Hypoxia PET Research Articles

Evaluation of an automated synthesis method for [18F]Fluoromisonidazole using a Scintomics GRP® module

There is limited information on utilizing commercially supplied [18F]Fluoride from an off-site cyclotron for the synthesis of radiopharmaceuticals. This study explored the production of the PET hypoxia marker [18F]FMISO, using the Scintomics® GRP module and distantly produced [18F]Fluoride, which lacks published data. A radiochemical yield of 27.4 ± 3.6% (n = 5) and high radiochemical purity were achieved in synthesis time of 48 min. The [18F]FMISO met all Ph. Eur. standards, demonstrating the consistency and reliability of the module in generating a clinical-grade radiopharmaceutical using off-site produced [18F]Fluoride, but available patient doses were severely limited.

Development of 18F-Fluoromisonidazole Hypoxia PET/CT Diagnostic Interpretation Criteria and Validation of Interreader Reliability, Reproducibility, and Performance.

Tumor hypoxia, an integral biomarker to guide radiotherapy, can be imaged with 18F-fluoromisonidazole (18F-FMISO) hypoxia PET. One major obstacle to its broader application is the lack of standardized interpretation criteria. We sought to develop and validate practical interpretation criteria and a dedicated training protocol for nuclear medicine physicians to interpret 18F-FMISO hypoxia PET. Methods: We randomly selected 123 patients with human papillomavirus-positive oropharyngeal cancer enrolled in a phase II trial who underwent 123 18F-FDG PET/CT and 134 18F-FMISO PET/CT scans. Four independent nuclear medicine physicians with no 18F-FMISO experience read the scans. Interpretation by a fifth nuclear medicine physician with over 2 decades of 18F-FMISO experience was the reference standard. Performance was evaluated after initial instruction and subsequent dedicated training. Scans were considered positive for hypoxia by visual assessment if 18F-FMISO uptake was greater than floor-of-mouth uptake. Additionally, SUVmax was determined to evaluate whether quantitative assessment using tumor-to-background ratios could be helpful to define hypoxia positivity. Results: Visual assessment produced a mean sensitivity and specificity of 77.3% and 80.9%, with fair interreader agreement (κ = 0.34), after initial instruction. After dedicated training, mean sensitivity and specificity improved to 97.6% and 86.9%, with almost perfect agreement (κ = 0.86). Quantitative assessment with an estimated best SUVmax ratio threshold of more than 1.2 to define hypoxia positivity produced a mean sensitivity and specificity of 56.8% and 95.9%, respectively, with substantial interreader agreement (κ = 0.66), after initial instruction. After dedicated training, mean sensitivity improved to 89.6% whereas mean specificity remained high at 95.3%, with near-perfect interreader agreement (κ = 0.86). Conclusion: Nuclear medicine physicians without 18F-FMISO hypoxia PET reading experience demonstrate much improved interreader agreement with dedicated training using specific interpretation criteria.

18 F-Fluorination of Nitroimidazolyl-Containing Sultone: A Direct Access to a Highly Hydrophilic Radiotracer for High-Performance Positron Emission Tomography Imaging of Hypoxia.

Hypoxia, characterized by nonphysiological levels of oxygen tension, is a key phenomenon common to the majority of malignant tumors with poor prognosis. Many efforts have been made to develop hypoxia imaging for diagnosis, staging, and monitoring of diseases, as well as for evaluating therapies. PET Imaging using 18F-fluoronitroimidazoles (i.e., [18F]FMISO as a lead radiotracer) has demonstrated potential for clinical investigations, but the poor contrast and prolonged acquisition times (>2.5 h) strongly limit its accuracy and routine developments. Here, we report an original [18F]fluoronitroimidazole bearing a sulfo group ([18F]FLUSONIM) that displays highly hydrophilic properties and rapid clearance, providing high-performance hypoxia specific PET imaging. We describe the synthesis and radiosynthesis of [18F]FLUSONIM, its in vivo preclinical evaluation by PET imaging in healthy rats and a rhabdomyosarcoma rat model, as well as its radiometabolization and histological studies. [18F]FLUSONIM was prepared in a single step by high yielding radiofluorination of a sultone precursor, highlighting the advantages of this new radiolabeling approach not yet explored for radiopharmaceutical development. PET imaging experiments were conducted by systematically comparing [18F]FLUSONIM to [18F]FMISO as a reference. The overall results unequivocally demonstrate that the developed radiopharmaceutical meets the criteria of an ideal candidate for hypoxia PET imaging-rapid and efficient radiosynthesis, total stability, exclusive urinary elimination, high specificity for hypoxic regions, unprecedented tumor/background ratios, short acquisition delays (<60 min), and promising potential for further preclinical and clinical applications.

Imaging of Hypoxia in Liver Tumors With 18 F-FMISO PET Before Selective Internal Radiotherapy With 90 Y Microspheres.

