Validation of HPLC and TLC analytical methods to determine radiochemical purity of 99mTc-cAbVCAM1-5, a new experimental radiotracer.

Cardiovascular diseases, including fatal myocardial infarctions from atheromatous plaques, are the primary global mortality cause. Detecting stenotic atheromatous plaques is possible through coronary angiography, but vulnerable plaques with eccentric remodeling are undetectable with current diagnostic methods. Addressing this challenge, our group developed a radiopharmaceutical drug targeting vascular cell adhesion molecule 1 (VCAM-1), radiolabeled with technetium-99m. Given the absence of a monograph in the European Pharmacopoeia, and in order to draft the investigational medicinal product documentation, analytical methods had to be validated by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) to determine the radiochemical purity (RCP) of 99mTc-cAbVCAM1-5. This study therefore presents the results of the validation of analytical methods obtained in this context. The method validation followed the European Association of Nuclear Medicine (EANM) recommendations adapted from ICH Q2(R1), ensuring conformity with specificity, accuracy, repeatability and intermediate precision, linearity, robustness, quantification limit (LoQ), and range criteria. Regarding the results of specificity, both HPLC and TLC methods demonstrated excellent separation of 99mTc-cAbVCAM1-5 from impurities 99mTcO4-. Accuracy results indicated recovery percentages within the range of 99.52-101.40% for the HPLC and 99.51-101.97% for TLC, ensuring reliable measurements for each concentration of 99mTcO4-. Precision of the methods was validated by assessing repeatability and intermediate precision. Linearity was determined over the usual concentrations range and the correlation coefficient was greater than 0.99 for both methods. The limit of quantification was measured by diluting the 99mTcO4- to obtain a signal-to-noise ratio of around 10:1. Under these conditions, we obtained an LOQ of 2.10 MBq/mL for HPLC and 2Mbq/mL for TLC. In conclusion, the analytical methods developed in this study comply with EANM recommendations. This therefore allows us to correctly assess the radiochemical purity of 99mTc-cAbVCAM1-5, a new radiotracer targeting inflammation in vulnerable plaques.

A single-domain antibody for the detection of pathological Tau protein in the early stages of oligomerization.

BACKGROUND: Soluble oligomeric forms of Tau protein have emerged as crucial players in the propagation of Tau pathology in Alzheimer's disease (AD). Our objective is to introduce a single-domain antibody (sdAb) named 2C5 as a novel radiotracer for the efficient detection and longitudinal monitoring of oligomeric Tau species in the human brain. METHODS: The development and production of 2C5 involved llama immunization with the largest human Tau isoform oligomers of different maturation states. Subsequently, 2C5 underwent comprehensive in vitro characterization for affinity and specificity via Enzyme-Linked Immunosorbent Assay and immunohistochemistry on human brain slices. Technetium-99m was employed to radiolabel 2C5, followed by its administration to healthy mice for biodistribution analysis. RESULTS: 2C5 exhibited robust binding affinity towards Tau oligomers (Kd = 6.280 nM ± 0.557) and to Tau fibers (Kd = 5.024 nM ± 0.453), with relatively weaker binding observed for native Tau protein (Kd = 1791 nM ± 8.714) and amyloid peptide (Kd > 10,000 nM). Remarkably, this SdAb facilitated immuno-histological labeling of pathological forms of Tau in neurons and neuritic plaques, yielding a high-contrast outcome in AD patients, closely mirroring the performance of reference antibodies AT8 and T22. Furthermore, 2C5 SdAb was successfully radiolabeled with 99mTc, preserving stability for up to 6 h post-radiolabeling (radiochemical purity > 93%). However, following intravenous injection into healthy mice, the predominant uptake occurred in kidneys, amounting to 115.32 ± 3.67, 97.70 ± 43.14 and 168.20 ± 34.52% of injected dose per gram (% ID/g) at 5, 10 and 45 min respectively. Conversely, brain uptake remained minimal at all measured time points, registering at 0.17 ± 0.03, 0.12 ± 0.07 and 0.02 ± 0.01% ID/g at 5, 10 and 45 min post-injection respectively. CONCLUSION: 2C5 demonstrates excellent affinity and specificity for pathological Tau oligomers, particularly in their early stages of oligomerization. However, the current limitation of insufficient blood-brain barrier penetration necessitates further modifications before considering its application in nuclear medicine imaging for humans.

Bacterial survival in radiopharmaceutical solutions: a critical impact on current practices.

