation using ¹⁸F-DPA-714 translocator protein (TSPO) positron emission tomography (PET), and to explore the relationship between these maps and individual trajectories of disability worsening in patients with multiple sclerosis (MS). Methods: Patients with MS (n = 37), whose trajectories of disability worsening over the 2 years preceding study entry were calculated, and healthy controls (n = 19) underwent magnetic resonance magnetic and ¹⁸F-DPA-714 PET. A threshold of significant activation of ¹⁸F-DPA-714 binding was calculated with a voxel-wise randomized permutation-based comparison between patients and controls, and used to classify each WM voxel in patients as characterized by a significant activation of innate immune cells (DPA+) or not. Individual maps of innate immune cell activation in the WM were employed to calculate the extent of activation in WM regions-of-interests and to classify each WM lesion as "DPA-active", "DPA-inactive" or "unclassified". Results: Compared with the WM of healthy controls, patients with MS had a significantly higher percentage of DPA+ voxels in the normal-appearing WM, (NAWM in patients=24.9±9.7%; WM in controls=14.0±7.8%, p<0.001). In patients with MS, the percentage of DPA+ voxels showed a significant increase from NAWM, to perilesional areas, T2 hyperintense lesions and T1 hypointense lesions (38.1±13.5%, 45.0±17.9%, and 51.9±22.9%, respectively, p<0.001). Among the 1379 T2 lesions identified, 512 were defined as DPA-active and 258 as DPA-inactive. A higher number of lesions classified as DPA-active (OR=1.13, P = 0.009), a higher percentage of DPA+ voxels in the NAWM (OR=1.16, P = 0.009) and in T1-spin-echo lesions (OR=1.06, P = 0.036), were significantly associated with a retrospective more severe clinical trajectory in patients with MS. Conclusion: A more severe trajectory of disability worsening in MS is associated with an innate immune cells activation inside and around WM lesions. ¹⁸F-DPA-714 PET may provide a promising biomarker to identify patients at risk of severe clinical trajectory.

Respiratory motion during the CT and PET parts of a PET/CT scan leads to imperfect alignment of anatomical features seen by the two modalities. In this work, we concentrate on the effects of motion during CT. We propose a novel approach for improving the alignment. Methods: Respiratory waveform data were gathered during the CT and PET parts of 28 PET/CT scans of cancer patients with 40 lesions up to 3 cm size in the lung or upper abdomen. PET list-mode data were reconstructed by three reconstruction methods: PET/static, PET/EX or end of expiration (OncoFreeze), and a novel PET/matched method that used both waveforms. The three methods were compared. The distance between tumor positions in PET and CT were characterized in visual interpretation by physicians as well as quantitatively. Tumor standardized uptake values (SUV_max and SUVpeak) were determined relative to SUV based on the static method. Image noise was evaluated in the liver and compared to PET/static. Results: In visual interpretation, the rate of good alignment was 13/21, 13/23 and 18/21 for PET/static, PET/EX and PET/matched methods, respectively, and the mean PET-CT distances were 3.5, 5.1 and 2.8 mm. In visual comparison with PET/EX, the rate of good alignment was increased in 1/10 and 7/10 cases for PET/static and PET/matched. SUV_max was on average 21% higher than PET/static when either PET/EX or PET/matched was used. SUVpeak was 12% higher. Image noise in the liver was 15% higher than static for the PET/EX method, and 40% higher for PET/matched; that is, noise was much lower than in gated PET. Conclusion: Acquiring respiratory waveforms both in PET (as in the current state of the art) and in CT (an unusual key step in this approach) has the potential to improve the alignment of PET and CT images. A proposed method for using this information was tested. Improved alignment was demonstrated.

Studies demonstrate that the investigational ⁶⁴Cu-DOTATATE radiopharmaceutical may provide diagnostic and logistical benefits over available imaging agents for patients with somatostatin receptor (SSTR)-positive neuroendocrine tumors (NETs). Accordingly, we aimed to prospectively determine the lowest dose of ⁶⁴Cu-DOTATATE that facilitates diagnostic quality scans and evaluated the diagnostic performance and safety in a phase III study of patients with SSTR-expressing NETs. Methods: A dose-ranging study was conducted in 12 patients divided into 3 dose groups (111 MBq [3.0 mCi], 148 MBq [4.0 mCi], and 185 MBq [5.0 mCi] ± 10%) to determine the lowest dose of ⁶⁴Cu-DOTATATE that produced diagnostic quality PET/CT images. Using the ⁶⁴Cu-DOTATATE dose identified in the dose-ranging study, 3 independent nuclear medicine physicians who were blinded to all clinical information read PET/CT scans from 21 healthy volunteers and 42 NET-positive patients to determine those with "Disease" and "No Disease," as well as "Localized" versus "Metastatic" status. Blinded-reader evaluations were compared to a patient-specific standard of truth (SOT), which was established by an independent oncologist who used all previously available pathology, clinical, and conventional imaging data. Diagnostic performance calculated for ⁶⁴Cu-DOTATATE included sensitivity, specificity, negative predictive value, positive predictive value, and accuracy. Inter- and intra-reader reliability, as well as ability to differentiate between localized and metastatic disease, was also determined. Adverse events (AEs) were recorded from ⁶⁴Cu-DOTATATE injection through 48 hours post-injection. Results: The dose-ranging study identified 148 MBq (4.0 mCi) as the optimal dose to obtain diagnostic quality PET/CT images. Following database lock, diagnostic performance from an initial majority read of the 3 independent readers showed a significant 90.9% sensitivity (P = 0.0042) and 96.6% specificity (P < 0.0001) for detecting NETs, which translated to a 100.0% sensitivity and 96.8% specificity after correcting for an initial SOT misread. Excellent inter- and intra-reader reliability, as well as ability to distinguish between localized and metastatic disease, was also noted. No AEs were related to ⁶⁴Cu-DOTATATE, and no serious AEs were observed. Conclusion: ⁶⁴Cu-DOTATATE PET/CT is a safe imaging technique that provides high-quality and accurate images at a dose of 148 MBq (4.0 mCi) for the detection of somatostatin-expressing NETs.

