Multiparametric MRI and 18F-PSMA-1007 PET/CT for the Detection of Clinically Significant Prostate Cancer.

Background Multiparametric MRI (mpMRI) is effective for detecting prostate cancer (PCa); however, there is a high rate of equivocal Prostate Imaging Reporting and Data System (PI-RADS) 3 lesions and false-positive findings. Purpose To investigate whether fluorine 18 (18F) prostate-specific membrane antigen (PSMA) 1007 PET/CT after mpMRI can help detect localized clinically significant PCa (csPCa), particularly for equivocal PI-RADS 3 lesions. Materials and Methods This prospective study included participants with elevated prostate-specific antigen (PSA) levels referred for prostate mpMRI between September 2020 and February 2022. 18F-PSMA-1007 PET/CT was performed within 30 days of mpMRI and before biopsy. PI-RADS category and level of suspicion (LOS) were assessed. PI-RADS 3 or higher lesions at mpMRI and/or LOS 3 or higher lesions at 18F-PSMA-1007 PET/CT underwent targeted biopsies. PI-RADS 2 or lower and LOS 2 or lower lesions were considered nonsuspicious and were monitored during a 1-year follow-up by means of PSA testing. Diagnostic accuracy was assessed, with histologic examination serving as the reference standard. International Society of Urological Pathology (ISUP) grade 2 or higher was considered csPCa. Results Seventy-five participants (median age, 67 years [range, 52-77 years]) were assessed, with PI-RADS 1 or 2, PI-RADS 3, and PI-RADS 4 or 5 groups each including 25 participants. A total of 102 lesions were identified, of which 80 were PI-RADS 3 or higher and/or LOS 3 or higher and therefore underwent targeted biopsy. The per-participant sensitivity for the detection of csPCa was 95% and 91% for mpMRI and 18F-PSMA-1007 PET/CT, respectively, with respective specificities of 45% and 62%. 18F-PSMA-1007 PET/CT was used to correctly differentiate 17 of 26 PI-RADS 3 lesions (65%), with a negative and positive predictive value of 93% and 27%, respectively, for ruling out or detecting csPCa. One additional significant and one insignificant PCa lesion (PI-RADS 1 or 2) were found at 18F-PSMA-1007 PET/CT that otherwise would have remained undetected. Two participants had ISUP 2 tumors without PSMA uptake that were missed at PET/CT. Conclusion 18F-PSMA-1007 PET/CT showed good sensitivity and moderate specificity for the detection of csPCa and ruled this out in 93% of participants with PI-RADS 3 lesions. Clinical trial registration no. NCT04487847 © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Turkbey in this issue.

Qualitative flow metabolic phenotype of pancreatic cancer. A new prognostic biomarker?

BACKGROUND: Retrospective analysis to investigate the relationship between the flow-metabolic phenotype and overall survival (OS) of pancreatic ductal adenocarcinoma (PDAC) and its potential clinical utility. METHODS: Patients with histopathologically proven PDAC between 2005 and 2014 using tumor attenuation on routine pre-operative CECT as a surrogate for the vascularity and [18F]FDG-uptake as a surrogate for metabolic activity on [18F]FDG-PET. RESULTS: In total, 93 patients (50 male, 43 female, median age 63) were included. Hypoattenuating PDAC with high [18F]FDG-uptake has the poorest prognosis (median OS 7 ± 1 months), compared to hypoattenuating PDAC with low [18F]FDG-uptake (median OS 11 ± 3 months; p = 0.176), iso- or hyperattenuating PDAC with high [18F]FDG-uptake (median OS 15 ± 5 months; p = 0.004) and iso- or hyperattenuating PDAC with low [18F]FDG-uptake (median OS 23 ± 4 months; p = 0.035). In multivariate analysis, surgery combined with tumor differentiation, tumor stage, systemic therapy and flow metabolic phenotype remained independent predictors for overall survival. DISCUSSION: The novel qualitative flow-metabolic phenotype of PDAC using a combination of CECT and [18F]FDG-PET features, predicted significantly worse survival for hypoattenuating-high uptake pancreatic cancers compared to the other phenotypes.

Importance of beta cell mass for glycaemic control in people with type 1 diabetes.

AIMS/HYPOTHESIS: The role of beta cell mass in the balance of glucose control and hypoglycaemic burden in people with type 1 diabetes is unclear. We applied positron emission tomography (PET) imaging with radiolabelled exendin to compare beta cell mass among people with type 1 diabetes and either low glucose variability (LGV) or high glucose variability (HGV). METHODS: All participants with either LGV (n=9) or HGV (n=7) underwent a mixed-meal tolerance test to determine beta cell function and wore a blinded continuous glucose monitor for a week. After an i.v. injection with [68Ga]Ga-NODAGA-exendin-4, PET images were acquired for the quantification of pancreatic uptake of radiolabelled exendin. The mean standardised uptake value (SUVmean) of the pancreas was used to determine the amount of beta cell mass. RESULTS: Participants with LGV had lower HbA1c (46.0 mmol/mol [44.5-52.5] [6.4% (6.3-7)] vs 80 mmol/mol [69.0-110] [9.5% (8.5-12.2)], p=0.001) and higher time in range (TIR) (75.6% [73.5-90.3] vs 38.7% [25.1-48.5], p=0.002) than those with HGV. The SUVmean of the pancreas was higher for the LGV than for the HGV group (5.1 [3.6-5.6] vs 2.9 [2.1-3.4], p=0.008). The AUCC-peptide:AUCglucose ratio was numerically, but not statistically, higher in the LGV compared with the HGV group (2.7×10-2 [6.2×10-4-5.3×10-2] vs 9.3×10-4 [4.7×10-4-5.2×10-3], p=0.21). SUVmean correlated with the AUCC-peptide:AUCglucose ratio (Pearson r=0.64, p=0.01), as well as with the TIR (r=0.64, p=0.01) and the SD of interstitial glucose levels (r=-0.66, p=0.007). CONCLUSION/INTERPRETATION: Our data show higher beta cell mass in people with type 1 diabetes and LGV than in those with HGV, independent of beta cell function.