Hypoxia is a known cause of resistance to radiotherapy and chemotherapy. Although there are multiple studies in external radiation therapies based on hypoxia PET, the effect of hypoxia in radioembolization is largely unknown. Here we present 2 cases of hepatocellular carcinoma patients from a prospective study with different lesion characteristics on pretreatment 18 F-FMISO PET and varying responses on 18 F-FDG PET.

2‐Nitroimidazole Based PET Tracers: Development of a General Synthetic Approach and Fluorination Methodology

Abstract2‐Nitroimidazole based PET tracers are widely used for non‐invasive hypoxic tumor diagnosis. In this context, we report here a general method based on a central Mitsunobu reaction to easily access 2‐nitroimidazole precursors. Optimization of fluorination conditions is described and successfully applied to the radiofluorination of the 2‐nitroimidazole precursors. Moreover, the radiosynthesis of a new potential hypoxia PET tracer is also described. Finally, this article presents a SFC analytical method allowing both an easy monitoring of the fluorination reactions and the measurement of partition coefficients (logP).

Hypoxia adapted relative biological effectiveness models for proton therapy: a simulation study

In proton therapy, a constant relative biological effectiveness (RBE) factor of 1.1 is applied although the RBE has been shown to depend on factors including the Linear Energy Transfer (LET). The biological effectiveness of radiotherapy has also been shown to depend on the level of oxygenation, quantified by the oxygen enhancement ratio (OER). To estimate the biological effectiveness across different levels of oxygenation the RBE-OER-weighted dose (ROWD) can be used. To investigate the consistency between different approaches to estimate ROWD, we implemented and compared OER models in a Monte Carlo (MC) simulation tool. Five OER models were explored: Wenzl and Wilkens 2011 (WEN), Tinganelli et al 2015 (TIN), Strigari et al 2018 (STR), Dahle et al 2020 (DAH) and Mein et al 2021 (MEI). OER calculations were combined with a proton RBE model and the microdosimetric kinetic model for ROWD calculations. ROWD and OER were studied for a water phantom scenario and a head and neck cancer case using hypoxia PET data for the OER calculation. The OER and ROWD estimates from the WEN, MEI and DAH showed good agreement while STR and TIN gave higher OER values and lower ROWD. The WEN, STR and DAH showed some degree of OER-LET dependency while this was negligible for the MEI and TIN models. The ROWD for all implemented models is reduced in hypoxic regions with an OER of 1.0–2.1 in the target volume. While some variations between the models were observed, all models display a large difference in the estimated dose from hypoxic and normoxic regions. This shows the potential to increase the dose or LET in hypoxic regions or reduce the dose to normoxic regions which again could lead to normal tissue sparing. With reliable hypoxia imaging, RBE-OER weighting could become a useful tool for proton therapy plan optimization.

Kinetic Evaluation of the Hypoxia Radiotracers [18F]FMISO and [18F]FAZA in Dogs with Spontaneous Tumors Using Dynamic PET/CT Imaging

PurposeWe evaluated the kinetics of the hypoxia PET radiotracers, [18F]fluoromisonidazole ([18F]FMISO) and [18F]fluoroazomycin-arabinoside ([18F]FAZA), for tumor hypoxia detection and to assess the correlation of hypoxic kinetic parameters with static imaging measures in canine spontaneous tumors.MethodsSixteen dogs with spontaneous tumors underwent a 150-min dynamic PET scan using either [18F]FMISO or [18F]FAZA. The maximum tumor-to-muscle ratio (TMRmax) > 1.4 on the last image frame was used as the standard threshold to determine tumor hypoxia. The tumor time-activity curves were analyzed using irreversible and reversible two-tissue compartment models and graphical methods. TMRmax was compared with radiotracer trapping rate (k3), influx rate (Ki), and distribution volume (VT).ResultsTumor hypoxia was detected in 7/8 tumors in the [18F]FMISO group and 4/8 tumors in the [18F]FAZA group. All hypoxic tumors were detected at > 120 min with [18F]FMISO and at > 60 min with [18F]FAZA. [18F]FAZA showed better fit with the reversible model. TMRmax was strongly correlated with the irreversible parameters (k3 and Ki) for [18F]FMISO at > 90 min and with the reversible parameter (VT) for [18F]FAZA at > 120 min.ConclusionsOur results showed that [18F]FAZA provided a promising alternative radiotracer to [18F]FMISO with detecting the presence of tumor hypoxia at an earlier time (60 min), consistent with its favorable faster kinetics. The strong correlation between TMRmax over the 90–150 min and 120–150 min timeframes with [18F]FMISO and [18F]FAZA, respectively, with kinetic parameters associated with tumor hypoxia for each radiotracer, suggests that a static scan measurement (TMRmax) is a good alternative to quantify tumor hypoxia.