Peptide receptor radiotherapy using ¹⁷⁷Lu-labeled somatostatin ligand analogs is a well-established treatment for neuroendocrine tumors (NET), with ¹⁷⁷Lu-DOTATATE having acquired marketing authorization in Europe and the USA. The investigation of the pharmacokinetics of those radiopharmaceuticals in vivo in humans is crucial for personalized treatment management and understanding of treatment effects. It requires input data on the in vivo stability of the radiopharmaceuticals in blood and plasma. The work presented here is devoted to the investigation of in vivo stability of ¹⁷⁷Lu-DOTATATE in humans affected by NET. Unexpectedly, fast metabolism of the radiopharmaceutical was observed, with fraction of intact ¹⁷⁷Lu-DOTATATE in plasma decreasing rapidly to 23±5% (mean ± SD) at 24 h and 1.7±0.9% at 96 h after injection.

Objectives: Esthesioneuroblastoma (ENB) is rare with limited therapeutic options when unresectable or metastatic; however, expression of somatostatin receptors qualifies it for peptide receptor radionuclide therapy (PRRT). We report outcomes of PRRT in ENB from two referral centers. Methods: Using PRRT databases at two European Neuroendocrine Tumour Society Centers of Excellence, case finding was undertaken between 2004-2018 for patients who had PRRT with recurrent/metastatic ENB deemed unsuitable for further conventional therapies. Evaluations of response using a composite reference standard and for survival were performed. Results: Of seven patients, four had partial response, two had disease stabilization and one had early progression. Possible side effects include worsening CSF-leaks. Median progression-free survival was 17 months (range, 0-30), and median overall survival was 32 months (range, 4–53). Conclusion: PRRT shows promising efficacy and moderate survival duration in unresectable locally advanced or metastatic ENB warranting larger cohort studies incorporating measures of quality of life.

Rationale: The effects of tobacco smoking on the brain’s immune system are not well elucidated. While nicotine is immunosuppressive, other constituents in tobacco smoke have inflammatory effects. Positron Emission Tomography (PET) imaging of the 18-kDa translocator protein (TSPO) provide a biomarker for microglia, the brain’s primary immunocompetent cells. This work compared brain TSPO levels in 20 tobacco smokers (abstinent for at least 2 hours) and 20 nonsmokers using a fully quantitative modeling approach for the first time. Methods: [¹¹C]PBR28 PET scans were acquired with arterial blood sampling to estimate the metabolite-corrected input function. [¹¹C]PBR28 volumes of distribution (V_T) were estimated throughout the brain with multilinear analysis. Results: Statistical analyses revealed no evidence for significant differences in regional [¹¹C]PBR28 V_T between smokers and non-smokers (whole-brain Cohen’s d=0.09) despite adequate power to detect medium effect sizes. Conclusion: These findings inform previous PET studies reporting lower TSPO radiotracer concentrations in brain (measured as standardized uptake value, SUV) of tobacco smokers compared to nonsmokers by demonstrating the importance of accounting for radiotracer concentrations in plasma. These findings suggest that compared to nonsmokers, smokers have comparable TSPO levels in brain. Additional work with other biomarkers is needed to fully characterize effects of tobacco smoking on the brain’s immune system.

Introduction: To use positron emission tomography coupled with computed tomography (¹⁸FDG-PET/CT) to identify a high-risk subgroup requiring therapeutic intensification among patients with locally advanced cervical cancer (LACC) and para-aortic lymph node (PALN) involvement. Methods: In this retrospective multicentric study, patients with LACC and PALN involvement concurrently treated with chemoradiotherapy and extended-field radiotherapy (EFR) between 2006 and 2016 were included. A senior nuclear medicine specialist in PET for gynaecologic oncology reviewed all ¹⁸FDG-PET/CT scans. Metabolic parameters including maximum standardised uptake value (SUV_max), metabolic tumour volume (MTV) and total lesion glycolysis (TLG) were determined for the primary tumour, pelvic lymph nodes and PALN. Associations between these parameters and overall survival (OS) were assessed with Cox's proportional hazards model. Results: Sixty-eight patients were enrolled in the study. Three-year OS was 55.5% (95% CI (40.8-68.0)). When adjusted for age, stage and histology, pelvic lymph node TLG, PALN TLG and PALN SUV_max were significantly associated with OS (p<0.005). Conclusion: FDG-PET/CT was able to identify predictors of survival in the homogeneous subgroup of patients with LACC and PALN involvement, thus allowing therapeutic intensification to be proposed.

Targeting tumor-expressed receptors using selective molecules for diagnostic, therapeutic or both diagnostic and therapeutic (theragnostic) purposes is a promising approach in oncological applications. Such approaches have increased significantly over the past decade. Peptides such as gastrin-releasing peptide receptors (GRPR) targeting radiopharmaceuticals are small molecules with fast blood clearance and urinary excretion. They demonstrate good tissue diffusion, low immunogenicity, and highly selective binding to their target cell-surface receptors. They are also easily produced. GRPR, part of the bombesin (BBN) family, are overexpressed in many tumors, including breast and prostate cancer, and therefore represent an attractive target for future development.