BACKGROUND: The aim of this brief communication is to highlight the potential bacteriological risk linked to the processes control of radiopharmaceutical preparations made in a radiopharmacy laboratory. Survival rate of Pseudomonas aeruginosa (ATCC: 27853) or Staphylococcus aureus (ATCC: 25923) or Staphylococcus epidermidis (ATCC: 1228) in multidose technetium-99 m solution was studied. RESULTS: Depending on the nature and level of contamination by pathogenic bacteria, the lethal effect of radioactivity is not systematically observed. We found that P. aeruginosa was indeed affected by radioactivity. However, this was not the case for S. epidermidis, as the quantity of bacteria found in both solutions (radioactive and non-radioactive) was rapidly reduced, probably due to a lack of nutrients. Finally, the example of S. aureus is an intermediate case where we observed that high radioactivity affected the bacteria, as did the absence of nutrients in the reaction medium. The results were discussed in the light of current practices on the sterility test method, which recommends waiting for radioactivity to decay before carrying out the sterility test. CONCLUSION: In terms of patient safety, the results run counter to current practice and the latest EANM recommendation of 2021 that radiopharmaceutical preparations should be decayed before sterility testing.

TrisOxine abiotic siderophores for technetium complexation: radiolabeling and biodistribution studies.

BACKGROUND: Despite the development of positron emission tomography (PET), single photon emission computed tomography (SPECT) still accounts for around 80% of all examinations performed in nuclear medicine departments. The search for new radiotracers or chelating agents for Technetium-99m is therefore still ongoing. O-TRENSOX and O-TRENOX two synthetic siderophores would be good candidates for this purpose as they are hexadentate ligands based on the very versatile and efficient 8-hydroxyquinoline chelating subunit. First, the radiolabeling of O-TRENOX and O-TRENSOX with 99mTc was investigated. Different parameters such as the quantity of chelating agent, type of reducing agent, pH and temperature of the reaction mixture were adjusted in order to find the best radiolabeling conditions. Then an assessment of the partition coefficient by measuring the distribution of each radiosynthesized complex between octanol and phosphate-buffered saline was realized. The complex's charge was evaluated on three different celluloses (neutral, negatively charged P81 and positively charged DE81), and finally in vivo studies with biodistribution and SPECT imaging of [99mTc]Tc-O-TRENOX and [99mTc]Tc-O-TRENSOX were performed. RESULTS: The radiolabeling studies showed a rapid and efficient complexation of 99mTc with both chelating agents. Using tin pyrophosphate as the reducing agent and a minimum of 100 nmol of ligand, we obtained the [99mTc]Tc-O-TRENOX complex with a radiochemical purity of more than 98% and the [99mTc]Tc-O-TRENSOX complex with one above 97% at room temperature within 5 min. [99mTc]Tc-O-TRENOX complex was lipophilic and neutral, leading to a hepatobiliary elimination in mice. On the contrary, the [99mTc]Tc-O-TRENSOX complex was found to be hydrophilic and negatively charged. This was confirmed by a predominantly renal elimination in mice. CONCLUSIONS: These encouraging results allow us to consider the O-TRENOX/99mTc and O-TRENSOX/99mTc complexes as serious candidates for SPECT imaging chelators. This study should be continued by conjugating these tris-oxine ligands to peptides or antibodies and comparing them with the other bifunctional agents used with Tc.

Regional CZT myocardial perfusion reserve for the detection of territories with simultaneously impaired CFR and IMR in patients without obstructive coronary artery disease: a pilot study.

OBJECTIVES: To assess the diagnostic performances of CZT myocardial perfusion reserve (MPR) for the detection of territories with simultaneous impaired coronary flow reserve (CFR) and index of microcirculatory resistance (IMR) in patients without obstructive coronary artery disease. METHODS: Patients were prospectively included before being referred for coronary angiography. All patients underwent CZT MPR before invasive coronary angiography (ICA) and coronary physiology assessment. Rest and dipyridamole-induced stress myocardial blood flow (MBF) and MPR were quantified using 99mTc-SestaMIBI and a CZT camera. Fractional flow reserve (FFR), Thermodilution CFR, and IMR were assessed during ICA. RESULTS: Between December 2016 and July 2019, 36 patients were included. 25/36 patients presented no obstructive coronary artery disease. A complete functional assessment was performed in 32 arteries. No territory presented a significant ischemia on CZT myocardial perfusion imaging. A moderate yet significant correlation was observed between regional CZT MPR and CFR (r = 0.4, P = .03). Sensitivity, specificity, positive and negative predictive value, and accuracy of regional CZT MPR versus the composite invasive criterion (impaired CFR and IMR) were 87 [47% to 99%], 92% [73% to 99%], 78% [47% to 93%], 96% [78% to 99%], and 91% [75% to 98%], respectively. All territories with a regional CZT MPR ≤ 1.8 showed a CFR < 2. Regional CZT MPR values were significantly higher in arteries with CFR ≥ 2 and IMR < 25 (negative composite criterion, n = 14) than in those with CFR < 2 and IMR ≥ 25 (2.6 [2.1 to 3.6] versus 1.6 [1.2 to 1.8]), P < .01). CONCLUSION: Regional CZT MPR presented excellent diagnostic performances for the detection of territories with simultaneously impaired CFR and IMR reflecting a very high cardiovascular risk in patients without obstructive coronary artery disease.

