Elisabeth von Guggenberg

68Ga-DOTA-Tyr3-Octreotide PET in Neuroendocrine Tumors: Comparison with Somatostatin Receptor Scintigraphy and CT

The aim of this study was to evaluate the diagnostic value of a new somatostatin analog, 68Ga-labeled 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid-d-Phe1-Tyr3-octreotide (68Ga-DOTA-TOC), for PET in patients with known or suspected neuroendocrine tumors. PET was compared with conventional scintigraphy and dedicated CT. Methods: Eighty-four patients (48 men, 36 women; age range, 28–79 y; mean age ± SD, 58.2 ± 12.2 y) were prospectively studied. For analysis, patients were divided into 3 groups: detection of unknown primary tumor in the presence of clinical or biochemical suspicion of neuroendocrine malignancy (n = 13 patients), initial tumor staging (n = 36 patients), and follow-up after therapy (n = 35 patients). Each patient received 100–150 MBq 68Ga-DOTA-TOC. Imaging results of PET were compared with 99mTc-labeled hydrazinonicotinyl-Tyr3-octreotide (99mTc-HYNIC-TOC) and 111In-DOTA-TOC. CT was also performed on every patient using a multidetector scanner. Each imaging modality was interpreted separately by observers who were unaware of imaging findings before comparison with PET. The gold standard for defining true-positive (TP), true-negative (TN), false-positive (FP), and false-negative (FN) results was based on all available histologic, imaging, and follow-up findings. Results: PET was TP in 69 patients, TN in 12 patients, FP in 1 patient, and FN in 2 patients, indicating a sensitivity of 97%, a specificity of 92%, and an accuracy of 96%. The FP finding was caused by enhanced tracer accumulation in the pancreatic head, and the FN results were obtained in patients with a tumor of the gastrointestinal tract displaying liver metastases. 68Ga-DOTA-TOC showed higher diagnostic efficacy compared with SPECT (TP in 37 patients, TN in 12 patients, FP in 1 patient, and FN in 34 patients) and diagnostic CT (TP in 41 patients, TN in 12 patients, FP in 5 patients, and FN in 26 patients). This difference was of statistical significance (P < 0.001). However, the combined use of PET and CT showed the highest overall accuracy. Conclusion: 68Ga-DOTA-TOC PET shows a significantly higher detection rate compared with conventional somatostatin receptor scintigraphy and diagnostic CT with clinical impact in a considerable number of patients.

Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells

Radiation therapy plays an important role in the management of prostate carcinoma. However, the problem of radioresistance and molecular mechanisms by which prostate carcinoma cells overcome cytotoxic effects of radiation therapy remains to be elucidated. In order to investigate possible intracellular mechanisms underlying the prostate carcinoma recurrences after radiotherapy, we have established three radiation‐resistant prostate cancer cell lines, LNCaP‐IRR, PC3‐IRR, and Du145‐IRR derived from the parental LNCaP, PC3, and Du145 prostate cancer cells by repetitive exposure to ionizing radiation. LNCaP‐IRR, PC3‐IRR, and Du145‐IRR cells (prostate carcinoma cells recurred after radiation exposure (IRR cells)) showed higher radioresistance and cell motility than parental cell lines. IRR cells exhibited higher levels of androgen and epidermal growth factor (EGF) receptors and activation of their downstream pathways, such as Ras‐mitogen‐activated protein kinase (MAPK) and phosphatidyl inositol 3‐kinase (PI3K)‐Akt and Jak‐STAT. In order to define additional mechanisms involved in the radioresistance development, we determined differences in the proteome profile of parental and IRR cells using 2‐D DIGE followed by computational image analysis and MS. Twenty‐seven proteins were found to be modulated in all three radioresistant cell lines compared to parental cells. Identified proteins revealed capacity to interact with EGF and androgen receptors related signal transduction pathways and were involved in the regulation of intracellular routs providing cell survival, increased motility, mutagenesis, and DNA repair. Our data suggest that radioresistance development is accompanied by multiple mechanisms, including activation of cell receptors and related downstream signal transduction pathways. Identified proteins regulated in the radioresistant prostate carcinoma cells can significantly intensify activation of intracellular signaling that govern cell survival, growth, proliferation, invasion, motility, and DNA repair. In addition, such analyses may be utilized in predicting cellular response to radiotherapy.