Impact of tyrosine kinase inhibitors on glucose control and insulin regulation in patients with chronic myeloid leukemia.

Treatment with tyrosine kinase inhibitors (TKIs), especially nilotinib, often results in hyperglycemia, which may further increase cardiovascular disease risk in patients with chronic myeloid leukemia (CML). The mechanism underlying the TKI-induced glucose dysregulation is not clear. TKIs are suggested to affect insulin secretion but also insulin sensitivity of peripheral tissue has been proposed to play a role in the pathogenesis of TKI-induced hyperglycemia. Here, we aimed to assess whether skeletal muscle glucose uptake and insulin responses are altered in nondiabetic patients with CML receiving TKI treatment. After a glycogen-depleted exercise bout, an intravenous glucose bolus (0.3 g/kg body weight) was administered to monitor 2-h glucose tolerance and insulin response in 14 patients with CML receiving nilotinib, 14 patients with CML receiving imatinib, and 14 non-CML age- and gender-matched controls. A dynamic [18F]-FDG PET scan during a hyperinsulinemic-euglycemic clamp was performed in a subgroup of 12 male patients with CML to assess m. quadriceps glucose uptake. We showed that patients with CML treated with nilotinib have an increased insulin response to intravenous glucose administration after muscle glycogen-depleted exercise. Despite the increased insulin response to glucose administration in patients with CML receiving nilotinib, glucose disappearance rates were significantly slower in nilotinib-treated patients when compared with controls in the first 15 min after glucose administration. Although [18F]-FDG uptake in m. quadriceps was not different, patients receiving nilotinib showed a trend toward decreased glucose infusion rates during euglycemic clamping when compared with patients receiving imatinib. Together, these findings indicate disturbed skeletal muscle glucose handling in patients with CML receiving nilotinib therapy.NEW & NOTEWORTHY In this study, we have shown that non-diabetic patients with CML receiving nilotinib therapy show early signs of disturbed skeletal muscle glucose handling, which was not observed in imatinib-treated patients. These observations in nilotinib users may reflect decreased muscle insulin sensitivity, which could serve as a potential target to counteract glycemic dysregulation, and is of clinical importance since these patients have an increased cardiovascular disease risk.

Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39).

PURPOSE: Exendin, an analogue of the glucagon-like peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9-39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9-39) could turn it into a useful alternative tracer with less side-effects than exendin-4. METHODS: We conjugated exendin-4 and exendin(9-39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vitro and their biodistribution in vivo. RESULTS: Exendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9-39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo tumour uptake and retention of exendin(9-39). CONCLUSION: We demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9-39), opening new avenues for antagonist-based in vivo imaging of GLP1R.

Imaging and photodynamic therapy of prostate cancer using a theranostic PSMA-targeting ligand.

PURPOSE: Incomplete resection of prostate cancer (PCa) results in increased risk of disease recurrence. Combined fluorescence-guided surgery with tumor-targeted photodynamic therapy (tPDT) may help to achieve complete tumor eradication. We developed a prostate-specific membrane antigen (PSMA) ligand consisting of a DOTA chelator for 111In labeling and a fluorophore/photosensitizer IRDye700DX (PSMA-N064). We evaluated the efficacy of PSMA-tPDT using PSMA-N064 in cell viability assays, a mouse xenograft model and in an ex vivo incubation study on fresh human PCa tissue. METHODS: In vitro, therapeutic efficacy of PSMA-N064 was evaluated using PSMA-positive LS174T cells and LS174T wild-type cells. In vivo, PSMA-N064-mediated tPDT was tested in immunodeficient BALB/c mice-bearing PSMA-positive LS174T xenografts. Tumor growth and survival were compared to control mice that received either NIR light or ligand injection only. Ex vivo tPDT efficacy was evaluated in excised fresh human PCa tissue incubated with PSMA-N064. RESULTS: In vitro, tPDT led to a PSMA-specific light- and ligand dose-dependent loss in cell viability. In vivo, tPDT-induced tumor cell apoptosis, delayed tumor growth, and significantly improved survival (p = 0.004) of the treated PSMA-positive tumor-bearing mice compared with the controls. In fresh ex vivo human PCa tissue, apoptosis was significantly increased in PSMA-tPDT-treated samples compared to non-treated control samples (p = 0.037). CONCLUSION: This study showed the feasibility of PSMA-N064-mediated tPDT in cell assays, a xenograft model and excised fresh human PCa tissue. This paves the way to investigate the impact of in vivo PSMA-tPDT on surgical outcome in PCa patients.

