Fully Automated Preparation and Conjugation of <Emphasis Type=Italic>N</Emphasis>-Succinimidyl 4-[<Superscript>18</Superscript>F]Fluorobenzoate ([<Superscript>18</Superscript>F]SFB) with RGD Peptide Using a GE FASTlab™ Synthesizer

Purpose  

The aim of this work was to automate the radiosynthesis of [¹⁸F]SFB, a widely used reagent for the labeling of biomolecules with ¹⁸F on a new generation commercial synthesis module (FASTLab™, GE Healthcare).     

Comparison of [<Superscript>99m</Superscript>Tc]3PRGD<Subscript>2</Subscript> Imaging and [<Superscript>18</Superscript>F]FDG PET/CT in Breast Cancer and Expression of Integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> in Breast Cancer Vascular Endothelial Cells

Purpose

This study aimed to investigate the value of ^99mtechnetium-three polyethylene glycol spacers-arginine-glycine-aspartic acid ([^99mTc]3PRGD₂) imaging in diagnosis and staging of breast cancer compared with 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) imaging, and to explore the expression of integrin α_vβ₃ in tumor vascular endothelial cells.

Procedures

Forty-two women with suspected breast cancer underwent both [^99mTc]3PRGD₂ imaging and [¹⁸F]FDG imaging. Visual analysis was used to assess primary breast lesion, axillary lymph node, and distant metastasis. The tumor-blood (T/B) ratios from [^99mTc]3PRGD₂ imaging and the maximum standardized uptake value (SUVmax) from [¹⁸F]FDG imaging were analyzed for breast lesions. Integrin α_vβ₃ was analyzed through immunohistochemistry.

Results

Forty-five breast lesions were found (malignant, n?=?38; benign, n?=?7). The sensitivity, specificity, and accuracy of [^99mTc]3PRGD₂ and [¹⁸F]FDG imaging in visual analysis for the breast lesion were 97.4, 87.5, and 95.6 % and 97.4, 71.4, and 93.3 %, respectively (P?>?0.05). For semi-quantitative analysis, no significant difference of the area under the curves (AUC) was found in the imaging using the two radiopharmaceuticals (0.880 and 0.955; Z?=?0.88, P?>?0.05). The sensitivity, specificity, and accuracy for axillary lymph node metastasis with [^99mTc]3PRGD₂ and [¹⁸F]FDG were 78.05, 99.36, and 94.92 % and 85.37, 98.72, and 95.64 %, respectively (P?>?0.05). Nine patients with distant metastases were all detected with the two radiopharmaceuticals. The expression of integrin α_vβ₃ was correlated with [^99mTc]3PRGD₂ uptake (r?=?0.582, P?=?0.001), which were significantly higher in the HER2-positive and stage III–IV patients (P?<?0.05).

Conclusions

The prospective study demonstrated that [^99mTc]3PRGD₂ imaging seems to be valuable for diagnosis of breast cancer and its staging. It may be less sensitive for detecting small lymph node metastatic lesions when compared with [¹⁸F]FDG imaging. Integrin α_vβ₃ in tumor microvessels was associated with the breast cancer subtype and its staging.

     

Comparison of Ga-68-Labeled Fusarinine C-Based Multivalent RGD Conjugates and [<Superscript>68</Superscript>Ga]NODAGA-RGD—<Emphasis Type="Italic">In Vivo</Emphasis> Imaging Studies in Human Xenograft Tumors

Purpose

Multimeric arginine-glycine-aspartic acid (RGD) peptides have advantages for imaging integrin α_vβ₃ expression. Here, we compared the in vitro and in vivo behavior of three different Ga-68-labeled multimeric Fusarinine C-RGD (FSC-RGD) conjugates, whereby RGD was coupled directly, via a succinic acid or PEG linker (FSC(RGDfE)₃, FSC(succ-RGD)₃, FSC(Mal-RGD)₃). The positron emission tomography/X-ray computed tomography (PET/CT) imaging properties were further compared using [⁶⁸Ga]FSC(succ-RGD)₃ with the monomeric [⁶⁸Ga]NODAGA-RGD in a murine tumor model.

