Molecular Imaging and Biology

Whole Body PET Imaging with a Norepinephrine Transporter Probe 4-[ 18 F]Fluorobenzylguanidine: Biodistribution and Radiation Dosimetry Abstract Purpose 4-[18F]Fluorobenzylguanidine ([18F]PFBG) is a positron emission tomography (PET) probe for non-invasive targeting of the norepinephrine transporter. The aim of this study was to assess uptake and distribution characteristics of this PET probe. Procedures Three cynomolgus monkeys were injected with 269 ± 51 MBq (7.3 ± 1.4 mCi) of [18F]PFBG and 21 whole body PET scans were acquired over 165 min. s around organs to generate time-activity curves. The absorbed doses to individual organs and the effective dose to the whole body were estimated. Results Favorable distribution of [18F]PFBG was noted with a fast wash-in and wash-out of radioactivity from several tissues. [18F]PFBG rapidly distributed in the heart, liver, kidneys, and adrenal glands. The uptake presented as %ID in the brain, lung, and spleen was 1.06 ± 0.45, 6.28 ± 0.33, and 1.39 ± 0.35 at 1 min and decreased to 0.29 ± 0.02, 1.78 ± 0.31, and 0.66 ± 0.22 by 112 min. In general, a two- to fourfold reduction was noted from peak radioactivity levels. Rapid uptake and significant retention of radioactivity was noted in the heart and the septal wall was distinctly visible by 20 min. Fast wash-in and washout kinetics for [18F]PFBG resulted in shorter residence times. The residence time for the liver, lungs, kidneys, and spleen were 28.01 ± 7.73 min, 2.97 ± 0.56 min, 6.04 ± 3.41 min, and 1.09 ± 0.33 min, respectively. The mean effective dose for the 70-kg male was 0.04 ± 0.00 mSv/MBq. The organs receiving the highest radiation dose in the 70-kg male model were the testes (0.11 ± 0.02 mGy/MBq), adrenals (0.08 ± 0.01 mGy/MBq), and urinary bladder wall (0.08 ± 0.01 mGy/MBq). Conclusions [18F]PFBG shows a favorable biodistribution pattern. Rapid and persistent uptake was noted in innervated organs. Renal clearance was the major path for elimination of [18F]PFBG. The estimated radiation burden from [18F]PFBG was significantly lower than that from [124I]MIBG.

Development of a Fluorinated Analogue of Erlotinib for PET Imaging of EGFR Mutation–Positive NSCLC Abstract Purpose Positron emission tomography (PET) using [11C]erlotinib identifies non-small cell lung carcinoma (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFRm). The short half-life of C-11, however, limits its clinical utility to centers with a nearby cyclotron. We therefore developed a F-18–labeled analogue of erlotinib for imaging EGFRm NSCLC. Procedures 6-O-Fluoroethylerlotinib (6-O-FEE) was synthesized and its anti-proliferative activity was tested using human NSCLC cell lines. The F-18–labeled compound, 6-O-[18F]FEE, was obtained in a two-step synthesis, and PET acquisitions were carried out following its injection to NSCLC tumor–bearing mice. Results In vitro, 6-O-FEE had maintained the selectivity and potency of erlotinib to EGFRm NSCLC. In vivo, 6-O-[18F]FEE accumulation in EGFRm tumors at 60 min after injection was 2- and 3.3-fold higher than in erlotinib-resistant or erlotinib-insensitive tumors, respectively. Conclusions 6-O-[18F]FEE holds promise for imaging EGFRm NSCLC, warranting further investigation to fully explore its potential for stratifying NSCLC patients.