New Radiolabeled Exendin Analogues Show Reduced Renal Retention.

PET imaging of the glucagon-like peptide-1 receptor (GLP-1R) using radiolabeled exendin is a promising imaging method to detect insulinomas. However, high renal accumulation of radiolabeled exendin could hamper the detection of small insulinomas in proximity to the kidneys and limit its use as a radiotherapeutic agent. Here, we report two new exendin analogues for GLP-1R imaging and therapy, designed to reduce renal retention by incorporating a cleavable methionine-isoleucine (Met-Ile) linker. We examined the renal retention and insulinoma targeting properties of these new exendin analogues in a nude mouse model bearing subcutaneous GLP-1R-expressing insulinomas. NOTA or DOTA was conjugated via a methionine-isoleucine linker to the C-terminus of exendin-4 (NOTA-MI-exendin-4 or DOTA-MI-exendin-4). NOTA- and DOTA-exendin-4 without the linker were used as references. The affinity for GLP-1R was determined in a competitive binding assay using GLP-1R transfected cells. Biodistribution of [68Ga]Ga-NOTA-exendin-4, [68Ga]Ga-NOTA-MI-exendin-4, [177Lu]Lu-DOTA-exendin-4, and [177Lu]Lu-DOTA-MI-exendin-4 was determined in INS-1 tumor-bearing BALB/c nude mice, and PET/CT was acquired to visualize renal retention and tumor targeting. For all tracers, dosimetric calculations were performed to determine the kidney self-dose. The affinity for GLP-1R was in the low nanomolar range (<11 nM) for all peptides. In vivo biodistribution revealed a significantly lower kidney uptake of [68Ga]Ga-NOTA-MI-exendin-4 at 4 h post-injection (p.i.) (34.2 ± 4.2 %IA/g), compared with [68Ga]Ga-NOTA-exendin-4 (128 ± 10 %IA/g). Accumulation of [68Ga]Ga-NOTA-MI-exendin-4 in the tumor was 25.0 ± 8.0 %IA/g 4 h p.i., which was similar to that of [68Ga]Ga-NOTA-exendin-4 (24.9 ± 9.3 %IA/g). This resulted in an improved tumor-to-kidney ratio from 0.2 ± 0.0 to 0.8 ± 0.3. PET/CT confirmed the findings in the biodistribution studies. The kidney uptake of [177Lu]Lu-DOTA-MI-exendin-4 was 39.4 ± 6.3 %IA/g at 24 h p.i. and 13.0 ± 2.5 %IA/g at 72 h p.i., which were significantly lower than those for [177Lu]Lu-DOTA-exendin-4 (99.3 ± 9.2 %IA/g 24 h p.i. and 45.8 ± 3.9 %IA/g 72 h p.i.). The uptake in the tumor was 7.8 ± 1.5 and 11.3 ± 2.0 %IA/g 24 h p.i. for [177Lu]Lu-DOTA-MI-exendin-4 and [177Lu]Lu-DOTA-exendin-4, respectively, resulting in improved tumor-to-kidney ratios for [177Lu]Lu-DOTA-MI-exendin-4. The new exendin analogues with a Met-Ile linker showed 2-3-fold reduced renal retention and improved tumor-to-kidney ratios compared with their reference without the Met-Ile linker. Future studies should demonstrate whether [68Ga]Ga-NOTA-MI-exendin-4 results in improved detection of small insulinomas in close proximity to the kidneys with PET/CT. [177Lu]Lu-DOTA-MI-exendin-4 might open a window of opportunity for exendin-based radionuclide therapy.

Fibroblast Activation Protein-Targeted Photodynamic Therapy of Cancer-Associated Fibroblasts in Murine Models for Pancreatic Ductal Adenocarcinoma.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5 year survival of 9%. One important limiting factor for treatment efficacy is the dense tumor-supporting stroma. The cancer-associated fibroblasts in this stroma deposit excessive amounts of extracellular matrix components and anti-inflammatory mediators, which hampers the efficacy of chemo- and immunotherapies. Systemic depletion of all activated fibroblasts is, however, not feasible nor desirable and therefore a local approach should be pursued. Here, we provide a proof-of-principle of using fibroblast activation protein (FAP)-targeted photodynamic therapy (tPDT) to treat PDAC. FAP-targeting antibody 28H1 and irrelevant control antibody DP47GS were conjugated to the photosensitizer IRDye700DX (700DX) and the chelator diethylenetriaminepentaacetic acid. In vitro binding and cytotoxicity were evaluated using the fibroblast cell-line NIH-3T3 stably transfected with FAP. Biodistribution of 111In-labeled antibody-700DX constructs was determined in mice carrying syngeneic tumors of the murine PDAC cell line PDAC299, and in a genetically engineered PDAC mouse model (CKP). Then, tPDT was performed by exposing the subcutaneous or the spontaneous PDAC tumors to 690 nm light. Induction of apoptosis after treatment was assessed using automated analyses of immunohistochemistry for cleaved caspase-3. 28H1-700DX effectively bound to 3T3-FAP cells and induced cytotoxicity upon exposure to 690 nm light, whereas no binding or cytotoxic effects were observed for DP47GS-700DX. Although both 28H1-700DX and DP47GS-700DX accumulated in subcutaneous PDAC299 tumors, autoradiography demonstrated that only 28H1-700DX reached the tumor core. On the contrary, control antibody DP47GS-700DX was only present at the tumor rim. In CKP mice, both antibodies accumulated in the tumor, but tumor-to-blood ratios of 28H1-700DX were higher than that of the control. Notably, in vivo FAP-tPDT caused upregulation of cleaved caspase-3 staining in both subcutaneous and in spontaneous tumors. In conclusion, we have shown that tPDT is a feasible approach for local depletion of FAP-expressing stromal cells in murine models for PDAC.