Procedure

FSC-RGD conjugates were labeled with Ga-68, and stability properties were studied. For in vitro characterization, the partition coefficient, integrin α_vβ₃ binding affinity, and cell uptake were determined. To characterize the in vivo properties, biodistribution studies and microPET/CT were carried out using mice bearing either human M21/M21-L melanoma or human U87MG glioblastoma tumor xenografts.

Results

All FSC-RGD conjugates were quantitatively labeled with Ga-68 within 10 min at RT. The [⁶⁸Ga]FSC-RGD conjugates exhibited high stability and hydrophilic character, with only minor differences between the different conjugates. In vitro and in vivo studies showed enhanced integrin α_vβ₃ binding affinity, receptor-selective tumor uptake, and rapid renal excretion resulting in good imaging properties.

Conclusions

The type of linker between FSC and RGD had no pronounced effect on targeting properties of [⁶⁸Ga]FSC-RGD trimers. In particular, [⁶⁸Ga]FSC(succ-RGD)₃ exhibited improved properties compared to [⁶⁸Ga]NODAGA-RGD, making it an alternative for imaging integrin α_vβ₃ expression.

     

<Superscript>131</Superscript>I-Tositumomab (Bexxar®) <Emphasis Type="Italic">vs.</Emphasis><Superscript>90</Superscript>Y-Ibritumomab (Zevalin®) Therapy of Low-Grade Refractory/Relapsed Non-Hodgkin Lymphoma

Introduction

The American Cancer Society estimated 66,120 new cases of non-Hodgkin lymphoma (NHL) in the USA in 2008. Radioimmunotherapy has been shown in clinical trials to be an effective treatment for refractory/relapsed NHL. The available agents are Bexxar®, a ¹³¹I radiolabeled murine monoclonal antibody and Zevalin®, a ⁹⁰Y radiolabeled murine antibody. Both target CD20 receptors present on the surface of lymphocytes. We present our clinical experience with Bexxar® and Zevalin® in the management of low-grade refractory or relapsed NHL.

Methods

This is a retrospective study (Jan 2000–Jul 2006) of 67 patients with NHL, who were treated with Bexxar® (31 patients, group A) or Zevalin® (36 patients, group B) for refractory/relapsed disease. Group A included 16 men and 15 women, 35–81 years old (average, 59.3?±?13.4). Group B included 27 men and nine women, 36–85 years old (average, 55.4?±?13.8). Therapeutic doses ranged 40–138 mCi (average, 78.1?±?28.2) for Bexxar® and 17–34 mCi (average, 28.8?±?4.37) for Zevalin®.

Results

Objective responses were induced in 22 of the 31 patients (70.9%) in group A and 28 of the 36 patients (77.8%) in group B. Complete response was noted in 11 patients (35.5%), partial response in seven patients (22.6%), and mixed response in four patients (12.9%) in group A. There were five patients (16.1%) with stable disease and four patients (12.9%) with disease progression in the same group. Complete response was noted in 15 patients (41.7%), partial response in nine patients (25%), and mixed response in four patients (11.1%) in group B. There were four patients (11.1%) with stable disease and another four patients (11.1%) with disease progression in the same group. The average decreases at posttherapy nadir were 36.9%?±?0.33 (group A) and 52.6%?±?0.32 (group B) for platelets, 27.8%?±?0.27 (group A) and 34.2%?±?0.38 (group B) for leukocytes, and 4.9%?±?0.15 (group A) and 7.6%?±?0.11 (group B) for hemoglobin. Grades 3 and 4 hematological toxicity occurred in 14 patients (45.2%) treated with Bexxar® and 22 patients (61.1%) treated with Zevalin®, but was reversible.

Conclusion

Our study suggests that clinical practice of Bexxar® and Zevalin® radioimmunotherapy is an effective and safe adjunctive treatment for patients with NHL refractory/relapsed to conventional treatment. However, due to the small number of subjects, it was not possible to determine whether differences in the outcomes or toxicities from the two agents were statistically significant.