P15.02.B Spatial habitat analysis in high-grade gliomas combining perfusion, diffusion, and hypoxia features derived from multiparametric MRI and 18F-FAZA PET

Abstract Background Heterogeneity represents one of the main issues in high-grade gliomas (HGGs) management and presumably is the key to understanding treatment failure. Spatial habitat imaging embodies a novel, non-invasive method to cluster different tumor biological microenvironments and characterize intratumoral heterogeneity through a quantitative radiomic approach. So far, habitat imaging has been chiefly explored on morphological magnetic resonance imaging (MRI): the aim of our study was to evaluate this technique on advanced imaging techniques as perfusion (PWI) and diffusion (dMRI) MRI, as well as 18F-FAZA PET, to subdivide the tumor into different biological compartments. Materials and Methods A preoperative PWI, dMRI, and 18F-FAZA PET acquisition was obtained in 17 HGG patients to assess intratumoral vascularization, cellularity, and hypoxia, respectively. A tumor mask was created including contrast-enhancing/necrotic regions on T1 imaging and surrounding FLAIR hyperintensity, and each parametric map (plasma volume from dynamic contrast-enhanced PWI, mean diffusivity from diffusion tensor imaging, and standardized uptake value from 18F-FAZA PET) underwent OTSU’s binary clustering by grouping together voxels with high and low-intensity values. Cluster intersections were then generated by combining all possible high and low-intensity regions of each map, and the final habitat map was composed. Habitats’ quantitative features, spatial distribution, and histopathological characteristics were analyzed. Results The eight habitats generated were carefully inspected, and their distribution pattern, in relation to morphological landmarks (as contrast-enhancing, necrotic, infiltrative and edematous areas), showed an excellent reproducibility among different HGGs, offering complementary data to conventional imaging. A consistent correlation was noted between each habitat’s awaited microenvironment and the corresponding histopathological characteristics among the ten patients for whom precise coordinates of stereotactic biopsies were available. Clusters considered more aggressive were more represented in WHO grade IV (vs. grade III) tumors. The habitat possibly representing edematous areas was significantly larger in IDH-1 wild-type (vs. IDH-1 mutated) tumors. Conclusions: PET and MRI hypoxia, perfusion, and tissue diffusion habitat maps proved an excellent reproducibility among HGGs and correlation with expected histopathological features, making it a way to characterize intratumoral heterogeneity. However, further study is needed to elucidate clinical implications and confirm the spatio-temporal evolution of the identified habitats. Funding Italian Ministry of Health, grant number GR-2018-12365670; Italian Association for Cancer Research (grant IG 2014 Id.1524).

Correlation of hypoxia PET tracer uptake with hypoxic radioresistance in cancer cells: PET biomarkers of resistance to stereotactic radiation therapy?

PurposeThe pO2 threshold of an ideal PET hypoxia tracer for radiotherapy planning in cancer would match those observed in clinically and biologically relevant processes such as radioresistance and HIF1α expression. To identify such tracers, we directly compared uptake in vitro of hypoxia PET tracers ([18F]FMISO, [64Cu]CuATSM, and analogues [64Cu]CuATS, [64Cu]CuATSE, [64Cu]CuCTS, [64Cu]CuDTS, [64Cu]CuDTSE, [64Cu]CuDTSM) with levels of radioresistance and HIF1α expression in cultured cancer cells under identical hypoxic conditions ranging from extreme hypoxia to normoxia. Pimonidazole uptake was also compared as a marker of hypoxia. MethodsA custom-built hypoxia apparatus enabled all experiments to be performed under identical hypoxic conditions with constant measurement of pO2 in media using an OxyLab pO2™ probe. HCT116 human colonic carcinoma and MCF-7 human Caucasian breast adenocarcinoma cells were irradiated using a cobalt teletherapy unit. Clonogenic assays were used to assess survival. HIF1α expression was determined by western blotting, tracer uptake by gamma counting and pimonidazole binding by flow cytometry. ResultsRadioresistance, pimonidazole binding and HIF1α expression increased gradually as pO2 decreased between 25 mmHg and 0 mmHg. In contrast, all the PET hypoxia tracers showed a sharp increase in uptake only when pO2 levels fell below 1 mmHg. Above this threshold, tracer uptake was not elevated above that in normoxic cells. ConclusionThis study highlights an important mismatch in pO2 thresholds between these PET tracers and other markers of hypoxia: tracer uptake only occurred at oxygen levels that were well below levels that induced radioresistance, pimonidazole uptake and HIF1α expression. Although their pO2 thresholds do not match the threshold for resistance to conventionally fractionated radiotherapy (pO2 2.5–10 mmHg), their specificity for extreme hypoxia (pO2 ≪ 1 mmHg) suggests these PET tracers may be of particular use to predict outcomes in stereotactic radiation therapy where these maximally resistant cells play a key role in determining the biological effect.