Respiratory gating is the standard to overcome respiration effects degrading image quality in positron emission tomography (PET). Data-driven gating (DDG) using signals derived from PET raw data are promising alternatives to gating approaches requiring additional hardware. However, continuous bed motion (CBM) scans require dedicated DDG approaches for axially-extended PET, compared to DDG for conventional step-and-shoot scans. In this study, a CBM-capable DDG algorithm was investigated in a clinical cohort, comparing it to hardware-based gating using gated and fully motion-corrected reconstructions. Methods: 56 patients with suspected malignancies in thorax or abdomen underwent whole-body ¹⁸F-FDG CBM-PET/CT imaging using DDG and hardware-based respiratory gating (pressure-sensitive belt gating, BG). Correlation analyses were performed on both gating signals. Besides static reconstructions, BG and DDG were used for optimally-gated PET (BG-OG, DDG-OG) and fully motion-corrected PET (elastic motion correction; BG-EMOCO, DDG-EMOCO). Metabolic volumes, SUV_max and SUVmean of lesions were compared amongst the reconstructions. Additionally, the quality of lesion delineation in different PET reconstructions was independently evaluated by three experts. Results: Global correlation coefficients between BG and DDG signals amounted to 0.48±0.11, peaking at 0.89±0.07 when scanning the kidney and liver region. In total, 196 lesions were analyzed. SUV measurements were significantly higher in BG-OG, DDG-OG, BG-EMOCO and DDG-EMOCO compared to static images (P<0.001; median SUV_max: static, 14.3±13.4; BG-EMOCO, 19.8±15.7; DDG-EMOCO, 20.5±15.6; BG-OG, 19.6±17.1; DDG-OG, 18.9±16.6). No significant differences between BG-OG and DDG-OG, and BG-EMOCO and DDG-EMOCO, respectively, were found. Visual lesion delineation was significantly better in BG-EMOCO and DDG-EMOCO than in static reconstructions (P<0.001); no significant difference was found comparing BG and DDG (EMOCO, OG, respectively). Conclusion: DDG-based motion-compensation of CBM-PET acquisitions outperforms static reconstructions, delivering qualities comparable to hardware-based approaches. The new algorithm may be a valuable alternative for CBM-PET systems.

Depth-encoding detectors with single-ended readout provide a practical, cost-effective approach for constructing high resolution and high sensitivity PET scanners. However, the current iteration of such detectors utilizes a uniform glass light guide to achieve depth-encoding, resulting in non-uniform performance throughout the detector array due to suboptimal intercrystal light sharing. We introduce Prism-PET, a single-ended readout PET detector module with a segmented light guide composed of an array of prismatoids that introduces enhanced, deterministic light sharing. Methods: High resolution PET detector modules were fabricated with single-ended readout of polished multicrystal lutetium yttrium orthosilicate (LYSO) scintillator arrays directly coupled 4-to-1 and 9-to-1 to arrays of 3.2 x 3.2 mm² silicon photomultiplier pixels. Each scintillator array was coupled at the non-readout side to a light guide (one 4-to-1 module with a uniform glass light guide, one 4-to-1 Prism-PET module and one 9-to-1 Prism-PET module) to introduce intercrystal light sharing, which closely mimics the behavior of dual-ended readout with the additional benefit of improved crystal identification. Flood histogram data was acquired using a 3 MBq Na-22 source to characterize crystal identification and energy resolution. Lead collimation was used to acquire data at specific depths to determine depth-of-interaction (DOI) resolution. Results: The flood histogram measurements showed excellent and uniform crystal separation throughout the Prism-PET modules while the uniform glass light guide module had performance degradation at the edges and corners. A DOI resolution of 5.0 mm full width at half maximum (FWHM) and energy resolution of 13% were obtained in the uniform glass light guide module. By comparison, the 4-to-1 coupled Prism-PET module achieved 2.5 mm FWHM DOI resolution and 9% energy resolution. Conclusion: PET scanners based on our Prism-PET modules with segmented prismatoid light guide arrays can achieve high and uniform spatial resolution (9-to-1 coupling with ~ 1 mm crystals), high sensitivity, good energy and timing resolutions (using polished crystals and after applying DOI-correction), and compact size (depth-encoding eliminates parallax error and permits smaller ring-diameter).

The kappa opioid receptor (KOR) is implicated in various neuropsychiatric disorders. We previously evaluated an agonist tracer, ¹¹C-GR103545, for PET imaging of KOR in humans. Although ¹¹C-GR103545 showed high brain uptake, good binding specificity, and selectivity to KOR, it displayed slow kinetics and relatively large test-retest variability (TRV) of distribution volume (V_T) estimates (15%). Therefore we set out to develop two novel KOR agonist radiotracers, ¹¹C-EKAP and ¹¹C-FEKAP, and in nonhuman primates, both tracers exhibited faster kinetics and comparable binding parameters to ¹¹C-GR103545. The aim of this study was to assess their kinetic and binding properties in humans. Methods: Six healthy subjects underwent 120-min test-retest PET scans with both ¹¹C-EKAP and ¹¹C-FEKAP. Metabolite-corrected arterial input functions were measured. Regional time-activity curves (TACs) were generated for 14 regions of interest. One- and two-tissue compartment models (1TC, 2TC) and the multilinear analysis-1 (MA1) method were applied to the regional TACs to calculate V_T. Time-stability of V_T values and test-retest reproducibility were evaluated. Levels of specific binding, as measured by the non-displaceable binding potential (BP_ND) for the three tracers (¹¹C-EKAP, ¹¹C-FEKAP and ¹¹C-GR103545), were compared using a graphical method. Results: For both tracers, regional TACs were fitted well with the 2TC model and MA1 method (t*=20min), but not with the 1TC model. Given unreliably estimated parameters in several fits with the 2TC model and a good match between V_T values from MA1 and 2TC, MA1 was chosen as the appropriate model for both tracers. Mean MA1 V_T values were highest for ¹¹C-GR103545, followed by ¹¹C-EKAP, then ¹¹C-FEKAP. Minimum scan time for stable V_T measurement was 90 and 110min for ¹¹C-EKAP and ¹¹C-FEKAP, respectively, compared with 140min for ¹¹C-GR103545. The mean absolute TRV in MA1 V_T estimates was 7% and 18% for ¹¹C-EKAP and ¹¹C-FEKAP, respectively. BP_ND levels were similar for ¹¹C-FEKAP and ¹¹C-GR103545, but ~25% lower for ¹¹C-EKAP. Conclusion: The two novel KOR agonist tracers showed faster tissue kinetics than ¹¹C-GR103545. Even with slightly lower BP_ND, ¹¹C-EKAP is judged to be a better tracer for imaging and quantification of KOR in humans, based on the shorter minimum scan time and excellent test-retest.