18F-FDG PET-CT in rheumatoid arthritis patients tapering TNFi: reliability, validity and predictive value.

OBJECTIVES: To investigate the reliability and validity of fluorine-18 fluorodeoxyglucose (18F-FDG) PET-CT scanning (FDG-PET) in RA patients with low disease activity tapering TNF inhibitors (TNFis) and its predictive value for successful tapering or discontinuation. METHODS: Patients in the tapering arm of the Dose REduction Strategies of Subcutaneous TNFi study, a randomized controlled trial of TNFi tapering in RA, underwent FDG-PET before tapering (baseline) and after maximal tapering. A total of 48 joints per scan were scored both visually [FDG-avid joint (FAJ), yes/no] and quantitatively [maximal and mean standardized uptake values (SUVmax and SUVmean)]. Interobserver agreement was calculated in 10 patients at baseline. Quantitative and visual FDG-PET scores were investigated for (multilevel) association with clinical parameters both on a joint and patient level and for the predictive value at baseline and the change between baseline and maximal tapering (Δ) for successful tapering and discontinuation at 18 months. RESULTS: A total of 79 patients underwent FDG-PET. For performance of identification of FAJs on PET, Cohen's κ was 0.49 (range 0.35-0.63). For SUVmax and SUVmean, intraclass correlation coefficients were 0.80 (range 0.77-0.83) and 0.96 (0.9-1.0), respectively. On a joint level, swelling was significantly associated with SUVmax and SUVmean [B coefficients 1.0 (95% CI 0.73, 1.35) and 0.2 (0.08, 0.32), respectively]. On a patient level, only correlation with acute phase reactants was found. FDG-PET scores were not predictive of successful tapering or discontinuation. CONCLUSIONS: Quantitative FDG-PET arthritis scoring in RA patients with low disease activity is reliable and has some construct validity. However, no predictive values were found for FDG-PET parameters for successful tapering and/or discontinuation of TNFi.

Targeting of fibroblast activation protein in rheumatoid arthritis patients: imaging and ex vivo photodynamic therapy.

OBJECTIVE: Activated synovial fibroblasts are key effector cells in RA. Selectively depleting these based upon their expression of fibroblast activation protein (FAP) is an attractive therapeutic approach. Here we introduce FAP imaging of inflamed joints using 68Ga-FAPI-04 in a RA patient, and aim to assess feasibility of anti-FAP targeted photodynamic therapy (FAP-tPDT) ex vivo using 28H1-IRDye700DX on RA synovial explants. METHODS: Remnant synovial tissue from RA patients was processed into 6 mm biopsies and, from several patients, into primary fibroblast cell cultures. Both were treated using FAP-tPDT. Cell viability was measured in fibroblast cultures and biopsies were evaluated for histological markers of cell damage. Selectivity of the effect of FAP-tPDT was assessed using flow cytometry on primary fibroblasts and co-cultured macrophages. Additionally, one RA patient intravenously received 68Ga-FAPI-04 and was scanned using PET/CT imaging. RESULTS: In the RA patient, FAPI-04 PET imaging showed high accumulation of the tracer in arthritic joints with very low background signal. In vitro, FAP-tPDT induced cell death in primary RA synovial fibroblasts in a light dose-dependent manner. An upregulation of cell damage markers was observed in the synovial biopsies after FAP-tPDT. No significant effects of FAP-tPDT were noted on macrophages after FAP-tPDT of neighbouring fibroblasts. CONCLUSION: In this study the feasibility of selective FAP-tPDT in synovium of rheumatoid arthritis patients ex vivo is demonstrated. Furthermore, this study provides the first indication that FAP-targeted PET/CT can be used to image arthritic joints, an important step towards application of FAP-tPDT as a targeted locoregional therapy for RA.

Intra-therapeutic dosimetry of [177Lu]Lu-PSMA-617 in low-volume hormone-sensitive metastatic prostate cancer patients and correlation with treatment outcome.