     

3′-Deoxy-3′-[<Superscript>18</Superscript>F]Fluorothymidine Uptake Is Related to Thymidine Phosphorylase Expression in Various Experimental Tumor Models

Purpose

We recently reported that high thymidine phosphorylase (TP) expression is accompanied by low tumor thymidine concentration and high 3′-deoxy-3′-[¹⁸F]fluorothymidine ([¹⁸F]FLT) uptake in four untreated lung cancer xenografts. Here, we investigated whether this relationship also holds true for a broader range of tumor models.

Procedures

Lysates from n = 15 different tumor models originating from n = 6 institutions were tested for TP and thymidylate synthase (TS) expression using western blots. Results were correlated to [¹⁸F]FLT accumulation in the tumors as determined by positron emission tomography (PET) measurements in the different institutions and to previously published thymidine concentrations.

Results

Expression of TP correlated positively with [¹⁸F]FLT SUV_max (ρ = 0.549, P < 0.05). Furthermore, tumors with high TP levels possessed lower levels of thymidine (ρ = ??0.939, P < 0.001).

Conclusions

In a broad range of tumors, [¹⁸F]FLT uptake as measured by PET is substantially influenced by TP expression and tumor thymidine concentrations. These data strengthen the role of TP as factor confounding [¹⁸F]FLT uptake.

     

Synthesis of 2′-Deoxy-2′-[<Superscript>18</Superscript>F]Fluoro-9-β-<Emphasis Type="SmallCaps">D</Emphasis>-Arabinofuranosylguanine: a Novel Agent for Imaging T-Cell Activation with PET

Purpose  

9-(β-D-Arabinofuranosyl)guanine (AraG) is a guanosine analog that has a proven efficacy in the treatment of T-cell lymphoblastic disease. To test the possibility of using a radiofluorinated AraG as an imaging agent, we have synthesized 2′-deoxy-2′-[¹⁸F]fluoro-9-β-D-arabinofuranosylguanine ([¹⁸F]F-AraG) and investigated its uptake in T cells.     

The Synergistic Effect of Selumetinib/Docetaxel Combination Therapy Monitored by [<Superscript>18</Superscript> F]FDG/[<Superscript>18</Superscript> F]FLT PET and Diffusion-Weighted Magnetic Resonance Imaging in a Colorectal Tumor Xenograft Model

Purpose

Positron emission tomography (PET) and diffusion-weighted MRI (DW-MRI) were used to characterize the treatment effects of the MEK1/2 inhibitor selumetinib (AZD6244), docetaxel, and their combination in HCT116 tumor-bearing mice on the molecular level.

Procedures

Mice were treated with vehicle, selumetinib (25 mg/kg), docetaxel (15 mg/kg), or a combination of both drugs for 7 days and imaged at four time points with 2-deoxy-2-[¹⁸?F]fluoro-D-glucose ([¹⁸?F]FDG) or 3′-deoxy-3′-[¹⁸?F]fluorothymidine ([¹⁸?F]FLT) followed by DW-MRI to calculate the apparent diffusion coefficient (ADC). Data was cross-validated using the Pearson correlation coefficient (PCC) and compared to histology (IHC).

Results

Each drug led to tumor growth inhibition but their combination resulted in regression. Separate analysis of PET or ADC could not provide significant differences between groups. Only PCC combined with IHC analysis revealed the highest therapeutic impact for combination therapy.

Conclusion

Combination treatment of selumetinib/docetaxel was superior to the respective mono-therapies shown by PCC of PET and ADC in conjunction with histology.

     

A Novel <Emphasis Type="Italic">In Vitro</Emphasis> Assay to Assess Phosphorylation of 3′-[<Superscript>18</Superscript>F]fluoro-3′-Deoxythymidine

Purpose  

3′-[¹⁸F]fluoro-3'-deoxythymidine ([¹⁸F]FLT) is phosphorylated by thymidine kinase 1 (TK-1), a cell cycle regulated enzyme. Appropriate use of [¹⁸F]FLT tracer requires validation of the TK-1 activity. Here, we report development of a novel phosphoryl-transfer assay to assess phosphorylation of [¹⁸F]FLT both in tumor cell lysates and tumor cells.     