Investigation of atovaquone-induced spatial changes in tumour hypoxia assessed by hypoxia PET/CT in non-small cell lung cancer patients

BackgroundTumour hypoxia promotes an aggressive tumour phenotype and enhances resistance to anticancer treatments. Following the recent observation that the mitochondrial inhibitor atovaquone increases tumour oxygenation in NSCLC, we sought to assess whether atovaquone affects tumour subregions differently depending on their level of hypoxia.MethodsPatients with resectable NSCLC participated in the ATOM trial (NCT02628080). Cohort 1 (n = 15) received atovaquone treatment, whilst cohort 2 (n = 15) did not. Hypoxia-related metrics, including change in mean tumour-to-blood ratio, tumour hypoxic volume, and fraction of hypoxic voxels, were assessed using hypoxia PET imaging. Tumours were divided into four subregions or distance categories: edge, outer, inner, and centre, using MATLAB.ResultsAtovaquone-induced reduction in tumour hypoxia mostly occurred in the inner and outer tumour subregions, and to a lesser extent in the centre subregion. Atovaquone did not seem to act in the edge subregion, which was the only tumour subregion that was non-hypoxic at baseline. Notably, the most intensely hypoxic tumour voxels, and therefore the most radiobiologically resistant areas, were subject to the most pronounced decrease in hypoxia in the different subregions.ConclusionsThis study provides insights into the action of atovaquone in tumour subregions that help to better understand its role as a novel tumour radiosensitiser.Trial registration: ClinicalTrials.gov, NCT0262808. Registered 11th December 2015, https://clinicaltrials.gov/ct2/show/NCT02628080

Development of a Hypoxia-Immune Prognostic Classifier for Head-and-Neck Cancer Patients Undergoing Radiotherapy ― Results From a Prospective Imaging Trial

Tumor-infiltrating lymphocytes (TILs) correlate with an improved outcome of head-and-neck squamous cell cancer (HNSCC) patients undergoing radiotherapy and are influenced by the tumor microenvironment and tumor-associated hypoxia. The present analysis is based on a prospective imaging trial and analyzes the value of TILs and tumor-associated hypoxia to stratify HNSCC patients according to their response to radiotherapy.A total of 49 patients with locoregionally advanced HNSCCs were prospectively enrolled in this trial and underwent longitudinal hypoxia PET imaging using fluoro-18 misonidazole ([18F]FMISO) in weeks 0, 2 and 5 during chemoradiation. Tumor hypoxia was assumed for a tumor-to-background SUV (contralateral sternocleidomastoid muscle) exceeding 1.4, and an early hypoxia response was defined as a decrease in the normalized maximum intratumoral SUV (FMISO-SUVindex) between weeks 0 and 2. Pre-treatment tumor biopsies were analyzed for TILs and the hypoxia tissue marker carbonic anhydrase IX (CAIX). Trial patients were stratified into 4 subgroups based on their TIL numbers (> 100 vs. < 100 TILs/HPF) and expression of CAIX (above vs. below median H-score), and locoregional control (LRC) and progression-free survival (PFS) rates for all subgroups were assessed using Cox and subsequent concordance analyses (Harrell's C).High TIL levels correlated with improved LRC (HR = 0.279, P = 0.011) and PFS (HR = 0.276, P = 0.006). Similarly, a decrease in the FMISO-SUVindex within the first 2 weeks of treatment corresponded with better LRC (HR = 0.321, P = 0.015) and PFS (HR = 0.402, P = 0.043). Harrell's C was 0.68, when TILs and early hypoxia PET response were combined. The hypoxia PET-based hypoxia-immune classifier separated 3 distinct prognostic patient subgroups, a favorable (TILhigh/ early PET response), and intermediate (TILhigh/no early PET response or TILlow/early PET response) and a poor (TILlow/no early PET response) prognostic group with 2-year LRC of 71%, 33% and 0%, respectively. Low pre-treatment tissue levels of CAIX were also prognostic for improved LRC (HR = 0.352, P = 0.050) but not PFS (HR = 0,468, P = 0,087). Harrell's C was 0.66 for CAIX and TILs separately and 0.71 for the combination. The immunohistochemistry-based immune-hypoxia classifier similarly stratified between a favorable (CAIXlow/TILhigh), an intermediate (CAIXlow/TILlow or CAIXhigh/TILhigh) and a poor (CAIXhigh/TILlow) subgroup with 2-year LRC rates of 73%, 62% and 11%, respectively.We developed a hypoxia PET-based hypoxia-immune classifier that was able to separate HNSCC patients into 3 distinct prognostic subgroups based on their tumor biology. These subgroups could also be stratified in a clinically feasible, immunohistochemistry-based classifier with comparable model accuracy. Therefore, this hypoxia-immune classifier could help to stratify prognoses of HNSCC patients undergoing radiotherapy pending validation in an external cohort.

First Comparison between [18f]-FMISO and [18f]-Faza for Preoperative Pet Imaging of Hypoxia in Lung Cancer.