Impact of Tumor Burden on Quantitative [ 68 Ga] DOTATOC Biodistribution Abstract Purpose As has been previously reported, the somatostatin receptor (SSTR) imaging agent [68Ga]-labeled 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid-d-Phe(1)-Tyr(3)-octreotate ([68Ga]DOTATATE) demonstrates lower uptake in normal organs in patients with a high neuroendocrine tumor (NET) burden. Given the higher SSTR affinity of [68Ga] DOTATATE, we aimed to quantitatively investigate the biodistribution of [68Ga]-labeled 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid-d-Phe(1)-Tyr(3)-octreotide ([68Ga]DOTATOC) to determine a potential correlation between uptake in normal organs and NET burden. Procedures Of the 44 included patients, 36/44 (82 %) patients demonstrated suspicious radiotracer uptake on [68Ga] DOTATOC positron emission tomography (PET)/X-ray computed tomography (CT). Volumes of interest (VOIs) were defined for tumor lesions and normal organs (spleen, liver, kidneys, adrenals). Mean body weight corrected standardized uptake value (SUVmean) for normal organs was assessed and was used to calculate the corresponding mean specific activity uptake (Upt: fraction of injected activity per kg of tissue). For the entire tumor burden, SUVmean, maximum standardized uptake value (SUVmax), and the total mass (TBM) was calculated and the decay corrected tumor fractional uptake (TBU) was assessed. A Spearman’s rank correlation coefficient was used to determine the correlations between normal organ uptake and tumor burden. Results The median SUVmean was 18.7 for the spleen (kidneys, 9.2; adrenals, 6.8; liver, 5.6). For tumor burden, the median values were SUVmean 6.9, SUVmax 35.5, TBM 42.6 g, and TBU 1.2 %. With increasing volume of distribution, represented by lean body mass and body surface area (BSA), Upt decreased in kidneys, liver, and adrenal glands and SUVmean increased in the spleen. Correlation improved only for both kidneys and adrenals when the influence of the tumor uptake on the activity available for organ uptake was taken into account by the factor 1/(1-TBU). TBU was neither predictive for SUVmean nor for Upt in any of the organs. The distribution of organ Upt vs. BSA/(1-TBU) were not different for patients with minor TBU (<3 %) vs. higher TBU (>7 %), indicating that the correlations observed in the present study are explainable by the body size effect. High tumor mass and uptake mitigated against G1 NET. Conclusions There is no significant impact on normal organ biodistribution with increasing tumor burden on [68Ga] DOTATOC PET/CT. Potential implications include increased normal organ dose with [177Lu-DOTA]0-D-Phe1-Tyr3-Octreotide and decreased absolute lesion detection with [68Ga] DOTATOC in high NET burden.

Spatial Concordance of Tumor Proliferation and Accelerated Repopulation from Pathologic Images to 3′-[ 18 F]Fluoro-3′-Deoxythymidine PET Images: a Basic Study Guided for PET-Based Radiotherapy Dose Painting Abstract Purpose To assess tumor cell proliferation and repopulation during fractionated radiotherapy and investigate the spatial concordance of cell proliferation and repopulation according to the uptake of 3′-[18F]fluoro-3′-deoxythymidine ([18F]FLT). Procedures Mice bearing A549 xenograft tumors were assigned to five irradiated groups, including 3 fraction (f)/6 days (d), 6f/12d, 9f/18d, 12f/24d, and 18f/36d with 2 Gy/f irradiations performed every other day and one non-irradiated group. Serial [18F]FLT positron emission tomography (PET) scans were performed at different time points as the groups finished the radiotherapy. The maximum of standard uptake values (SUVmax) were measured to confirm the likely time of tumor repopulation. A layer-by-layer comparison between SUVmax of PET images and Ki-67 LI of pathology images, including the thresholds at which maximum overlap occurred between FLT-segmented areas and cell proliferation areas were conducted to evaluate the spatial correlation. Results The SUVmax decreased in the 3f/6d group (P = 0.000) compared to the non-irradiated group, increased in the 6f/12d group and then gradually reduced with prolonged treatment. Proliferation changes in 6f/12d group on pathology images were also confirmed. Significant correlations were found between the SUVmax and Ki-67 LI in each in vitro tumor of cell proliferation group and accelerated repopulation group (both of the P < 0.001). Furthermore, the mean overlap region rates (ORRs) were 56.21 % and 57.82 % in the proliferation group and repopulation group, respectively. The data represented the preferable registration. Conclusions [18F]FLT PET is a promising imaging surrogate of tumor proliferative response to fractionated radiotherapy and may help make an adaptive radiation oncology treatment plan to realize radiotherapy dose painting.