INTRODUCTION: While [177Lu]Lu-PSMA radioligand therapy is currently only applied in end-stage metastatic castrate-resistant prostate cancer (mCRPC) patients, also low-volume hormone-sensitive metastatic prostate cancer (mHSPC) patients can benefit from it. However, there are toxicity concerns related to the sink effect in low-volume disease. This prospective study aims to determine the kinetics of [177Lu]Lu-PSMA in mHSPC patients, analyzing the doses to organs at risk (salivary glands, kidneys, liver, and bone marrow) and tumor lesions < 1 cm diameter. METHODS: Ten mHSPC patients underwent two cycles of [177Lu]Lu-PSMA therapy. Three-bed position SPECT/CT was performed at 5 time points after each therapy. Organ dosimetry and lesion dosimetry were performed using commercial software and a manual approach, respectively. Correlation between absorbed index lesion dose and treatment response (PSA drop of > 50% at the end of the study) was calculated and given as Spearman's r and p-values. RESULTS: Kinetics of [177Lu]Lu-PSMA in mHSPC patients are comparable to those in mCRPC patients. Lesion absorbed dose was high (3.25 ± 3.19 Gy/GBq) compared to organ absorbed dose (salivary glands: 0.39 ± 0.17 Gy/GBq, kidneys: 0.49 ± 0.11 Gy/GBq, liver: 0.09 ± 0.01 Gy/GBq, bone marrow: 0.017 ± 0.008 Gy/GBq). A statistically significant correlation was found between treatment response and absorbed index lesion dose (p = 0.047). CONCLUSIONS: We successfully performed small lesion dosimetry and showed that the tumor sink effect in mHSPC patients is of less concern than was expected. Tumor-to-organ ratio of absorbed dose was high and tumor uptake correlates with PSA response. Additional treatment cycles are legitimate in terms of organ toxicity and could lead to better tumor response.

[68Ga]Ga-PSMA-11 PET imaging as a predictor for absorbed doses in organs at risk and small lesions in [177Lu]Lu-PSMA-617 treatment.

INTRODUCTION: Patient eligibility for [177Lu]Lu-PSMA therapy remains a challenge, with only 40-60% response rate when patient selection is done based on the lesion uptake (SUV) on [68Ga]Ga-PSMA-PET/CT. Prediction of absorbed dose based on this pre-treatment scan could improve patient selection and help to individualize treatment by maximizing the absorbed dose to target lesions while adhering to the threshold doses for the organs at risk (kidneys, salivary glands, and liver). METHODS: Ten patients with low-volume hormone-sensitive prostate cancer received a pre-therapeutic [68Ga]Ga-PSMA-11 PET/CT, followed by 3 GBq [177Lu]Lu-PSMA-617 therapy. Intra-therapeutically, SPECT/CT was acquired at 1, 24, 48, 72, and 168 h. Absorbed dose in organs and lesions (n = 22) was determined according to the MIRD scheme. Absorbed dose prediction based on [68Ga]Ga-PSMA-PET/CT was performed using tracer uptake at 1 h post-injection and the mean tissue effective half-life on SPECT. Predicted PET/actual SPECT absorbed dose ratios were determined for each target volume. RESULTS: PET/SPECT absorbed dose ratio was 1.01 ± 0.21, 1.10 ± 0.15, 1.20 ± 0.34, and 1.11 ± 0.29 for kidneys (using a 2.2 scaling factor), liver, submandibular, and parotid glands, respectively. While a large inter-patient variation in lesion kinetics was observed, PET/SPECT absorbed dose ratio was 1.3 ± 0.7 (range: 0.4-2.7, correlation coefficient r = 0.69, p < 0.01). CONCLUSION: A single time point [68Ga]Ga-PSMA-PET scan can be used to predict the absorbed dose of [177Lu]Lu-PSMA therapy to organs, and (to a limited extent) to lesions. This strategy facilitates in treatment management and could increase the personalization of [177Lu]Lu-PSMA therapy.

89Zr-labeled PSMA ligands for pharmacokinetic PET imaging and dosimetry of PSMA-617 and PSMA-I&T: a preclinical evaluation and first in man.

RATIONALE: Prolonged in vivo evaluation of PSMA tracers could improve tumor imaging and patient selection for 177Lu-PSMA-617 and 177Lu-PSMA-I&T. In this study, we present the radiolabeling method of PSMA-617 and PSMA-I&T with the long-lived positron emitter 89Zr to enable PET imaging up to 7 days post-injection. We compared the biodistribution of 89Zr-PSMA-617 and 89Zr-PSMA-I&T to those of 177Lu-PSMA-617 and 177Lu-PSMA-I&T, respectively, in a PSMA+ xenograft model. Moreover, we provide the first human 89Zr-PSMA-617 images. MATERIALS AND METHODS: PSMA ligands were labeled with 50-55 MBq [89Zr]ZrCl4 using a two-step labeling protocol. For biodistribution, BALB/c nude mice bearing PSMA+ and PSMA- xenografts received 0.6 µg (0.6-1 MBq) of 89Zr-PSMA-617, 89Zr-PSMA-I&T, 177Lu-PSMA-617, or 177Lu-PSMA-I&T intravenously. Ex vivo biodistribution and PET/SPECT imaging were performed up to 168 h post-injection. Dosimetry was performed from the biodistribution data. The patient received 90.5 MBq 89Zr-PSMA-617 followed by PET/CT imaging. RESULTS: 89Zr-labeled PSMA ligands showed a comparable ex vivo biodistribution to its respective 177Lu-labeled counterparts with high tumor accumulation in the PSMA+ xenografts. However, using a dose estimation model for 177Lu, absorbed radiation dose in bone and kidneys differed among the 177Lu-PSMA and 89Zr-PSMA tracers. 89Zr-PSMA-617 PET in the first human patient showed high contrast of PSMA expressing tissues up to 48 h post-injection. CONCLUSION: PSMA-617 and PSMA-I&T were successfully labeled with 89Zr and demonstrated high uptake in PSMA+ xenografts, which enabled PET up to 168 h post-injection. The biodistribution of 89Zr-PSMA-I&T and 89Zr-PSMA-617 resembled that of 177Lu-PSMA-I&T and 177Lu-PSMA-617, respectively. The first patient 89Zr-PSMA-617 PET images were of high quality warranting further clinical investigation.