The objective of this retrospective study was to determine the role of ¹⁸F-FDG PET/CT in a large cohort of 495 patients with metastatic neuroendocrine neoplasms (NENs) who were treated with peptide receptor radionuclide therapy (PRRT) with a long-term follow-up. Methods: The 495 patients were treated with ¹⁷⁷Lu- and/or ⁹⁰Y- DOTATOC/DOTATATE PRRT between 2/2002 and 7/2018. All subjects received both ⁶⁸Ga-DOTATOC/TATE/NOC and ¹⁸F-FDG PET/CT prior to treatment and were followed 3-189 months. Kaplan-Meier analysis, log-rank test (Mantel-Cox), and Cox regression analysis were performed for overall survival (OS) and progression-free survival (PFS). Results: 199 patients (40.2%) presented with pancreatic NEN, 49 with CUP (cancer of unknown primary), 139 with midgut NEN, whereas the primary tumor was present in the rectum in 20, in the lung in 38, in the stomach in 8 and other locations in 42 patients. FDG-PET/CT was positive in 382 (77.2%) patients and 113 (22.8%) were FDG-negative before PRRT, while 100% were ⁶⁸Ga-DOTATOC/TATE/NOC positive. For all patients, the median PFS and OS, defined from start of PRRT, were 19.6 mo and 58.7 mo, respectively. Positive FDG predicted shorter PFS (18.5 mo vs 24.1 mo; P = 0.0015) and OS (53.2 mo vs 83.1 mo; P < 0.001) than negative FDG. Amongst the pancreatic NEN, the median OS was 52.8 mo in FDG positive and 114.3 mo in FDG negative subjects (P = 0.0006). For all patients with positive ¹⁸F-FDG uptake, and a ratio of the highest SUV_max on ⁶⁸Ga-SSTR PET to the most ¹⁸F-FDG-avid tumor lesions >2, the median OS was 53.0 mo, compared to 43.4 mo in those patients with a ratio <2 (P = 0.030). For patients with no ¹⁸F-FDG uptake (complete "mismatch" imaging pattern), the median OS was 108.3 mo vs 76.9 mo for SUV_max >15.0 and ≤15.0 on ⁶⁸Ga-SSTR PET/CT, respectively. Conclusion: The presence of positive lesions on ¹⁸F-FDG PET is an independent prognostic factor in patients with NEN treated with PRRT. Metabolic imaging with ¹⁸F-FDG PET/CT compliments the molecular imaging aspect of ⁶⁸Ga-SSTR PET/CT for the prognosis of survival after PRRT. High SSTR expression combined with negative ¹⁸F-FDG PET/CT imaging is associated with the most favorable long-term prognosis.

A data-driven method for respiratory gating in PET has recently been commercially developed. We sought to compare the performance of the algorithm to an external, device-based system for oncological [¹⁸F]-FDG PET/CT imaging. Methods: 144 whole-body [¹⁸F]-FDG PET/CT examinations were acquired using a Discovery D690 or D710 PET/CT scanner (GE Healthcare), with a respiratory gating waveform recorded by an external, device based respiratory gating system. In each examination, two of the bed positions covering the liver and lung bases were acquired with duration of 6 minutes. Quiescent period gating retaining ~50% of coincidences was then able to produce images with an effective duration of 3 minutes for these two bed positions, matching the other bed positions. For each exam, 4 reconstructions were performed and compared: data driven gating (DDG-retro), external device-based gating (RPM Gated), no gating but using only the first 3 minutes of data (Ungated Matched), and no gating retaining all coincidences (Ungated Full). Lesions in the images were quantified and image quality was scored by a radiologist, blinded to the method of data processing. Results: The use of DDG-retro was found to increase SUVmax and to decrease the threshold-defined lesion volume in comparison to each of the other reconstruction options. Compared to RPM-gated, DDG-retro gave an average increase in SUV_max of 0.66 ± 0.1 g/mL (n=87, p<0.0005). Although results from the blinded image evaluation were most commonly equivalent, DDG-retro was preferred over RPM gated in 13% of exams while the opposite occurred in just 2% of exams. This was a significant preference for DDG-retro (p=0.008, n=121). Liver lesions were identified in 23 exams. Considering this subset of data, DDG-retro was ranked superior to Ungated Full in 6/23 (26%) of cases. Gated reconstruction using the external device failed in 16% of exams, while DDG-retro always provided a clinically acceptable image. Conclusion: In this clinical evaluation, the data driven respiratory gating technique provided superior performance as compared to the external device-based system. For the majority of exams the performance was equivalent, but data driven respiratory gating had superior performance in 13% of exams, leading to a significant preference overall.