Predicting Glioblastoma Response to Bevacizumab Through MRI Biomarkers of the Tumor Microenvironment Abstract Purpose Glioblastoma (GB) is one of the most vascularized of all solid tumors and, therefore, represents an attractive target for antiangiogenic therapies. Many lesions, however, quickly develop escape mechanisms associated with changes in the tumor microenvironment (TME) resulting in rapid treatment failure. To prevent patients from adverse effects of ineffective therapy, there is a strong need to better predict and monitor antiangiogenic treatment response. Procedures We utilized a novel physiological magnetic resonance imaging (MRI) method combining the visualization of oxygen metabolism and neovascularization for classification of five different TME compartments: necrosis, hypoxia with/without neovascularization, oxidative phosphorylation, and aerobic glycolysis. This approach, termed TME mapping, was used to monitor changes in tumor biology and pathophysiology within the TME in response to bevacizumab treatment in 18 patients with recurrent GB. Results We detected dramatic changes in the TME by rearrangement of its compartments after the onset of bevacizumab treatment. All patients showed a decrease in active tumor volume and neovascularization as well as an increase in hypoxia and necrosis in the first follow-up after 3 months. We found that recurrent GB with a high percentage of neovascularization and active tumor before bevacizumab onset showed a poor or no treatment response. Conclusions TME mapping might be useful to develop strategies for patient stratification and response prediction before bevacizumab onset.

Histogram Analysis Parameters Derived from Conventional T1- and T2-Weighted Images Can Predict Different Histopathological Features Including Expression of Ki67, EGFR, VEGF, HIF-1α, and p53 and Cell Count in Head and Neck Squamous Cell Carcinoma Abstract Purpose To analyze associations between histogram analysis parameters derived from conventional magnetic resonance imaging (MRI) and different histopathological features in head and neck squamous cell carcinoma (HNSCC). Procedures Thirty-four patients with histologically proven primary HNSCC were prospectively acquired. Histogram analysis was derived from pre-contrast T1-weighted (T1w) and T2-weighted (T2w) images. In all cases, expression of HIF-1α, VEGF, EGFR, p53, Ki67, and p16 as well as tumor cell count was analyzed. Results In the overall sample, inverse correlation between entropy derived from T1w images and p53 expression (p = − 0.458, P = 0.01) was found. Furthermore, p10 derived from T1w images correlated with VEGF expression (p = 0.371, P = 0.04). In the p16-positive tumors, VEGF expression correlated with several parameters derived from T1w images: mean (p = 0.481, P = 0.032), p10 (p = 0.489, P = 0.029), p25 (p = 0.475, P = 0.034), median (p = 0.468, P = 0.037), and mode (p = 0.492, P = 0.028). Several T2w parameters were associated with p53 expression: mean (p = 0.569, P = 0.007), p25 (p = 0.508, P = 0.019), p75 (p = 0.479, P = 0.028), median (p = 0.555, P = 0.009), and mode (p = 0.468, P = 0.033). Kurtosis derived from T2w images correlated with cell count (p = 0.534, P = 0.013). In p16-negative carcinomas, T2w parameters correlated with p53 expression: max (p = 0.736, P = 0.015), p90 (p = 0.687, P = 0.028), and standard deviation (p = 0.760, P = 0.011). T2w p10 (p = − 0.709, P = 0.022) and T2w p25 (p = − 0.733, P = 0.016) correlated also with HIF-1α expression. Conclusions Multiple associations between histogram parameters derived from T1w and T2w images and clinically relevant histopathological features were found in HNSCC. Therefore, imaging parameters can be also used as surrogate markers for tumor cellularity, proliferation, and vascularization in HNSCC. The identified correlations differed significantly between p16-positive and p16-negative cancers.