Simple SummaryThe definition of the tumor hypoxia is important in oncology because this characteristic is linked to a poor prognosis. In this context, we compared two hypoxia tracers, FMISO and FAZA, before surgery for lung cancer. Hypoxia tracers correlate well with each other and FMISO is superior to FAZA in defining the hypoxia volume of lung cancers. However, there is no correlation with immunohistochemical findings (GLUT-1, CAIX, LDH-5, and HIF1-Alpha).Hypoxic areas are typically resistant to treatment. However, the fluorine-18-fluoroazomycin-arabinoside (FAZA) and fluorine 18 misonidazole (FMISO) tracers have never been compared in non small cell lung cancer (NSCLC). This study compares the capability of 18F-FAZA PET/CT with that of 18F-FMISO PET/CT for detecting hypoxic tumour regions in early and locally advanced NSCLC patients. We prospectively evaluated patients who underwent preoperative PET scans before surgery for localised NSCLC (i.e., fluorodeoxyglucose (FDG)-PET, FMISO-PET, and FAZA-PET). The PET data of the three tracers were compared with each other and then compared to immunohistochemical analysis (GLUT-1, CAIX, LDH-5, and HIF1-Alpha) after tumour resection. Overall, 19 patients with a mean age of 68.2 ± 8 years were included. There were 18 lesions with significant uptake (i.e., SUVmax >1.4) for the F-MISO and 17 for FAZA. The mean SUVmax was 3 (±1.4) with a mean volume of 25.8 cc (±25.8) for FMISO and 2.2 (±0.7) with a mean volume of 13.06 cc (±13.76) for FAZA. The SUVmax of F-MISO was greater than that of FAZA (p = 0.0003). The SUVmax of F-MISO shows a good correlation with that of FAZA at 0.86 (0.66–0.94). Immunohistochemical results are not correlated to hypoxia PET regardless of the staining. The two tracers show a good correlation with hypoxia, with FMISO being superior to FAZA. FMISO, therefore, remains the reference tracer for defining hypoxic volumes.

18F-FAZA PET/CT in pretreatment assessment of hypoxic status in high-grade glioma: correlation with hypoxia immunohistochemical biomarkers.

To investigate the correlation between 18F-labeled fluoroazomycinarabinoside (18F-FAZA) PET data and hypoxia immunohistochemical markers in patients with high-grade glioma (HGG). Prospective study including 20 patients with brain MRI suggestive for HGG and undergoing 18F-FAZA PET/CT before treatment for hypoxia assessment. For each 18F-FAZA PET scan SUVmax, SUVmean and 18F-FAZA tumour volume (FTV) at 40, 50 and 60% threshold of SUVmax were calculated; hypoxic volume was estimated by applying different thresholds (1.2, 1.3 and 1.4) to tumour/blood ratio. Seventeen patients were analysed. The immunohistochemical analysis assessed the following parameters: hypoxia-inducible factor 1α, carbonic anhydrase IX (CA-IX), glucose transporter-1, tumour vascularity and Ki-67. 18F-FAZA PET showed a single lesion in 15/17 patients and multiple lesions in 2/17 patients. Twelve/17 patients had grade IV glioma and 5/17 with grade III glioma. Bioptic and surgical samples have been analysed separately. In the surgical subgroup (n = 7) a positive correlation was observed between CA-IX and SUVmax (P = 0.0002), SUVmean40 (P = 0.0058), SUVmean50 (P = 0.009), SUVmean60 (P = 0.0153), FTV-40-50-60 (P = 0.0424) and hypoxic volume1.2-1.3-1.4 (P = 0.0058). In the bioptic group (n = 10) tumour vascularisation was inversely correlated with SUVmax (P = 0.0094), SUVmean40 (P = 0.0107), SUVmean50 (P = 0.0094) and SUVmean60 (P = 0.0154). The correlation of 18F-FAZA PET parameters with CD31 and CA-IX represents a reliable method for assessing tumour hypoxia in HGG. The inverse correlation between tumour vascularisation, SUVmax and SUVmean suggest that highly vascularized tumours might present more oxygen supply than hypoxia.

Synthesis, Radiosynthesis, and in vitro Studies on Novel Hypoxia PET Tracers Incorporating [18F]FDR

AbstractWe report the synthesis of five radiotracers incorporating different oxyamine spacers between the hypoxia‐reactive 2‐nitroimidazole moiety and the 5‐[18F]‐fluorodeoxyribose ([18F]FDR, 12) prosthetic group: three linear alkyl chains with 3, 5, 7 carbon atoms (15 a–c), a cyclopropyl ring (15 d) and a 1,4‐disubstituted‐1,2,3‐triazole (15 e). Experiments in hypoxic cells showed that 15 d displays superior uptake kinetics – and similar selectivity for hypoxic cells – relative to the gold standard hypoxia tracers [18F]fluoroazomycin arabinoside ([18F]FAZA) and [18F]fluoromisonidazole ([18F]FMISO). Lipophilicity and structural rigidity have strong influence on the selectivity of tracers 15 towards hypoxic cells: the lead tracer 15 d displays a logP=0.38 and the most rigid spacer. A sixth radiotracer (15 f), with a 2‐H‐imidazole replacing the 2‐nitroimidazole moiety of 15 d, was used to demonstrate that the cyclopropyl group does not play a meaningful role in the sensitivity towards hypoxia.