Rationale: Currently available imaging techniques have limited specificity for the detection of active myocardial inflammation. Aluminum fluoride-18-labeled 1,4,7-triazacyclononane-N,N',N''-triacetic acid conjugated folate (¹⁸F-FOL) is a positron emission tomography (PET) tracer targeting folate receptor β (FR-β) that is expressed on activated macrophages at sites of inflammation. We evaluated ¹⁸F-FOL PET for the detection of myocardial inflammation in rats with autoimmune myocarditis and studied expression of FR-β in human cardiac sarcoidosis specimens. Methods: Myocarditis was induced by immunizing rats (n = 18) with porcine cardiac myosin in complete Freund’s adjuvant. Control rats (n = 6) were injected with Freund’s adjuvant alone. ¹⁸F-FOL was intravenously injected followed by imaging with a small animal PET/computed tomography (CT) scanner and autoradiography. Contrast-enhanced high-resolution CT or 2-deoxy-2-¹⁸F-fluoro-D-glucose (¹⁸F-FDG) PET images were used for co-registration. Rat tissue sections and myocardial autopsy samples of 6 patients with cardiac sarcoidosis were studied for macrophages and FR-β. Results: The myocardium of 10 out of 18 immunized rats showed focal macrophage-rich inflammatory lesions with FR-β expression occurring mainly in M1-polarized macrophages. PET images showed focal myocardial ¹⁸F-FOL uptake co-localizing with inflammatory lesions (SUVmean, 2.1 ± 1.1), whereas uptake in the remote myocardium of immunized rats and controls was low (SUVmean, 0.4 ± 0.2 and 0.4 ± 0.1, respectively; P < 0.01). Ex vivo autoradiography of tissue sections confirmed uptake of ¹⁸F-FOL in myocardial inflammatory lesions. Uptake of ¹⁸F-FOL to inflamed myocardium was efficiently blocked by a non-labeled FR-β ligand folate glucosamine in vivo. The myocardium of patients with cardiac sarcoidosis showed many FR-β-positive macrophages in inflammatory lesions. Conclusion: In a rat model of autoimmune myocarditis, ¹⁸F-FOL shows specific uptake in inflamed myocardium containing macrophages expressing FR-β, which were also present in human cardiac sarcoid lesions. Imaging of FR-β expression is a potential approach for the detection of active myocardial inflammation.

Bone is the most common site of distant metastatic spread in prostate adenocarcinoma. Prostate-specific membrane antigen uptake has been described in both benign and malignant bone lesions, which can lead to false-positive findings on ⁶⁸Ga-prostate-specific membrane antigen-11 positron emission tomography (⁶⁸Ga-PSMA-11 PET). The purpose of this study was to evaluate the diagnostic accuracy of ⁶⁸Ga-PSMA-11 PET for osseous prostate cancer metastases and improve bone uptake interpretation using semi-quantitative metrics. METHODS: 56 prostate cancer patients (18 pre-prostatectomy, 38 biochemical recurrence) who underwent ⁶⁸Ga-PSMA-11 PET/MRI or PET/CT examinations with osseous PSMA-ligand uptake were included in the study. Medical records were reviewed retrospectively by board-certified nuclear radiologists to determine true or false positivity based on a composite endpoint. For each avid osseous lesion, biological volume, size, PSMA-RADS rating, maximum standardized uptake value (SUV_max), and ratio of lesion SUV_max to liver, blood pool, and background bone SUV_max were measured. Differences between benign and malignant lesions were evaluated for statistical significance, and cut-off values for these parameters were determined to maximize diagnostic accuracy. RESULTS: Among 56 participants, 13 patients (22.8%) had false-positive osseous ⁶⁸Ga-PSMA-11 findings and 43 patients (76.8%) had true-positive osseous ⁶⁸Ga-PSMA-11 findings. Twenty-two patients (39%) had 1 osseous lesion, 18 (32%) had 2-4 lesions, and 16 (29%) had 5 or more lesions. Cut-off values resulting in statistically significant (p<0.005) differences between benign and malignant lesions were: PSMA-RADS ≥4, SUV_max ≥4.1, SUV_max ratio of lesion to blood pool ≥2.11, to liver ≥0.55, and to bone ≥4.4. These measurements corresponded to lesion-based ⁶⁸Ga-PSMA-11 PET lesion detection rate for malignancy of 80%, 93%, 89%, 21%, 89%, and a specificity of 73%, 73%, 73%, 93%, 60%, respectively. CONCLUSION: PSMA-RADS rating, SUV_max, and SUV_max ratio of lesion to blood pool can help differentiate benign from malignant lesions on ⁶⁸Ga-PSMA-11 PET. SUV_max ratio to blood pool above 2.2 is a reasonable parameter to support image interpretation and presented superior lesion detection rate and specificity when compared to visual interpretation by PSMA RADS. These parameters hold clinical value by improving diagnostic accuracy for metastatic prostate cancer on ⁶⁸Ga-PSMA-11 PET/MRI and PET/CT.