Does Measurement of First-Order and Heterogeneity Parameters Improve Response Assessment of Bone Metastases in Breast Cancer Compared to SUV max in [ 18 F]fluoride and [ 18 F]FDG PET? Abstract Purpose To establish whether first-order statistical features from [18F]fluoride and 2-deoxy-2-[18F] fluoro-d-glucose ([18F]FDG) positron emission tomography/x-ray computed tomography (PET/CT) demonstrate incremental value in skeletal metastasis response assessment compared with maximum standardised uptake value (SUVmax). Procedures Sixteen patients starting endocrine treatment for de novo or progressive breast cancer bone metastases were prospectively recruited to undergo [18F]fluoride and [18F]FDG PET/CT scans before and 8 weeks after treatment. Percentage changes in SUV parameters, metabolic tumour volume (MTV), total lesion metabolism (TLM), standard deviation (SD), entropy, uniformity and absolute changes in kurtosis and skewness, from the same ≤ 5 index lesions, were measured. Clinical response to 24 weeks, assessed by two experienced oncologists blinded to PET/CT imaging findings, was used as a reference standard and associations were made between parameters and progression free and overall survival. Results [18F]fluoride PET/CT: In four patients (20 lesions) with progressive disease (PD), TLM and kurtosis predicted PD better than SUVmax on a patient basis (4, 4 and 3 out of 4, respectively) and TLM, entropy, uniformity and skewness on a lesion basis (18, 16, 16, 18 and 15 out of 20, respectively). Kurtosis was independently associated with PFS (p = 0.033) and OS (p = 0.008) on Kaplan-Meier analysis. [18F]FDG PET: No parameter provided incremental value over SUVmax in predicting PD or non-PD. TLM was significantly associated with OS (p = 0.041) and skewness with PFS (p = 0.005). Interlesional heterogeneity of response was seen in 11/16 and 8/16 patients on [18F]fluoride and [18F]FDG PET/CT, respectively. Conclusion With [18F]fluoride PET/CT, some first-order features, including those that take into account lesion volume but also some heterogeneity parameters, provide incremental value over SUVmax in predicting clinical response and survival in breast cancer patients with bone metastases treated with endocrine therapy. With [18F]FDG PET/CT, no first-order parameters were more accurate than SUVmax although TLM and skewness were associated with OS and PFS, respectively. Intra-patient heterogeneity of response occurs commonly between metastases with both tracers and most parameters.

A Pilot Study of Texture Analysis of Primary Tumor [ 18 F]FDG Uptake to Predict Recurrence in Surgically Treated Patients with Non-small Cell Lung Cancer Abstract Purpose To examine whether the heterogeneous texture parameters in primary tumor can predict prognosis of patients with non-small cell lung cancer (NSCLC) received surgery after 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET)/X-ray computed tomography (CT). Procedure This retrospective study included 55 patients with NSCLC who underwent [18F]FDG-PET/CT before surgery from January 2011 and December 2015. SUV-related (SUVmax and SUVmean), volumetric (metabolic tumor volume [SUV ≥ 2.5], and total lesion glycolysis) and texture parameters (local parameters; entropy, homogeneity, and dissimilarity and regional parameters; intensity variability [IV], size-zone variability [SZV], and zone percentage [ZP]) were obtained. Tumor size, TNM stage, SUV-related, volumetric, and texture parameters were compared between the patients with progression and without progression using Mann-Whitney’s U or χ2 test and progression-free survival (PFS) and prognostic significance were assessed by Kaplan-Meier method and Cox regression analysis, respectively. Results Nineteen patients eventually showed progression, and 36 patients were alive without progression during clinical follow-up (median follow-up PFS; 23 months [range, 1–71]). The patients with progression showed significantly larger tumor size (p < 0.001), higher IV (p = 0.010), and higher SZV (p = 0.007) than those without progression. PFS was significantly shorter in patients with large tumor size (p = 0.008), high T stage (p = 0.009), high stage (p = 0.013), high IV (p = 0.012), and high SZV (p = 0.015) at univariate analysis. At multivariate analysis, stage (hazard ratio [HR] 1.62, p = 0.035) and IV (hazard ratio 6.19, p = 0.048) were only remained independent predictors for PFS. Conclusions The regional heterogeneity texture parameters IV and SZV can predict tumor progression, and IV has the potential to predict prognosis of surgically treated NSCLC patients.