A fully automated synthesis for the preparation of 68Ga-labelled peptides.

BackgroundGenerator-produced 68Ga has attracted increasing interest for radiolabelling peptides used in PET applications. So far, the synthesis of 68Ga-peptide radiopharmaceuticals is mainly based on semi-automated systems. Here we describe a fully automated approach for the synthesis of 68Ga-labelled peptides. MethodA commercially available 68Ga generator was eluted with 0.1 mol · l−1 HCl. Reaction parameters such as buffer conditions, pH range, reaction temperature and time, volume of reaction solution and generator fraction were optimized for labelling DOTA-Tyr3-octreotide (DOTATOC). Reaction yields, pH, radiochemical purity, sterility, endotoxins, breakthrough of 68Ge and final 68Ge content were determined. A fully automated radiopharmaceutical synthesis device based on a modular concept for remote-controlled processing was developed and evaluated for a number of DOTA-derivatized peptides. ResultsDOTATOC could be labelled in almost quantitative yields by heating 10–50 nmol peptide at pH 3.5–4.0 for 5 min at 95°C in 1.5 ml. Purification using a reversed-phase cartridge was required to avoid any potential 68Ge breakthrough: final activities of 68Ge were below 100 Bq · ml−1. Automated synthesis resulted in overall decay-corrected reaction yields of about 60% within 10 min. Even after 1 year using a 1110 MBq generator more than 130 MBq 68Ga-DOTATOC could be obtained. Moreover, it was demonstrated that a variety of DOTA-derivatized peptides can be labelled using identical reaction conditions with high yields. ConclusionThe system described allows the fully automated, efficient and rapid preparation of 68Ga-DOTA-derivatized peptides. It has been used successfully and reliably for routine preparations in clinical studies.

Radiolabeling of lipid-based nanoparticles for diagnostics and therapeutic applications: a comparison using different radiometals.

Radiolabeling of nanoparticles (NPs) has been performed for a variety of reasons, such as for studying pharmacokinetics, for imaging, or for therapy. Here, we describe the in vitro and in vivo evaluation of DTPA-derivatized lipid-based NP (DTPA-NP) radiolabeled with different radiometals, including 111In and 99mTc, for single-photon emission computed tomography (SPECT), 68Ga for positron emission tomography (PET), and 177Lu for therapeutic applications. PEGylated DTPA-NP with varying DTPA amounts, different composition, and size were radiolabeled with 111In, 177Lu, and 68Ga, using various buffers. 99mTc-labeling was performed directly and by using the carbonyl aquaion, [99mTc(H2O)3(CO)3]+. Stability was tested and biodistribution evaluated. High labeling yields (>90%) were achieved for all radionuclides and different liposomal formulations. Specific activities (SAs) were highest for 111In (>4 MBq/μg liposome), followed by 68Ga and 177Lu; for 99mTc, high labeling yields and SA were only achieved by using [99mTc(H2O)3(CO)3]+. Stability toward DTPA/histidine and in serum was high (>80 % RCP, 24 hours postpreparation).). Biodistribution in Lewis rats revealed no significant differences between NP in terms of DTPA loading and particle composition; however, different uptake patterns were found between the radionuclides used. We observed lower retention in blood (<3.3 %ID/g) and lower liver uptake (< 2.7 %ID/g) for 99mTc- and 68Ga, compared to 111In-NP (blood, <4 %ID/g; liver, <3.6 %ID/g). Imaging potential was shown by both PET magnetic resonance imaging fusion imaging and SPECT imaging. Overall, our study shows that PEGylated DTPA-NP are suitable for radiolabeling studies with a variety of radiometals, thereby achieving high SA suitable for targeting applications.

Tumor targeting and imaging with dual-peptide conjugated multifunctional liposomal nanoparticles.