Synthesis and Preliminary Evaluation of a Novel 18F-Labeled 2-Nitroimidazole Derivative for Hypoxia Imaging.

ObjectiveHypoxia is prevalent in tumors and plays a pivotal role in resistance to chemoradiotherapy. 18F-MISO (18F-labeled fluoromisonidazole) is currently the preferred choice of PET hypoxia tracers in clinical practice, but has severe disadvantages involving complex labeling methods and low efficient imaging due to lipophilicity. We aimed to design a novel nitroimidazole derivative labeled by 18F via a chelation technique to detect hypoxic regions and provide a basis for planning radiotherapy.Materials and MethodsFirst, we synthesized a 2-nitroimidazole precursor, 2-[4-(carboxymethyl)-7-[2-(2-(2-nitro-1H-imidazol-1-yl)acetamido)ethyl]-1,4,7-triazanonan-1-yl]acetic acid (NOTA-NI). For 18F-labeling, a 18F solution was reacted with a mixture of AlCl3 and NOTA-NI at pH 3.5 and 100°C for 20 min, and the radiochemical purity and stability were evaluated. Biological behaviors of Al18F-NOTA-NI were analyzed by an uptake study in ECA109 normoxic and hypoxic cells, and a biodistribution study and microPET imaging in ECA109 xenografted mice.ResultsAl18F-NOTA-NI required a straightforward and efficient labeling procedure compared with 18F-MISO. The uptake values were distinctly higher in hypoxic tumor cells. Animal studies revealed that the imaging agent was principally excreted via the kidneys. Due to hydrophilicity, the radioactivities in blood and muscle were decreased, and we could clearly distinguish xenografted tumors from para-carcinoma tissue by PET imaging.ConclusionsThe nitroimidazole tracer Al18F-NOTA-NI steadily accumulated in hypoxic areas in tumors and was rapidly eliminated from normal tissue. It appears to be a promising candidate for hypoxia imaging with high sensitivity and resolution.

Correlative Analyses Between Immune Markers, Tumor Hypoxia And Outcomes Of Head-And-Neck Cancer Patients Undergoing Chemoradiation: Results From A Prospective Trial

Tumor-infiltrating lymphocytes (TILs) influence the response of patients with head-and-neck squamous cell carcinomas (HNSCCs) to chemoradiation and are modulated by the tumor microenvironment and tumor-associated hypoxia. This analysis aimed to assess the prognostic value of TILs and their association with the dynamic tumor hypoxia during chemoradiation in a prospective trial of HNSCC patients. 49 patients with locally advanced HNSCCs were enrolled in this prospective trial prior to undergoing definitive chemoradiation. Patients received tumor biopsies and [18F]-FDG-PET imaging at baseline and [18F]-FMISO hypoxia PET at baseline and in weeks 2 and 5 into treatment. Total lymphocytes, CD3-, CD4-, CD8-positive lymphocytes and expression of PD-1 and PD-L1 were quantified by immunohistochemistry in tumoral and stromal areas of the biopsies and correlated with the tissue hypoxia markers HIF1α, CAIX, CD34 and the longitudinal hypoxia PET/CT data. Potential correlations of immune markers with locoregional control and patient survival were assessed. Patients with high TIL numbers demonstrated increased locoregional control (p = 0.007) and progression-free survival (p = 0.003) compared to patients with lower levels in the biopsies. High numbers of CD3-positive T lymphocytes correlated with improved progression-free (p = 0.018) and overall survival (p = 0.037). In contrast, there was a trend towards impaired progression-free survival for high numbers of CD4-positive regulatory T lymphocytes (p = 0.091), while neither CD8-positive cytotoxic T lymphocytes nor PD-1 or PD-L1 expression correlated with patient outcomes. High levels of TILs were linked to an increased density of CD34-positive microvessels, but not increased levels of hypoxia markers HIF1α or CAIX. No significant correlations could be observed between TILs and dynamic hypoxia PET imaging parameters. In contrast, high expression of intratumoral and stromal PD-1 was associated with low initial tumor hypoxia levels (p = 0.023 for intratumoral PD-1, p = 0.004 for stromal PD-1) as well as a further decrease in the PET hypoxia signal between treatment initiation and week 5 (p = 0.033 for intratumoral PD-1, p = 0.001 for stromal PD-1). In this trial, infiltration of CD3-positive, but not CD4-positive or CD8-positive lymphocytes correlated with an improved locoregional control and increased survival of HNSCC patients after chemoradiation. While no clear associations were found between lymphocyte counts and tumor hypoxia, tumoral PD-1 expression was linked to low initial hypoxia levels and further reductions during treatment. The interplay between tumor hypoxia and tumor-associated immune patterns warrants further investigations to predict treatment response of HNSCC patients.