Pharmacokinetic Modeling of Targeted Ultrasound Contrast Agents for Quantitative Assessment of Anti-Angiogenic Therapy: a Longitudinal Case-Control Study in Colon Cancer Abstract Purpose To evaluate quantitative and semi-quantitative ultrasound molecular imaging (USMI) for antiangiogenic therapy monitoring in human colon cancer xenografts in mice. Procedures Colon cancer was established in 17 mice by injection of LS174T (Nr = 9) or CT26 (Nn = 8) cancer cells to simulate clinical responders and non-responders, respectively. Antiangiogenic treatment (bevacizumab; Nrt = Nnt = 5) or control treatment (saline; Nrc = 4, Nnc = 3) was administered at days 0, 3, and 7. Three-dimensional USMI was performed by injection at days 0, 1, 3, 7, and 10 of microbubbles targeted to the vascular endothelial growth factor receptor 2 (VEGFR2). Microbubble binding rate (kb), estimated by first-pass binding model fitting, and semi-quantitative parameters late enhancement (LE) and differential targeted enhancement (dTE) were compared at each day to evaluate their ability to assess and predict the response to therapy. Correlation analysis with the ex-vivo immunohistological quantification of VEGFR2 expression and the percentage blood vessel area was also performed. Results Significant changes in the USMI parameters during treatment were observed only in the responders treated with bevacizumab (p-value < 0.05). Prediction of the response to therapy as early as 1 day after treatment was achieved by the quantitative parameter kb (p-value < 0.01), earlier than possible by tumor volume quantification. USMI parameters could significantly distinguish between clinical responders and non-responders (p-value << 0.01) and correlated well with the ex-vivo quantification of VEGFR2 expression and the percentage blood vessels area (p-value << 0.01). Conclusion USMI (semi)quantitative parameters provide earlier assessment of the response to therapy compared to tumor volume, permit early prediction of non-responders, and correlate well with ex-vivo angiogenesis biomarkers.

Preclinical SPECT Imaging of Choroidal Neovascularization in Mice Using Integrin-Binding [ 99m Tc]IDA-D-[c(RGDfK)] 2 Abstract Purpose Integrin ɑvβ3, an adhesion molecule overexpressed in neovascular endothelial cells, is involved in ocular angiogenesis. Integrin ɑvβ3-binding arginine–glycine–aspartic acid (RGD) peptide has been used to target and visualize new vessels. We explored the use of integrin ɑvβ3-targeted RGD peptide ([99mTc]IDA-D-[c(RGDfK)]2) for in vivo molecular imaging of choroidal neovascularization (CNV). Procedures To induce CNV in animals, the right eyes of C57BL/6 mice were treated with retinal argon laser photocoagulation. CNV formation was confirmed on immunohistopathological examination of retinal and choroidal tissues. To explore the association of integrin with angiogenesis, integrin mRNA expression in the retinal and choroidal tissues was measured using real-time reverse transcriptase-polymerase chain reaction. For in vivo imaging, mice were intravenously injected with [99mTc]IDA-D-[c(RGDfK)]2 and single-photon emission computed tomography (SPECT) images of [99mTc]IDA-D-[c(RGDfK)]2 were obtained before laser induction (baseline) and at 1, 3, 7, and 14 days post-induction. CNV-induced regional alterations were measured using radiotracer uptake count. Results Immunohistopathological examination revealed that CNV lesions showed intense fluorescein isothiocyanate (FITC)-D-[c(RGDfK)]2 immunofluorescence, in contrast to the normal retina and choroid. Retinal integrin mRNA expression peaked at day 1 following CNV induction. On SPECT images using [99mTc]IDA-D-[c(RGDfK)]2, the radio-uptake count in eyes with CNV was significantly higher than in normal controls on days 1–7 (all p < 0.05), with a peak at day 3 representing the highest angiogenic activity. Our preclinical data demonstrated that [99mTc]IDA-D-[c(RGDfK)]2 can detect CNV and its associated angiogenesis in an animal model of CNV. Conclusions SPECT imaging using an integrin ɑvβ3-targeted RGD peptide radiotracer may be a useful tool for in vivo functional molecular imaging of CNV.