Development and Validation of Analytical Methods for Radiochemical Purity of 177Lu-PSMA-1.

Prostate Specific Membrane Antigen (PSMA) is a highly relevant target in nuclear medicine due to its overexpression in prostate cancer. The 68Ga/177Lu-PSMA-1 combination is a theranostic agent for the detection and treatment of tumors overexpressing the PSMA target. Specifically, 177Lu-PSMA-1 is used in the treatment of castration-resistant prostate cancer that is ineffective or intolerant to the latest generation of chemotherapy and/or hormone therapy. This radiopharmaceutical is manufactured in a radiopharmaceutical synthesizing unit and must pass a quality control where the radiochemical purity (RCP) is assessed prior to release of the batch. RCP evaluation is performed by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). Since there is no monograph for 177Lu-PSMA-1 in the European Pharmacopoeia, we validate the analytical methods according to the EANM recommendations adapted from ICH Q2. Specificity, linearity, accuracy, precision, intermediate precision, limit of quantification (LOQ) and robustness were described for HPLC and TLC in this study. The results obtained demonstrated the robustness and reliability of the HPLC and TLC analytical methods for the evaluation of the RCP of 177Lu-PSMA-1.

Outcome Prediction at Patient Level Derived from Pre-Treatment 18F-FDG PET Due to Machine Learning in Metastatic Melanoma Treated with Anti-PD1 Treatment.

(1) Background: As outcome of patients with metastatic melanoma treated with anti-PD1 immunotherapy can vary in success, predictors are needed. We aimed to predict at the patients' levels, overall survival (OS) and progression-free survival (PFS) after one year of immunotherapy, based on their pre-treatment 18F-FDG PET; (2) Methods: Fifty-six metastatic melanoma patients-without prior systemic treatment-were retrospectively included. Forty-five 18F-FDG PET-based radiomic features were computed and the top five features associated with the patient's outcome were selected. The analyzed machine learning classifiers were random forest (RF), neural network, naive Bayes, logistic regression and support vector machine. The receiver operating characteristic curve was used to compare model performances, which were validated by cross-validation; (3) Results: The RF model obtained the best performance after validation to predict OS and PFS and presented AUC, sensitivities and specificities (IC95%) of 0.87 ± 0.1, 0.79 ± 0.11 and 0.95 ± 0.06 for OS and 0.9 ± 0.07, 0.88 ± 0.09 and 0.91 ± 0.08 for PFS, respectively. (4) Conclusion: A RF classifier, based on pretreatment 18F-FDG PET radiomic features may be useful for predicting the survival status for melanoma patients, after one year of a first line systemic treatment by immunotherapy.

Decrease in the cortex/striatum metabolic ratio on [18F]-FDG PET: a biomarker of autoimmune encephalitis.

PURPOSE: The aim of this [18F]-FDG PET study was to determine the diagnostic value of the cortex/striatum metabolic ratio in a large cohort of patients suffering from autoimmune encephalitis (AE) and to search for correlations with the course of the disease. METHODS: We retrospectively collected clinical and paraclinical data of patients with AE, including brain 18F-FDG PET/CT. Whole-brain statistical analysis was performed using SPM8 software after activity parametrization to the striatum in comparison to healthy subjects. The discriminative performance of this metabolic ratio was evaluated in patients with AE using receiver operating characteristic curves against 44 healthy subjects and a control group of 688 patients with MCI. Relationship between cortex/striatum metabolic ratios and clinical/paraclinical data was assessed using univariate and multivariate analysis in patients with AE. RESULTS: Fifty-six patients with AE were included. In comparison to healthy subjects, voxel-based statistical analysis identified one large cluster (p-cluster < 0.05, FWE corrected) of widespread decreased cortex/striatum ratio in patients with AE. The mean metabolic ratio was significantly lower for AE patients (1.16 ± 0.13) than that for healthy subjects (1.39 ± 0.08; p < 0.001) and than that for MCI patients (1.32 ± 0.11; p < 0.001). A ratio threshold of 1.23 allowed to detect AE patients with a sensitivity of 71% and a specificity of 82% against MCI patients, and 98% against healthy subjects. A lower cortex/striatum metabolic ratio had a trend towards shorter delay before 18F-FDG PET/CT (p = 0.07) in multivariate analysis. CONCLUSION: The decrease in the cortex/striatal metabolic ratio has a good early diagnostic performance for the differentiation of AE patients from controls.