Influence of FAZA PET hypoxia and HPV-status for the outcome of head and neck squamous cell carcinoma (HNSCC) treated with radiotherapy: Long-term results from the DAHANCA 24 trial (NCT01017224)

PurposeHypoxic tumor volumes can be visualized with 18F-FAZA PET/CT. In head and neck squamous cell carcinoma (HNSCC), hypoxia is important for the clinical outcome after primary radiotherapy (RT). The outcome is furthermore heavily influenced by the HPV/p16-positivity of oropharyngeal tumors (OPCp16+ tumors). The study purposes were (1) to report on locoregional failures within five years after primary RT in a prospective cohort stratified by both HPV/p16-status and PET hypoxia and (2) to characterize the failure site and the spatial association to PET hypoxia. Material and methodsFrom 2009 to 2011, 38 patients with non-metastatic SCC of the larynx, oro-, hypo- and nasopharynx completing primary RT were included in the prospective DAHANCA 24 trial (NCT01017224). Fifteen patients had OPCp16+ tumors. All were imaged with a static FAZA PET/CT prior to treatment. The hypoxia threshold was determined by a tumor-to-muscle ratio (TMR) of 1.6. Recurrences were documented histologically. Imaging of the recurrence was deformable fused with the pre-treatment FAZA PET/CT. The spatial information of recurrence- and hypoxic volumes were compared visually. ResultsSixteen patients had more hypoxic tumors (high tracer uptake, TMR ≥1.6) before treatment (42%). With a median follow-up of 7.8 years, nine locoregional recurrences were observed, of which seven were in patients with high-uptake tumors (44% and 9%, respectively, HR 5.8 [1.2–28.2]). The risk of locoregional recurrence was highest among patients with more hypoxic, non-OPCp16+ tumors (57% [21–94%]), with a risk difference of 45% [4–86%], when comparing to less hypoxic, non-OPCp16+ tumors. Eight patients had sufficient imaging of the recurrence for co-registration with the FAZA PET/CT. Six had hypoxic primary tumors, and in two, the recurrence was overlapping the baseline hypoxic subvolume. ConclusionHNSCC demonstrating a TMR ≥1.6 at baseline is significantly associated with treatment failure after primary RT. In addition to HPV/p16-status, FAZA PET/CT has potential for the selection of tumors requiring treatment intensification.

Abstract 1648: Multimodal imaging of tumor hypoxia with 18F-misonidazole PET, EPR, and MRI

Abstract Introduction: In tumor mouse models, electron paramagnetic resonance (EPR) imaging has been used to quantify pO2 in-vivo for oxygen-guided radiation therapy. Irradiating the hypoxic tumor sub-regions with a boost of radiation significantly increased survival probability when compared to the boosted radiation of normoxic tumor sub-regions. These results indicate 1) the importance of accurate oxygen imaging in-vivo and 2) an advantage to dose-painting hypoxic tumor sub-regions to improve radiation therapy outcomes of hypoxic tumor treatment. The purpose of this study is to evaluate the accuracy of hypoxia imaging using 18F-misonidazole (FMISO) with PET imaging while using EPR imaging as the reference standard for true hypoxia. We include T2-weighted MRI to define tumor anatomy and dynamic contrast enhanced (DCE)-MRI imaging to model vasculature properties to make FMISO PET more accurate in its measurement of hypoxia. Methods: We used six SCC7 tumor mouse models in C3H mice, grown in the leg in the range of 200-500 mm3. Under minimal anesthesia, each mouse leg was set in a soft vinylpolysiloxane cast with embedded fiducials for registered images. A custom-built PET insert was inserted into a 720 MHz EPR imager for near-simultaneous imaging with PET and EPR, acquired two hours post-injection of FMISO to allow the radiotracer to bind to hypoxic tumor cells. Then, T2-weighted and gadodiamide DCE-MRI images were acquired and Ktrans and ve parameters were fitted to model tumor vasculature properties using the Tofts model. Data from all modalities were registered using fiducials, and resampled to isotropic (0.5 mm)3 voxels. The thresholds for hypoxia were defined as tumor to muscle ratio (TMR) ≥ 2 and pO2 ≤ 10 torr for PET and EPR, respectively. To correct the PET image using DCE-MRI parameters, we first modeled FMISO retention as a logistic function of pO2 to map the EPR image to PET TMR image, so that its sigmoidal point of inflection was at the threshold of retention. Then we estimated optimal weighting coefficients of DCE-MRI parameters to add or subtract voxel by voxel to the PET data so that its definition of hypoxia is more similar to the EPR image's hypoxia definition. The quality of overlap between hypoxic tumor regions as defined by PET and EPR was assessed using the Dice Similarity Coefficient (DSC) and the Hausdorff Distance (HD), before and after applying a correction to the PET data. Results: The DSC between hypoxic regions as defined by PET and EPR, before and after applying a correction to PET data, was 0.53 ± 0.2 and 0.80 ± 0.2, respectively. The HD was respectively 3.8 ± 0.5 and 2.1 ± 0.5 mm. Conclusion: These results indicate that there is poor agreement between FMISO PET hypoxia measurements when compared to hypoxia as defined by EPR pO2 images. Applying our correction method significantly improved the overlap between PET and EPR hypoxic tumor regions. Citation Format: Inna Gertsenshteyn, Boris Epel, Lara Leoni, Xiaobing Fan, Richard Friefelder, Eugene Barth, Heejong Kim, Marta Zamora, Erica Markiewicz, Darwin Bodero, Mohammed Bhuiyan, Anna Kucharski, Hsiu-Ming Tsai, Mellissa Grana, Subramanian V. Sundramoorthy, Gregory S. Karczmar, Chien-Min Kao, Chin-Tu Chen, Howard Halpern. Multimodal imaging of tumor hypoxia with 18F-misonidazole PET, EPR, and MRI [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1648.