Novel VHH-Based Tracers with Variable Plasma Half-Lives for Imaging of CAIX-Expressing Hypoxic Tumor Cells.

Hypoxic areas are present in the majority of solid tumors, and hypoxia is associated with resistance to therapies and poor outcomes. A transmembrane protein that is upregulated by tumor cells that have adapted to hypoxic conditions is carbonic anhydrase IX (CAIX). Therefore, noninvasive imaging of CAIX could be of prognostic value, and it could steer treatment strategies. The aim of this study was to compare variants of CAIX-binding VHH B9, with and without a C-terminal albumin-binding domain with varying affinity (ABDlow and ABDhigh), for SPECT imaging of CAIX expression. The binding affinity and internalization of the various B9-variants were analyzed using SK-RC-52 cells. Biodistribution studies were performed in mice with subcutaneous SCCNij153 human head and neck cancer xenografts. Tracer uptake was determined by ex vivo radioactivity counting and visualized by SPECT/CT imaging. Furthermore, autoradiography images of tumor sections were spatially correlated with CAIX immunohistochemistry. B9-variants demonstrated a similar moderate affinity for CAIX in vitro. Maximal tumor uptake and acceptable tumor-to-blood ratios were found in the SCCNij153 model at 4 h post injection for [111In]In-DTPA-B9 (0.51 ± 0.08%ID/g and 8.1 ± 0.85, respectively), 24 h post injection for [111In]In-DTPA-B9-ABDlow (2.39 ± 0.44%ID/g and 3.66 ± 0.81, respectively) and at 72 h post injection for [111In]In-DTPA-B9-ABDhigh (8.7 ± 1.34%ID/g and 2.43 ± 0.15, respectively). An excess of unlabeled monoclonal anti-CAIX antibody efficiently inhibited tumor uptake of [111In]In-DTPA-B9, while only a partial reduction of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh uptake was found. Immunohistochemistry and autoradiography images showed colocalization of all B9-variants with CAIX expression; however, [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh also accumulated in non-CAIX expressing regions. Tumor uptake of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh, but not of [111In]In-DTPA-B9, could be visualized with SPECT/CT imaging. In conclusion, [111In]In-DTPA-B9 has a high affinity to CAIX and shows specific targeting to CAIX in head and neck cancer xenografts. The addition of ABD prolonged plasma half-life, increased tumor uptake, and enabled SPECT/CT imaging. This uptake was, however, partly CAIX- independent, precluding the ABD-tracers for use in hypoxia quantification in this tumor type.

Multiscale imaging of therapeutic anti-PD-L1 antibody localization using molecularly defined imaging agents.

BACKGROUND: While immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics. RESULTS: To introduce imaging modalities, a peptide containing a near-infrared dye (sulfo-Cy5), a chelator (DTPA), an azide, and a sortase-recognition motif was synthesized. This peptide and a non-fluorescent intermediate were used for site-specific functionalization of c-terminally sortaggable mouse IgG1 (mIgG1) and Fab anti-PD-L1. To increase the half-life of the Fab fragment, a 20 kDa PEG chain was attached via strain-promoted azide-alkyne cycloaddition (SPAAC). Biodistribution and imaging studies were performed with 111In-labeled constructs in 4T1 tumor-bearing mice. Comparing our site-specific antibody-conjugates with randomly conjugated antibodies, we found that antibody clone, isotype and method of DTPA conjugation did not change tumor uptake. Furthermore, addition of sulfo-Cy5 did not affect the biodistribution. PEGylated Fab fragment displayed a significantly longer half-life compared to unPEGylated Fab and demonstrated the highest overall tumor uptake of all constructs. PD-L1 in tumors was clearly visualized by SPECT/CT, as well as whole body fluorescence imaging. Immunohistochemistry staining of tumor sections demonstrated that PD-L1 co-localized with the fluorescent and autoradiographic signal. Intratumoral localization of the imaging agent could be determined with cellular resolution using fluorescent microscopy. CONCLUSIONS: A set of molecularly defined multimodal antibody-based PD-L1 imaging agents were synthesized and validated for multiscale monitoring of PD-L1 expression and localization. Our modular approach for site-specific functionalization could easily be adapted to other targets.

Development and Validation of an Analytical HPLC Method to Assess Chemical and Radiochemical Purity of [68Ga]Ga-NODAGA-Exendin-4 Produced by a Fully Automated Method.