<Superscript>18</Superscript>F-Labeled Galacto and PEGylated RGD Dimers for PET Imaging of α<Subscript>v</Subscript>β<Subscript>3</Subscript> Integrin Expression

Purpose

In vivo imaging of α_vβ₃ has important diagnostic and therapeutic applications. ¹⁸F-Galacto-arginine–glycine–aspartic acid (RGD) has been developed for positron emission tomography (PET) imaging of integrin α_vβ₃ expression and is now being tested on humans. Dimerization and multimerization of cyclic RGD peptides have been reported to improve the integrin α_vβ₃-binding affinity due to the polyvalency effect. Here, we compared a number of new dimeric RGD peptide tracers with the clinically used ¹⁸F-galacto-RGD.

Procedures

RGD monomers and dimers were coupled with galacto or PEG₃ linkers, and labeled with ¹⁸F using 4-nitrophenyl 2-¹⁸F-fluoropropionate (¹⁸F-NFP) or N-succinimidyl 4-¹⁸F-fluorobenzoate as a prosthetic group. The newly developed tracers were evaluated by cell-based receptor-binding assay, biodistribution, and small-animal PET studies in a subcutaneous U87MG glioblastoma xenograft model.

Results

Starting with ¹⁸F-F^?, the total reaction time for ¹⁸F-FP-SRGD2 and ¹⁸F-FP-PRGD2 is about 120 min. The decay-corrected radiochemical yields for ¹⁸F-FP-SRGD2 and ¹⁸F-FP-PRGD2 are 52?±?9% and 80?±?7% calculated from ¹⁸F-NFP. Noninvasive small-animal PET and direct tissue sampling experiments demonstrated that the dimeric RGD peptides had significantly higher tumor uptake as compared to ¹⁸F-galacto-RGD.

Conclusion

Dimeric RGD peptide tracers with relatively high tumor integrin-specific accumulation and favorable in vivo kinetics may have the potential to be translated into clinic for integrin α_vβ₃ imaging.

     

Imaging α4β2 Nicotinic Acetylcholine Receptors (nAChRs) in Baboons with [<Superscript>18</Superscript>F]XTRA,a Radioligand with Improved Specific Binding in Extra-Thalamic Regions

Purpose

Currently available positron-emitting radiotracers for imaging of the α4β2 subtype of nicotinic acetylcholine receptors (nAChRs) exhibit high and moderate specific binding in the thalamus and extra-thalamic brain regions, respectively. In many neuropsychiatric disorders, α4β2-nAChRs are altered in the extra-thalamic brain regions, but not necessarily in the thalamus. The purpose of this study was to evaluate [¹⁸F]XTRA, a new α4β2-nAChR positron emission tomography (PET) radioligand with improved specific binding in extra-thalamic brain regions, in non-human primates.

Procedures

The regional distribution of [¹⁸F]XTRA in the brain of Papio anubis baboons was evaluated in baseline and blocking experiments. Various PET modeling procedures were used for determination of volume of distribution (V _T), binding potential (BP_ND), and receptor occupancy. Radiation dosimetry for [¹⁸F]XTRA was studied in male CD-1 mice and extrapolated to human dosimetry estimates using OLINDA/EXM software.

Results

[¹⁸F]XTRA was synthesized using an automated radiochemistry module with 25 % decay-corrected radiochemical yield. [¹⁸F]XTRA readily enters the baboon brain and specifically labels α4β2-nAChRs. Mathematical modeling demonstrates high binding potential values (BP_ND = 7 and 1.3 in the thalamus and frontal cortex, respectively). A PET scanning time of 90–120 min was sufficient to obtain stable V _T values in the extra-thalamic regions. The extrapolated human effective dose was 0.041 mSv/MBq (0.15 Rem/mCi).

Conclusion

[¹⁸F]XTRA exhibits improved specific binding in the baboon brain including extra-thalamic regions and it is considered radiologically acceptable for human studies. Further evaluations of [¹⁸F]XTRA in human subjects are under way.