Introduction: Gastrin Releasing peptide receptors (GRPRs) are potential molecular imaging targets in a variety of tumors. Recently, a ⁶⁸Ga-labelled antagonist to GRPRs, NeoBOMB1, was developed for PET. We report on the outcome of a Phase I/IIa clinical trial (EudraCT 2016-002053-38) within the EU-FP7 project Closed-loop Molecular Environment for Minimally Invasive Treatment of Patients with Metastatic Gastrointestinal Stromal Tumours (‘MITIGATE’) (grant agreement number 602306) in patients with oligometastatic gastrointestinal stromal tumors (GIST). Materials and Methods: The main objectives were evaluation of safety, biodistribution, dosimetry and preliminary tumor targeting of ⁶⁸Ga-NeoBOMB1 in patients with advanced TKI-treated GIST using PET/CT. Six patients with histologically confirmed GIST and unresectable primary or metastases undergoing an extended protocol for detailed pharmacokinetic analysis were included. ⁶⁸Ga-NeoBOMB1 was prepared using a kit procedure with a licensed ⁶⁸Ge/⁶⁸Ga generator. 3 MBq/kg body-weight were injected intravenously and safety parameters were assessed. PET/CT included dynamic imaging at 5 min, 11 min and 19 min as well as static imaging at 1, 2 and 3-4 h p.i. for dosimetry calculations. Venous blood samples and urine were collected for pharmacokinetics. Tumor targeting was assessed on a per-lesion and per-patient basis. Results: ⁶⁸Ga-NeoBOMB1 (50 µg) was prepared with high radiochemical purity (yield >97%). Patients received 174 ± 28 MBq of the radiotracer, which was well tolerated in all patients over a follow-up period of 4 weeks. Dosimetry calculations revealed a mean adsorbed effective dose of 0.029 ± 0.06 mSv/MBq with highest organ dose to the pancreas (0.274 ± 0.099 mSv/MBq). Mean plasma half-life was 27.3 min with primarily renal clearance (mean 25.7 ± 5.4% of injected dose 4h p.i.). Plasma metabolite analyses revealed high stability, metabolites were only detected in the urine. In three patients a significant uptake with increasing maximum standard uptake values (SUV_max at 2h p.i.: 4.3 to 25.9) over time was found in tumor lesions. Conclusion: This Phase I/IIa study provides safety data for ⁶⁸Ga-NeoBOMB1, a promising radiopharmaceutical for targeting GRPR-expressing tumors. Safety profiles and pharmacokinetics are suitable for PET imaging and absorbed dose estimates are comparable to other ⁶⁸Ga-labelled radiopharmaceuticals used in clinical routine.

Fibroblast activation protein (FAP) has emerged as an interesting molecular target used in the imaging and therapy of various types of cancers. Gallium-68–labeled chelator-linked FAP inhibitors (FAPIs) have been successfully applied to positron emission tomography (PET) imaging of various tumor types. To broaden the spectrum of applicable PET tracers for extended imaging studies of FAP-dependent diseases, we herein report the radiosynthesis and preclinical evaluation of an ¹⁸F–labeled glycosylated FAP inhibitor ([¹⁸F]FGlc-FAPI). Methods: An alkyne-bearing precursor was synthesized and subjected to click chemistry–based radiosynthesis of [¹⁸F]FGlc-FAPI by two-step ¹⁸F-fluoroglycosylation. FAP-expressing HT1080hFAP cells were used to study competitive binding to FAP, cellular uptake, internalization, and efflux of [¹⁸F]FGlc-FAPI in vitro. Biodistribution studies and in vivo small animal PET studies of [¹⁸F]FGlc-FAPI compared to [⁶⁸Ga]Ga-FAPI-04 were conducted in nude mice bearing HT1080hFAP tumors or U87MG xenografts. Results: [¹⁸F]FGlc-FAPI was synthesized with a 15% radioactivity yield and a high radiochemical purity of >99%. In HT1080hFAP cells, [¹⁸F]FGlc-FAPI showed specific uptake, a high internalized fraction, and low cellular efflux. Compared to FAPI-04 (IC50 = 32 nM), the glycoconjugate, FGlc-FAPI (IC50 = 167 nM), showed slightly lower affinity for FAP in vitro, while plasma protein binding was higher for [¹⁸F]FGlc-FAPI. Biodistribution studies revealed significant hepatobiliary excretion of [¹⁸F]FGlc-FAPI; however, small animal PET studies in HT1080hFAP xenografts showed higher specific tumor uptake of [¹⁸F]FGlc-FAPI (4.5 % injected dose per gram of tissue [ID/g]) compared to [⁶⁸Ga]Ga-FAPI-04 (2 %ID/g). In U87MG tumor–bearing mice, both tracers showed similar tumor uptake, but [¹⁸F]FGlc-FAPI showed a higher tumor retention. Interestingly, [¹⁸F]FGlc-FAPI demonstrated high specific uptake in bone structures and joints. Conclusion: [¹⁸F]FGlc-FAPI is an interesting candidate for translation to the clinic, taking advantage of the longer half-life and physical imaging properties of F-18. The availability of [¹⁸F]FGlc-FAPI may allow extended PET studies of FAP-related diseases, such as cancer, but also arthritis, heart diseases, or pulmonary fibrosis.