Background The significant progress in nanotechnology provides a wide spectrum of nanosized material for various applications, including tumor targeting and molecular imaging. The aim of this study was to evaluate multifunctional liposomal nanoparticles for targeting approaches and detection of tumors using different imaging modalities. The concept of dual-targeting was tested in vitro and in vivo using liposomes derivatized with an arginine-glycine-aspartic acid (RGD) peptide binding to αvβ3 integrin receptors and a substance P peptide binding to neurokinin-1 receptors. Methods For liposome preparation, lipids, polyethylene glycol building blocks, DTPA-derivatized lipids for radiolabeling, lipid-based RGD and substance P building blocks and imaging labels were combined in defined molar ratios. Liposomes were characterized by photon correlation spectroscopy and zeta potential measurements, and in vitro binding properties were tested using fluorescence microscopy. Standardized protocols for radiolabeling were developed to perform biodistribution and micro-single photon emission computed tomography/computed tomography (SPECT/CT) studies in nude mice bearing glioblastoma and/or melanoma tumor xenografts. Additionally, an initial magnetic resonance imaging study was performed. Results Liposomes were radiolabeled with high radiochemical yields. Fluorescence microscopy showed specific cellular interactions with RGD-liposomes and substance P-liposomes. Biodistribution and micro-SPECT/CT imaging of 111In-labeled liposomal nanoparticles revealed low tumor uptake, but in a preliminary magnetic resonance imaging study with a single-targeted RGD-liposome, uptake in the tumor xenografts could be visualized. Conclusion The present study shows the potential of liposomes as multifunctional targeted vehicles for imaging of tumors combining radioactive, fluorescent, and magnetic resonance signaling. Specific in vitro tumor targeting by fluorescence microscopy and radioactivity was achieved. However, biodistribution studies in an animal tumor model revealed only moderate tumor uptake and no additive effect using a dual-targeting approach.

Cyclic minigastrin analogues for gastrin receptor scintigraphy with technetium-99m: preclinical evaluation.

Two cyclized minigastrin analogues for gastrin receptor scintigraphy were synthesized and derivatized with HYNIC at the N-terminus for labeling with 99mTc. Radiolabeling efficiency, stability, cell internalization, and receptor binding on CCK-2 receptor expressing AR42J cells were studied and the biodistribution evaluated in tumor bearing nude mice, including NanoSPECT/CT imaging. Metabolites in urine, liver, and kidneys were analyzed by radio-HPLC. Radiolabeled cyclic MG showed high stability in vitro and receptor mediated uptake in AR42J cells. In the animal tumor model, fast renal clearance and low nonspecific uptake in most organs were observed. A tumor uptake >3% was calculated ex vivo 1 h p.i. for both 99mTc-EDDA-HYNIC-cyclo-MG1 and 99mTc-EDDA-HYNIC-cyclo-MG2. In an imaging study with 99mTc-EDDA-HYNIC-cyclo-MG1, the tumor was clearly visualized. The metabolite analysis indicated rapid enzymatic degradation in vivo.

Response of Recurrent High-Grade Glioma to Treatment with 90Y-DOTATOC

The treatment of patients with high-grade malignant glioma still represents an unsolved clinical problem. We report the treatment of 3 patients who had World Health Organization grade IV recurrent glioblastoma: a 23-y-old woman and 2 men aged 61 and 62 y. Methods: All 3 patients were treated with the somatostatin receptor radiopharmaceutical 90Y-labeled [1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid0-d-Phe1,Tyr3]octreotide (DOTATOC). A cumulated dose of 1.7–2.2 GBq given in 3 or 4 cycles was locally injected into a previously implanted catheter system. Results: Treatment was successful in all 3 patients, with only minor side effects reported. After treatment, MRI and PET showed complete remission in one patient and partial remission in the other patients. These findings correlated well with clinical improvement and improved quality of life. Conclusion: Receptor-mediated radionuclide therapy by locally injected 90Y-DOTATOC is feasible and well tolerated. This approach represents an attractive strategy for the treatment of locally recurring or progressing glioblastoma.

Influence of PEGylation and RGD loading on the targeting properties of radiolabeled liposomal nanoparticles.