     

Monitoring Tumor Response after Liposomal Doxorubicin in Combination with Liposomal Vinorelbine Treatment Using 3′-Deoxy-3′-[<Superscript>18</Superscript>F]Fluorothymidine PET

Purpose

Surgical resection is the standard treatment for localized colorectal cancer, which is the most common type of gastrointestinal cancer. However, over 40 % cases are diagnosed metastasized and apparently inoperable. Systemic chemotherapy provides an alternative to these patients. This study aims to evaluate the therapeutic potential of liposomal doxorubicin (lipoDox) in combination with liposomal vinorelbine (lipoVNB) in a CT-26 colon carcinoma-bearing mouse model.

Procedures

The in vitro cytotoxicity of Dox and VNB on CT-26 cancer cells was determined by MTT and colony formation assays. Mice were subcutaneously inoculated with 2 × 10⁵ of CT-26 cells in the right hind flank. When tumor size reached 200 ± 50 mm³, mice were assigned to receive different treatment protocols. The pharmacokinetics, micro single-photon emission computed tomography/x-ray computed tomography imaging, biodistribution, and immunohistochemical staining studies were performed to survey the therapeutic efficacy of each regimen.

Results

Based on the results of pharmacokinetic study, co-administration of lipoDox and lipoVNB did not affect their individual systemic distribution, while lipoDox retained longer in blood than lipoVNB did. Superior tumor growth retardation was observed in the group received lipoDox plus lipoVNB administration (1 mg/kg each, namely D₁V₁) than those injected with lipoDox plus VNB (1 mg/kg each, namely D₁fV₁). No severe side effects were detected in each group. The tumor-to-muscle ratio (T/M) derived from 3′-dexoy-3′-[¹⁸F]fluorothymidine ([¹⁸F]FLT) micro positron emission tomography (PET) images of D₁V₁- and D₁fV₁-treated mice and the controls on day 7 was 6.88 ± 0.54, 7.50 ± 0.84, and 9.87 ± 0.73, respectively, suggesting that D₁V₁ is a more efficacious regimen against CT-26 xenografts. The results of proliferating cell nuclear antigen (PCNA) immunohistochemical staining were consistent with those findings obtained from [¹⁸F]FLT microPET imaging.

Conclusion

This study demonstrated that lipoDox in combination with lipoVNB was more efficacious than clinically used regimen, lipoDox plus VNB, in the treatment of colon carcinoma and [¹⁸F]FLT-PET is a promising approach in monitoring the treatment outcome at early stage.

     

Increased circulating regulatory T cells (CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>CD127<Superscript>−</Superscript>) contribute to lymphocyte anergy in septic shock patients

Purpose  Sepsis syndrome represents the leading cause of death in intensive care unit. Patients present features consistent with a decline in immune responsiveness potentially contributing to mortality. We investigated whether CD4⁺CD25⁺ regulatory T cells (Treg) participate in the induction of lymphocyte anergy after sepsis. Method  Observational study in septic shock patients and experimental study in mice. Results  We took advantage of the recently described flow cytometric gating strategy using the measurement of CD25 and CD127 expressions for monitoring Treg (CD4⁺CD25⁺CD127⁻Foxp3⁺). In patients the increased circulating Treg percentage significantly correlated with a decreased lympho-proliferative response. In a murine model of sepsis mimicking these observations, the ex vivo downregulation of Foxp3 expression using siRNA was associated with a restoration of this response. Conclusion  The relative increase in circulating Treg might play a role in lymphocyte anergy described after septic shock and represent a standardizable surrogate marker of declining proliferative capacity after sepsis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cross-sectional and Test-Retest Characterization of PET with [<Superscript>18</Superscript>F]FP-(+)-DTBZ for β Cell Mass Estimates in Diabetes

Purpose

The vesicular monoamine transporter, type 2 (VMAT2) is expressed by insulin producing β cells and was evaluated as a biomarker of β cell mass (BCM) by positron emission tomography (PET) with [¹⁸F]fluoropropyl-dihydrotetrabenazine ([¹⁸F]FP-(+)-DTBZ).