Treatment of hyperinsulinemic hypoglycemia is challenging. Surgical treatment of insulinomas and focal lesions in congenital hyperinsulinism (CHI) is invasive and carries major risks of morbidity. Medication to treat nesidioblastosis and diffuse CHI has varying efficacy and causes significant side effects. Here, we describe a novel method for therapy of hyperinsulinemic hyperglycemia, highly selectively killing beta cells by targeted photodynamic therapy (tPDT) with exendin-4-IRDye700DX, targeting the glucagon-like peptide 1 receptor (GLP-1R). A competitive binding assay was performed using Chinese hamster lung (CHL) cells transfected with the GLP-1R. The efficacy and specificity of tPDT with exendin-4-IRDye700DX was examined in vitro in cells with different levels of GLP-1R expression. Tracer biodistribution was determined in BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts. Induction of cellular damage and the effect on tumor growth were analyzed to determine treatment efficacy. Exendin-4-IRDye700DX has a high affinity for the GLP-1R with an IC50 value of 6.3 nM. TPDT caused significant specific phototoxicity in GLP-1R positive cells (2.3 ± 0.8 % and 2.7 ± 0.3 % remaining cell viability in CHL-GLP-1R and INS-1 cells resp.). The tracer accumulates dose-dependently in GLP-1R positive tumors. In vivo tPDT induces cellular damage in tumors, shown by strong expression of cleaved-caspase-3 and leads to a prolonged median survival of the mice (36.5 vs. 22.5 days resp. p<0.05). These data show in vitro as well as in vivo evidence for the potency of tPDT using exendin-4-IRDye700DX. This could in the future provide a new, minimally invasive and highly specific treatment method for hyperinsulinemic hypoglycemia.

Research Event

Event participants

Professor Stig Jarle Hansen, Professor, Norwegian University of Life Sciences; Author, Horn, Sahel and Rift: Fault-lines of the African Jihad
Bulama Bukarti, PhD Candidate, SOAS, University of London; Analyst, Tony Blair Institute for Global Change
Chair: Aoife McCullough, PhD Candidate, LSE

Research Event

Event participants

Abdirahman Abdishakur Warsame, Leader, Wadajir Party, Federal Republic of Somalia
Chair: Ahmed Soliman, Research Fellow, Africa Programme, Chatham House

Research Event

Event participants

Tutu Alicante, Executive Director, EG Justice
Chair: Dr Alex Vines OBE, Managing Director, Ethics, Risk & Resilience; Director, Africa Programme, Chatham House

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein–coupled receptors. Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors and GTPase-activating proteins. Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function.

FcRIIIa (CD16a) and FcRIIa (CD32a) on monocytes are essential for proper effector functions including antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Indeed, therapeutic monoclonal antibodies (mAbs) that bind FcRs with greater affinity exhibit greater efficacy. Furthermore, post-translational modification impacts antibody binding affinity, most notably the composition of the asparagine(N)-linked glycan at N162 of CD16a. CD16a is widely recognized as the key receptor for the monocyte response, however the post-translational modifications of CD16a from endogenous monocytes are not described. Here we isolated monocytes from individual donors and characterized the composition of CD16a and CD32a N-glycans from all modified sites. The composition of CD16a N-glycans varied by glycosylation site and donor. CD16a displayed primarily complex-type biantennary N-glycans at N162, however some individuals expressed CD16a V158 with ~20% hybrid and oligomannose types which increased affinity for IgG1 Fc according to surface plasmon resonance binding analyses. The CD16a N45-glycans contain markedly less processing than other sites with >75% hybrid and oligomannose forms. N38 and N74 of CD16a both contain highly processed complex-type N-glycans with N-acetyllactosamine repeats and complex-type biantennary N-glycans dominate at N169. The composition of CD16a N-glycans isolated from monocytes included a higher proportion of oligomannose-type N-glycans at N45 and less sialylation plus greater branch fucosylation than we observed in a recent analysis of NK cell CD16a. The additional analysis of CD32a from monocytes revealed different features than observed for CD16a including the presence of a predominantly biantennary complex-type N-glycans with two sialic acids at both sites (N64 and N145).

Aberrantly high mTORC1 signaling is a known driver of many cancers and human disorders, yet pharmacological inhibition of mTORC1 rarely confers durable clinical responses. To explore alternative therapeutic strategies, herein we conducted a proteomics survey to identify cell surface proteins upregulated by mTORC1. A comparison of the surfaceome from Tsc1^–/– versus Tsc1^+/+ mouse embryonic fibroblasts revealed 59 proteins predicted to be significantly overexpressed in Tsc1^–/– cells. Further validation of the data in multiple mouse and human cell lines showed that mTORC1 signaling most dramatically induced the expression of the proteases neprilysin (NEP/CD10) and aminopeptidase N (APN/CD13). Functional studies showed that constitutive mTORC1 signaling sensitized cells to genetic ablation of NEP and APN, as well as the biochemical inhibition of APN. In summary, these data show that mTORC1 signaling plays a significant role in the constitution of the surfaceome, which in turn may present novel therapeutic strategies.

HNF4α is a nuclear receptor produced as 12 isoforms from two promoters by alternative splicing. To characterize the transcriptional capacities of all 12 HNF4α isoforms, stable lines expressing each isoform were generated. The entire transcriptome associated with each isoform was analyzed as well as their respective interacting proteome. Major differences were noted in the transcriptional function of these isoforms. The α1 and α2 isoforms were the strongest regulators of gene expression whereas the α3 isoform exhibited significantly reduced activity. The α4, α5, and α6 isoforms, which use an alternative first exon, were characterized for the first time, and showed a greatly reduced transcriptional potential with an inability to recognize the consensus response element of HNF4α. Several transcription factors and coregulators were identified as potential specific partners for certain HNF4α isoforms. An analysis integrating the vast amount of omics data enabled the identification of transcriptional regulatory mechanisms specific to certain HNF4α isoforms, hence demonstrating the importance of considering all isoforms given their seemingly diverse functions.