Background: Glucagon-like peptide 1 receptor (GLP-1R) is preferentially expressed in pancreatic islets, especially in ß-cells, and highly expressed in human insulinomas and gastrinomas. In recent years several GLP-1R-avid radioligands have been developed to image insulin-secreting tumors or to provide a tentative quantitative in vivo biomarker of pancreatic ß-cell mass. Exendin-4, a 39-amino acid peptide with high binding affinity to GLP-1R, has been labeled with Ga-68 for imaging with positron emission tomography (PET). Preparation conditions may influence the quality and in vivo behavior of tracers. Starting from a published synthesis and quality controls (QCs) procedure, we have developed and validated a new rapid and simple UV-Radio-HPLC method to test the chemical and radiochemical purity of [68Ga]Ga-NODAGA-exendin-4, to be used in the clinical routine. Methods: Ga-68 was obtained from a 68Ge/68Ga Generator (GalliaPharma®) and purified using a cationic-exchange cartridge on an automated synthesis module (Scintomics GRP®). NODAGA-exendin-4 contained in the reactor (10 µg) was reconstituted with HEPES and ascorbic acid. The reaction mixture was incubated at 100 °C. The product was purified through HLB cartridge, diluted, and sterilized. To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document released by the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH), adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. Therefore, a range of concentrations of Ga-NODAGA-exendin-4, NODAGA-exendin-4 (5, 4, 3.125, 1.25, 1, and 0.75 µg/mL) and [68Ga]Ga-NODAGA-exendin-4 were analyzed. To validate the entire production process, three consecutive batches of [68Ga]Ga-NODAGA-exendin-4 were tested. Results: Excellent linearity was found between 5-0.75 µg/mL for both the analytes (NODAGA-exendin-4 and 68Ga-NODAGA-exendin-4), with a correlation coefficient (R2) for calibration curves equal to 0.999, average coefficients of variation (CV%) < 2% (0.45% and 0.39%) and average per cent deviation value of bias from 100%, of 0.06% and 0.04%, respectively. The calibration curve for the determination of [68Ga]Ga-NODAGA-exendin-4 was linear with a R2 of 0.993 and CV% < 2% (1.97%), in accordance to acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed using 10 µg of peptide. The mean radiochemical yield was 45 ± 2.4% in all the three validation batches of [68Ga]Ga-NODAGA-exendin-4. The radiochemical purity of [68Ga]Ga-NODAGA-exendin-4 was >95% (97.05%, 95.75% and 96.15%) in all the three batches. Conclusions: The developed UV-Radio-HPLC method to assess the radiochemical and chemical purity of [68Ga]Ga-NODAGA-exendin-4 is rapid, accurate and reproducible like its fully automated production. It allows the routine use of this PET tracer as a diagnostic tool for PET imaging of GLP-1R expression in vivo, ensuring patient safety.

Targeted photodynamic therapy selectively kills activated fibroblasts in experimental arthritis.

OBJECTIVE: In RA, synovial fibroblasts become activated. These cells express fibroblast activation protein (FAP) and contribute to the pathogenesis by producing cytokines, chemokines and proteases. Selective depletion in inflamed joints could therefore constitute a viable treatment option. To this end, we developed and tested a new therapeutic strategy based on the selective destruction of FAP-positive cells by targeted photodynamic therapy (tPDT) using the anti-FAP antibody 28H1 coupled to the photosensitizer IRDye700DX. METHODS: After conjugation of IRDye700DX to 28H1, the immunoreactive binding and specificity of the conjugate were determined. Subsequently, tPDT efficiency was established in vitro using a 3T3 cell line stably transfected with FAP. The biodistribution of [111In]In-DTPA-28H1 with and without IRDye700DX was assessed in healthy C57BL/6N mice and in C57BL/6N mice with antigen-induced arthritis. The potential of FAP-tPDT to induce targeted damage was determined ex vivo by treating knee joints from C57BL/6N mice with antigen-induced arthritis 24 h after injection of the conjugate. Finally, the effect of FAP-tPDT on arthritis development was determined in mice with collagen-induced arthritis. RESULTS: 28H1-700DX was able to efficiently induce FAP-specific cell death in vitro. Accumulation of the anti-FAP antibody in arthritic knee joints was not affected by conjugation with the photosensitizer. Arthritis development was moderately delayed in mice with collagen-induced arthritis after FAP-tPDT. CONCLUSION: Here we demonstrate the feasibility of tPDT to selectively target and kill FAP-positive fibroblasts in vitro and modulate arthritis in vivo using a mouse model of RA. This approach may have therapeutic potential in (refractory) arthritis.

Lutetium-177-PSMA-I&T as metastases directed therapy in oligometastatic hormone sensitive prostate cancer, a randomized controlled trial.