The cellular basis of increased PET hypoxia tracer uptake in focal cerebral ischemia with comparison between [18F]FMISO and [64Cu]CuATSM

PET hypoxia imaging can assess tissue viability in acute ischemic stroke (AIS). [18F]FMISO is an established tracer but requires substantial accumulation time, limiting its use in hyperacute AIS. [64Cu]CuATSM requires less accumulation time and has shown promise as a hypoxia tracer. We compared these tracers in a M2-occlusion model (M2CAO) with preserved collateral blood flow. Rats underwent M2CAO and [18F]FMISO (n = 12) or [64Cu]CuATSM (n = 6) examinations. [64Cu]CuATSM animals were also examined with MRI. Pimonidazole was used as a surrogate for [18F]FMISO in an immunofluorescence analysis employed to profile levels of hypoxia in neurons (NeuN) and astrocytes (GFAP). There was increased [18F]FMISO uptake in the M2CAO cortex. No increase in [64Cu]CuATSM activity was found. The pimonidazole intensity of neurons and astrocytes was increased in hypoxic regions. The pimonidazole intensity ratio was higher in neurons than in astrocytes. In the majority of animals, immunofluorescence revealed a loss of astrocytes within the core of regions with increased pimonidazole uptake. We conclude that [18F]FMISO is superior to [64Cu]CuATSM in detecting hypoxia in AIS, consistent with an earlier study. [18F]FMISO may provide efficient diagnostic imaging beyond the hyperacute phase. Results do not provide encouragement for the use of [64Cu]CuATSM in experimental AIS.

Hypoxia PET imaging beyond 18F-FMISO in patients with high-grade glioma: 18F-FAZA and other hypoxia radiotracers

High-grade gliomas are aggressive primitive brain tumors presenting aberrant vasculature, regional necrosis, and areas of hypoxia. Tumor hypoxia is associated with resistance to conventional treatment and worse prognosis. [18F]-fluoromisonidazole (18F-FMISO) is the most extensively investigated radiotracer for the evaluation of hypoxia. However, the use of 18F-FMISO in clinical practice has been hampered mainly due to the slow clearance of the unbound tracer from normoxic tissue and its low tumor-to-background ratio (TBR). The research community has therefore investigated other radiotracers to overcome the drawbacks of 18F-FMISO. This mini-review aims to present an update on the most relevant PET studies published in the last 15 years evaluating the utility of radiotracers for hypoxia imaging other than 18F-FMISO in high-grade glioma (HGG) patients. A comprehensive computer literature search of studies was carried out in PubMed/MEDLINE database to identify the most relevant studies published in the last 15 years which investigated the utility of hypoxia PET tracers other than 18F-FMISO in the assessment of tumor hypoxia in patients with HGG. 18F-flouroazomycin arabinoside (18F-FAZA) has been proposed as a valid alternative to 18F-FMISO for the assessment of hypoxia, due to its improved biodistribution and enhanced tumor-to-background ratio. Also 1-(2-[18F]fluoro-1[hydroxymethyl]ethoxy)methyl-2-nitroimidazole(18F-FRP170) seems a valuable hypoxia tracer in patients with brain tumor. The value of copper-diacetyl-bis(N4-methylthiosemicarbazone)(Cu-ATSM) seems controversial. Few evidences still exist regarding the utility of 18F-2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (18F-EF5). Hypoxia PET imaging has the potential to provide useful information for the clinicians and to guide hypoxia tailored treatments. According to the present literature, the most promising hypoxic tracer seems to be 18F-FAZA, but well-designed and wide trials to validate hypoxia radiotracers and evaluate their clinical utility in daily practice are still lacking.