Previous studies have shown that sphingosine kinase interacting protein (SKIP) inhibits sphingosine kinase (SK) function in fibroblasts. SK phosphorylates sphingosine producing the potent signaling molecule sphingosine-1-phosphate (S1P). SKIP gene (SPHKAP) expression is silenced by hypermethylation of its promoter in acute myeloid leukemia (AML). However, why SKIP activity is silenced in primary AML cells is unclear. Here, we investigated the consequences of SKIP down-regulation in AML primary cells and the effects of SKIP re-expression in leukemic cell lines. Using targeted ultra-HPLC-tandem MS (UPLC-MS/MS), we measured sphingolipids (including S1P and ceramides) in AML and control cells. Primary AML cells had significantly lower SK activity and intracellular S1P concentrations than control cells, and SKIP-transfected leukemia cell lines exhibited increased SK activity. These findings show that SKIP re-expression enhances SK activity in leukemia cells. Furthermore, other bioactive sphingolipids such as ceramide were also down-regulated in primary AML cells. Of note, SKIP re-expression in leukemia cells increased ceramide levels 2-fold, inactivated the key signaling protein extracellular signal-regulated kinase, and increased apoptosis following serum deprivation or chemotherapy. These results indicate that SKIP down-regulation in AML reduces SK activity and ceramide levels, an effect that ultimately inhibits apoptosis in leukemia cells. The findings of our study contrast with previous results indicating that SKIP inhibits SK function in fibroblasts and therefore challenge the notion that SKIP always inhibits SK activity.

The peroxisome is a subcellular organelle that functions in essential metabolic pathways, including biosynthesis of plasmalogens, fatty acid β-oxidation of very-long-chain fatty acids, and degradation of hydrogen peroxide. Peroxisome biogenesis disorders (PBDs) manifest as severe dysfunction in multiple organs, including the central nervous system (CNS), but the pathogenic mechanisms in PBDs are largely unknown. Because CNS integrity is coordinately established and maintained by neural cell interactions, we here investigated whether cell-cell communication is impaired and responsible for the neurological defects associated with PBDs. Results from a noncontact co-culture system consisting of primary hippocampal neurons with glial cells revealed that a peroxisome-deficient astrocytic cell line secretes increased levels of brain-derived neurotrophic factor (BDNF), resulting in axonal branching of the neurons. Of note, the BDNF expression in astrocytes was not affected by defects in plasmalogen biosynthesis and peroxisomal fatty acid β-oxidation in the astrocytes. Instead, we found that cytosolic reductive states caused by a mislocalized catalase in the peroxisome-deficient cells induce the elevation in BDNF secretion. Our results suggest that peroxisome deficiency dysregulates neuronal axogenesis by causing a cytosolic reductive state in astrocytes. We conclude that astrocytic peroxisomes regulate BDNF expression and thereby support neuronal integrity and function.

Coenzyme Q (Qn) is a vital lipid component of the electron transport chain that functions in cellular energy metabolism and as a membrane antioxidant. In the yeast Saccharomyces cerevisiae, coq1–coq9 deletion mutants are respiratory-incompetent, sensitive to lipid peroxidation stress, and unable to synthesize Q6. The yeast coq10 deletion mutant is also respiratory-deficient and sensitive to lipid peroxidation, yet it continues to produce Q6 at an impaired rate. Thus, Coq10 is required for the function of Q6 in respiration and as an antioxidant and is believed to chaperone Q6 from its site of synthesis to the respiratory complexes. In several fungi, Coq10 is encoded as a fusion polypeptide with Coq11, a recently identified protein of unknown function required for efficient Q6 biosynthesis. Because “fused” proteins are often involved in similar biochemical pathways, here we examined the putative functional relationship between Coq10 and Coq11 in yeast. We used plate growth and Seahorse assays and LC-MS/MS analysis to show that COQ11 deletion rescues respiratory deficiency, sensitivity to lipid peroxidation, and decreased Q6 biosynthesis of the coq10Δ mutant. Additionally, immunoblotting indicated that yeast coq11Δ mutants accumulate increased amounts of certain Coq polypeptides and display a stabilized CoQ synthome. These effects suggest that Coq11 modulates Q6 biosynthesis and that its absence increases mitochondrial Q6 content in the coq10Δcoq11Δ double mutant. This augmented mitochondrial Q6 content counteracts the respiratory deficiency and lipid peroxidation sensitivity phenotypes of the coq10Δ mutant. This study further clarifies the intricate connection between Q6 biosynthesis, trafficking, and function in mitochondrial metabolism.

Heat shock factor 1 (HSF1) regulates cellular adaptation to challenges such as heat shock and oxidative and proteotoxic stresses. We have recently reported a previously unappreciated role for HSF1 in the regulation of energy metabolism in fat tissues; however, whether HSF1 is differentially expressed in adipose depots and how its levels are regulated in fat tissues remain unclear. Here, we show that HSF1 levels are higher in brown and subcutaneous fat tissues than in those in the visceral depot and that HSF1 is more abundant in differentiated, thermogenic adipocytes. Gene expression experiments indicated that HSF1 is transcriptionally regulated in fat by agents that modulate cAMP levels, by cold exposure, and by pharmacological stimulation of β-adrenergic signaling. An in silico promoter analysis helped identify a putative response element for activating transcription factor 3 (ATF3) at −258 to −250 base pairs from the HSF1 transcriptional start site, and electrophoretic mobility shift and ChIP assays confirmed ATF3 binding to this sequence. Furthermore, functional assays disclosed that ATF3 is necessary and sufficient for HSF1 regulation. Detailed gene expression analysis revealed that ATF3 is one of the most highly induced ATFs in thermogenic tissues of mice exposed to cold temperatures or treated with the β-adrenergic receptor agonist CL316,243 and that its expression is induced by modulators of cAMP levels in isolated adipocytes. To the best of our knowledge, our results show for the first time that HSF1 is transcriptionally controlled by ATF3 in response to classic stimuli that promote heat generation in thermogenic tissues.