BACKGROUND: In recent years, there is increasing evidence showing a beneficial outcome (e.g. progression free survival; PFS) after metastases-directed therapy (MDT) with external beam radiotherapy (EBRT) or targeted surgery for oligometastatic hormone sensitive prostate cancer (oHSPC). However, many patients do not qualify for these treatments due to prior interventions or tumor location. Such oligometastatic patients could benefit from radioligand therapy (RLT) with 177Lu-PSMA; a novel tumor targeting therapy for end-stage metastatic castration-resistant prostate cancer (mCRPC). Especially because RLT could be more effective in low volume disease, such as the oligometastatic status, due to high uptake of radioligands in smaller lesions. To test the hypothesis that 177Lu-PSMA is an effective treatment in oHSPC to prolong PFS and postpone the need for androgen deprivation therapy (ADT), we initiated a multicenter randomized clinical trial. This is globally, the first prospective study using 177Lu-PSMA-I&T in a randomized multicenter setting. METHODS & DESIGN: This study compares 177Lu-PSMA-I&T MDT to the current standard of care (SOC); deferred ADT. Fifty-eight patients with oHSPC (≤5 metastases on PSMA PET) and high PSMA uptake (SUVmax > 15, partial volume corrected) on 18F-PSMA PET after prior surgery and/or EBRT and a PSA doubling time of < 6 months, will be randomized in a 1:1 ratio. The patients randomized to the interventional arm will be eligible for two cycles of 7.4GBq 177Lu-PSMA-I&T at a 6-week interval. After both cycles, patients are monitored every 3 weeks (including adverse events, QoL- and xerostomia questionnaires and laboratory testing) at the outpatient clinic. Twenty-four weeks after cycle two an end of study evaluation is planned together with another 18F-PSMA PET and (whole body) MRI. Patients in the SOC arm are eligible to receive 177Lu-PSMA-I&T after meeting the primary study objective, which is the fraction of patients who show disease progression during the study follow up. A second primary objective is the time to disease progression. Disease progression is defined as a 100% increase in PSA from baseline or clinical progression. DISCUSSION: This is the first prospective randomized clinical study assessing the therapeutic efficacy and toxicity of 177Lu-PSMA-I&T for patients with oHSPC. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT04443062 .

PET imaging during hypoglycaemia to study adipose tissue metabolism.

BACKGROUND: Disturbances in adipose tissue glucose uptake may play a role in the pathogenesis of type 2 diabetes, yet its examination by 2-deoxy-2-[18 F]fluorodeoxyglucose ([18 F]FDG) PET/CT is challenged by relatively low uptake kinetics. We tested the hypothesis that performing [18 F]FDG PET/CT during a hypoglycaemic clamp would improve adipose tissue tracer uptake to allow specific comparison of adipose tissue glucose handling between people with or without type 2 diabetes. DESIGN: We enrolled participants with or without diabetes who were at least overweight, to undergo a hyperinsulinaemic hypoglycaemic clamp or a hyperinsulinaemic euglycaemic clamp (n = 5 per group). Tracer uptake was quantified using [18 F]FDG PET/CT. RESULTS: Hypoglycaemic clamping increased [18 F]FDG uptake in visceral adipose tissue of healthy participants (P = 0.002). During hypoglycaemia, glucose uptake in visceral adipose tissue of type 2 diabetic participants was lower as compared to healthy participants (P < 0.0005). No significant differences were observed in skeletal muscle, liver or pancreas. CONCLUSIONS: The present findings indicate that [18 F]FDG PET/CT during a hypoglycaemic clamp provides a promising new research tool to evaluate adipose tissue glucose metabolism. Using this method, we observed a specific impairment in visceral adipose tissue [18 F]FDG uptake in type 2 diabetes, suggesting a previously underestimated role for adipose tissue glucose handling in type 2 diabetes.

Exendin-4 Derivatives with an Albumin-Binding Moiety Show Decreased Renal Retention and Improved GLP-1 Receptor Targeting.

The glucagon-like peptide-1 receptor (GLP-1R) is highly and specifically expressed on the pancreatic ß-cells. It plays an important role in glucose metabolism as well as in ß-cell-derived diseases like diabetes, insulinoma, or congenital and adult hyperinsulinemic hypoglycemia. Radiolabeled exendin-4, a ligand of GLP-1R, has routinely been used in clinics to image insulinomas. However, its major drawback is the high kidney accumulation. Here, we show that the addition of an albumin-binding moiety (ABM) to radiolabeled exendin-4 results in a significant reduction of kidney uptake while retaining its high affinity and specificity to GLP-1R. The four tested peptides were shown to have high affinity to the GLP-1 receptor (IC50 of 3.7 ± 0.6 to 15.1 ± 0.8 nM). The radiolabeled derivatives were taken up into cells efficiently, internalizing between 39 ± 2 and 56 ± 2% after 2 h. Thus, the derivatives with ABM outperformed the reference peptide with its IC50 of 22.5 ± 2.9 nM and internalization of 41 ± 4%. Stability in human blood plasma was slightly enhanced by the addition of the albumin binder. In biodistribution studies, the radioligands exhibited an improved target-to-kidney ratio in comparison to the reference peptide of up to seven-fold. This was confirmed qualitatively in single-photon-emission computed tomography (SPECT)/CT imaging. This study demonstrated in vitro and in vivo that the addition of an ABM to radiolabeled exendin-4 strongly decreased kidney accumulation while retaining affinity to GLP-1R. Thus, exendin-4 derivatives with an albumin-binding moiety could present a viable class of diagnostic tracers for the detection of insulinomas and other GLP-1R-positive tissue in clinical application.