Pancreatic Ductal Adenocarcinoma: The Dawn of the Era of Nuclear Medicine?

Pancreatic ductal adenocarcinoma (PDAC), accounting for 90-95% of all pancreatic tumors, is a highly devastating disease associated with poor prognosis. The lack of accurate diagnostic tests and failure of conventional therapies contribute to this pejorative issue. Over the last decade, the advent of theranostics in nuclear medicine has opened great opportunities for the diagnosis and treatment of several solid tumors. Several radiotracers dedicated to PDAC imaging or internal vectorized radiotherapy have been developed and some of them are currently under clinical consideration. The functional information provided by Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) could indeed provide an additive diagnostic value and thus help in the selection of patients for targeted therapies. Moreover, the therapeutic potential of ß-- and α-emitter-radiolabeled agents could also overcome the resistance to conventional therapies. This review summarizes the current knowledge concerning the recent developments in the nuclear medicine field for the management of PDAC patients.

Prognostic value of SPECT myocardial perfusion entropy in high-risk type 2 diabetic patients.

PURPOSE: Risk stratification of patients with type 2 diabetes mellitus (T2D) remains suboptimal. We hypothesized that myocardial perfusion entropy (MPE) quantified from SPECT myocardial perfusion images may provide incremental prognostic value in T2D patients independently from myocardial ischemia. METHODS: T2D patients with very high and high cardiovascular risk were prospectively included (n = 166, 65 ± 12 years). Stress perfusion defect was quantified by visual evaluation of SPECT MPI. SPECT MPI was also used for the quantification of rest and stress MPE. The primary end point was major adverse cardiac events (MACEs) defined as cardiac death, myocardial infarction (MI), and myocardial revascularization > 3 months after SPECT. RESULTS: Forty-four MACEs were observed during a 4.6-year median follow-up. Significant differences in stress MPE were observed between patients with and without MACEs (4.19 ± 0.46 vs. 3.93 ± 0.40; P ≤ .01). By Kaplan-Meier analysis, the risk of MACEs was significantly higher in patients with higher stress MPE (log-rank P ≤ 01). Stress MPE and stress perfusion defect (SSS ≥ 4) were significantly associated with the risk of MACEs (hazard ratio 2.77 and 2.06, respectively, P < .05 for both) after adjustment for clinical and imaging risk predictors as identified from preliminary univariate analysis. MPE demonstrated incremental prognostic value over clinical risk factors, stress test EKG and SSS as evidenced by nested models showing improved Akaike information criterion (AIC), reclassification (global continuous net reclassification improvement [NRI]: 63), global integrated discrimination improvement (IDI: 6%), and discrimination (change in c-statistic: 0.66 vs 0.74). CONCLUSIONS: Stress MPE provided independent and incremental prognostic information for the prediction of MACEs in diabetic patients. TRIAL REGISTRATION NUMBER: NCT02316054 (12/12/2014).

Aggressive Bone Metastatic Prostate Cancer With Periosteal Reaction in 18F-Choline PET/CT.

ABSTRACT: Prostate cancer bone metastases usually appear as osteosclerotic lesions. However, atypical lesions have also been described. We report herein the case of a 65-year-old man treated since 2013 for prostate cancer with early bone metastases. This asymptomatic patient was referred for 18F-choline PET/CT due to a major elevation of prostate-specific antigen to >1500 ng/mL. The results indicated multiple bone lesions, disseminated on the axial skeleton, girdles, and upper extremities of femurs. Interestingly, we described the development of an intensely hypermetabolic spiculated periosteal reaction, evidencing a rapidly progressive disease.

Does whole-body bone SPECT/CT provide additional diagnostic information over [18F]-FCH PET/CT for the detection of bone metastases in the setting of prostate cancer biochemical recurrence?