Procedures

We evaluated the feasibility of longitudinal pancreatic PET VMAT2 quantification in the pancreas in two studies of healthy controls and patients with type 1 or 2 diabetes. VMAT2 binding potential (BP_ND) was estimated voxelwise using a reference tissue method in a cross-sectional study, followed by assessment of reproducibility using a test-retest paradigm. Metabolic function was evaluated by stimulated c-peptide measurements.

Results

Pancreatic BP_ND was significantly decreased in patients with type 1 diabetes relative to controls and the test-retest variability was 9.4 %.

Conclusions

Pancreatic VMAT2 content is significantly reduced in long-term diabetes patients relative to controls and repeat scans are sufficiently reproducible to suggest the feasibility clinically VMAT2 measurements in longitudinal studies of new onset diabetes.

     

Assessment of Progression and Treatment Response of Optic Pathway Glioma with Positron Emission Tomography using α-[<Superscript>11</Superscript>C]Methyl-<Emphasis Type="SmallCaps">l</Emphasis>-Tryptophan

Purpose To report the utility of positron emission tomography (PET) with α-[¹¹C]methyl-l-tryptophan (AMT) for monitoring progression and response to treatment of an isolated optic pathway glioma (OPG) in a 16-year-old girl. Procedures Positron emission tomography scanning of the brain was performed 20 minutes after intravenous administration of AMT. The AMT-PET images were reconstructed and examined for tumor uptake of the tracer in correlation with coregistered magnetic resonance images. Results The PET scan demonstrated increased uptake of AMT by OPG in a clinically symptomatic child whose magnetic resonance imaging (MRI) was inconclusive for morphological changes of the tumor. The tracer uptake was dramatically decreased on the images obtained after chemotherapy. Subsequently, AMT-PET revealed a new tumor lesion of increased AMT uptake when the patient developed vision problems and MRI showed no significant interval morphological changes. Significant vision improvement was observed after external beam radiotherapy for the newly identified tumor lesion. Conclusions Positron emission tomography with α-[¹¹C]methyl-l-tryptophan may be useful for monitoring progression and response to treatment of OPGs, which needs to be further investigated in a prospective study of more patients, including those with neurofibromatosis.     

PET Imaging of l-Type Amino Acid Transporter (LAT1) and Cystine-Glutamate Antiporter (xc−) with [18F]FDOPA and [18F]FSPG in Breast Cancer Models

Molecular Imaging and Biology - The present study describes the analysis of amino acid transporters ASCT1, ASCT2, LAT1, and xc? in breast cancer under normoxic and hypoxic conditions....     

Radiosynthesis and Initial <Emphasis Type="Italic">In Vitro</Emphasis> Evaluation of [<Superscript>18</Superscript>F]F-PEG<Subscript>6</Subscript>-IPQA—A Novel PET Radiotracer for Imaging EGFR Expression-Activity in Lung Carcinomas

Introduction  

Epidermal growth factor receptor (EGFR)-targeted therapies with antibodies and small molecular EGFR kinase inhibitors have shown poor efficacy in unselected populations of patients with advanced non-small cell lung carcinomas (NSCLC). In contrast, patients with overexpression of EGFR and activating mutations in EGFR kinase domain demonstrated improved responses to EGFR kinase inhibitors. Therefore, we have developed a novel radiotracer, [¹⁸F]F-PEG₆-IPQA for PET imaging of EGFR expression-activity in NSCLC, and have described its radiosynthesis and in vitro evaluation in two NSCLC cell lines with wild-type and L858R active mutant EGFR.     

Blood Clearance Kinetics,Biodistribution, and Radiation Dosimetry of a Kit-Formulated Integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript>-Selective Radiotracer <Superscript>99m</Superscript>Tc-3PRGD<Subscript>2</Subscript> in Non-Human Primates

Purpose  

^99mTc-3PRGD₂ is a ^99mTc-labeled dimeric cyclic RGD peptide with increased receptor binding affinity and improved kinetics for in vivo imaging of integrin α_vβ₃ expression in nude mouse model. To accelerate its clinical translation, we reported here the evaluation of the kit-formulated ^99mTc-3PRGD₂ in healthy cynomolgus primates for its blood clearance kinetics, biodistribution, and radiation dosimetry.