BACKGROUND: To assess whether whole-body (WB) bone SPECT/CT provides additional diagnostic information over [18F]-FCH PET/CT for the detection of bone metastases in the setting of prostate cancer biochemical recurrence (PC-BR). METHODS: Patients referred for a PC-BR and whom benefited from a WB bone SPECT/CT and FCH PET/CT were retrospectively included. Tests were classified as positive, equivocal, or negative for bone metastases. A best valuable comparator (BVC) strategy including imaging and follow-up data was used to determine the metastatic status in the absence of systematic histological evaluation. RESULTS: Between January 2011 and November 2017, 115 consecutive patients with a PC-BR were evaluated. According to the BVC, 30 patients had bone metastases and 85 patients did not present with bone lesions. The sensitivity, specificity, positive and negative predictive values were respectively 86.7% [69.3-96.2], 98.8% [93.6-100.0], 96.3% [78.7-99.5], and 95.5% [89.4-98.1] for WB bone SPECT/CT and 93.3% [77.9-99.2], 100.0% [95.8-100.0], 100.0 and 97.7% [91.8-99.4] for FCH PET/CT. There was no significant difference in diagnostic accuracy of bone metastases between WB Bone SPECT/CT (AUC 0.824 [0.74-0.90]) and FCH PET/CT (AUC 0.829 [0.75-0.90], p = 0.41). CONCLUSION: Despite good performances for the diagnosis of bone metastases in PC-BR, WB bone SPECT/CT does not provide additive diagnostic information over concomitant FCH PET/CT.

FDG PET/CT of a Supraclavicular Silicone Granuloma at Follow-up of a Breast Carcinoma.

We report herein the case of a 33-year-old woman who was referred for FDG PET/CT staging prior to pregnancy after a 4-year lost to follow-up for a breast invasive ductal carcinoma (pT2N1 SBRII). FDG PET/CT revealed right supraclavicular lymphadenopathy potentially caused by breast carcinoma recurrence. No additional site was involved. Supraclavicular ultrasonography showed typical "snowstorm" appearance. MRI revealed signs of breast implant intracapsular rupture and signal intensity of silicone within a supraclavicular node. Fine-needle aspiration and microbiopsy of adenopathy finally confirmed silicone granuloma and ruled out breast cancer recurrence.

Prospective diagnostic performance of semiconductor SPECT myocardial perfusion imaging: wall thickening analysis reduces the need for an additional prone acquisition.

PURPOSE: To determine whether the assessment of regional wall thickening (WT) in addition to myocardial perfusion from stress supine acquisitions could compensate for the lack of prone acquisition and the corresponding decrease in the diagnostic performance of SPECT myocardial perfusion imaging (MPI) in patients with known or suspected coronary artery disease (CAD). METHODS: The study group comprised 41 patients (123 vessels) with known or suspected CAD prospectively recruited for systematic prone and supine 201Tl stress SPECT MPI. The diagnostic performance of SPECT MPI was determined for various image sets including nongated supine images (supine NG), nongated combined prone and supine images (prone and supine NG) and gated supine images, allowing WT evaluation from NG images in addition to perfusion (supine NG + WT) using invasive coronary angiography and fractional flow reserve as the gold standards. RESULTS: The rate of false positives was significantly higher among the supine NG images (20.8%) than among either the prone and supine NG or the supine NG + WT images (3.3% and 2.7%, respectively, P < 0.05 vs. supine NG). Consequently, specificity was higher for the prone and supine NG images than for the supine NG images (96.1% vs. 76.1%, P < 0.01) and was highest for the supine NG + WT images (96.8%, P not significant vs. prone and supine NG), without significant differences in sensitivity (80.0%, 86.6% and 73.3%, respectively, P not significant for all comparisons). CONCLUSION: The diagnostic performance of supine stress SPECT MPI is improved when WT assessment of ischaemic segments is used as an additional diagnostic criterion to values not significantly different from those with combined prone and supine acquisitions.

18F-Choline and 18F-FDG PET/CT in a Patient With Diffuse Large B-Cell Lymphoma and Recurrent Prostate Cancer.

We report herein the case of an 80-year-old man who was referred for a biochemical recurrence of a high-risk prostate cancer. In addition to prostate cancer recurrence, F-choline allowed partial initial staging of an incidental diffuse large B-cell lymphoma which was further confirmed and staged using F-FDG and a biopsy. Two types of metabolic behavior were therefore identified using F-choline and F-FDG which corresponded to 2 different uptake patterns, that is, those of the prostate and lymphoma tumoral cell contingents.