Purpose Liposomes have been proposed to be a means of selectively targeting cancer sites for diagnostic and therapeutic applications. The focus of this work was the evaluation of radiolabeled PEGylated liposomes derivatized with varying amounts of a cyclic arginyl–glycyl–aspartic acid (RGD) peptide. RGD peptides are known to bind to αvβ3 integrin receptors overexpressed during tumor-induced angiogenesis. Methods Several liposomal nanoparticles carrying the RGD peptide targeting sequence (RLPs) were synthesized using a combination of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, cholesterol, diethylenetriaminepentaacetic acid-derivatized lipids for radiolabeling, a polyethylene glycol (PEG) building block, and a lipid-based RGD building block. Relative amounts of RGD and PEG building blocks were varied. In vitro binding affinities were determined using isolated αvβ3 integrin receptors incubated with different concentrations of RLPs in competition with iodine-125-labeled cyclo-(-RGDyV-). Binding of the indium-111-labeled RLPs was also evaluated. Biodistribution and micro single photon emission computed tomography/computed tomography imaging studies were performed in nude mice using different tumor xenograft models. Results RLPs were labeled with indium-111 with high radiochemical yields. In vitro binding studies of RLPs with different RGD/PEG loading revealed good binding to isolated receptors, which was dependent on the extent of RGD and PEG loading. Binding increased with higher RGD loading, whereas reduced binding was found with higher PEG loading. Biodistribution showed increased circulating time for PEGylated RLPs, but no dependence on RGD loading. Both biodistribution and micro single photon emission computed tomography/computed tomography imaging studies revealed low, nonspecific tumor uptake values. Conclusion In this study, RLPs for targeting angiogenesis were described. Even though good binding to αvβ3 integrin receptors was found in vitro, the balance between PEGylation and RGD loading clearly requires optimization to achieve targeting in vivo. These data form the basis for future development and provide a platform for the investigation of multimodal approaches.

Radiolabelling of peptides for PET, SPECT and therapeutic applications using a fully automated disposable cassette system.

ObjectivesRadiolabelled somatostatin analogues have found wide clinical use in nuclear medicine for both diagnostic and therapeutic applications. Here, we describe the development of a fully automated synthesis system allowing radiolabelling of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-derivatized peptides with 68Ga/111In/177Lu and 90Y, meeting radiation safety and pharmaceutical requirements. Materials and methodsThe system consists of a syringe pump, a holder for insertion of a single use multivalve cassette, a heater and a removable radiation shielding. 68Ga labelling was performed in acetate buffer and 177Lu, 90Y and 111In labelling in ascorbate buffer, respectively, followed by purification on a C18 cartridge and final sterile filtration. Cross-contamination was prevented by using disposable cassettes and also by ensuring pharmaceutical standards. Radiochemical purity (RCP) was determined by instant thin-layer chromatography on silica gel impregnated glass fibres and reversed-phase high performance liquid chromatography. Results68Ga-DOTA-peptides were prepared with high RCP (>91%) and radiochemical yields (RCY>80% decay corrected) and 68Ge content was less than 0.0001% in all cases. Synthesis time did not exceed 30 min. 111In, 177Lu and 90Y labelling of DOTA-peptides resulted again in high yields (approximately 90%) and RCP (approximately 95%) and total synthesis time of less than 45 min. Radiation dose to fingers was considerably reduced when compared with manual labelling procedures. ConclusionThe described system allows fully automated, aseptic preparation of DOTA-peptides radiolabelled with different radionuclides in high radiochemical yields and pharmaceutical quality suitable for clinical application.

Role of radiopharmaceuticals in the diagnosis and treatment of neuroendocrine tumours.

Neuroendocrine tumours (NETs) are a heterogeneous group of neoplasms characterised by their endocrine metabolism and histological pattern, originating mainly from the gastroenteropancreatic tract (GEP NETs). As opposed to other tumour entities GEP NETs are relatively rare tumours and their diagnosis requires a high index of suspicion. NETs characteristically synthesise, store and secrete a variety of peptides and neuroamines which may lead to clinical syndromes such as the carcinoid syndrome, the Zollinger Ellison syndrome, or the Verner Morrison syndrome [1,2]. However, most GEP NETs are clinically silent until late presentation with metastases. In fact, a delayed diagnosis of NET is typical, resulting in excessive morbidity and mortality and increased probability of metastatic disease. A multidisciplinary approach for therapeutic intervention is necessary. On initial clinical presentation, NETs may be difficult to diagnose because of the large variability in tumour location and the high frequency of small lesions. The proliferation marker Ki-67 (MIB-1) is important in determining tumour grade and prognosis. Once metastasised, the therapeutic options for patients with NETs are limited. Treatment with long-acting somatostatin (SST) analogues results in reduced hormonal (over)production, and thus, relief of symptoms. Surgery is the therapy of choice in localised disease. Chemoembolisation of primary tumour/liver metastases can be attempted in order to reduce the tumour mass for subsequent therapy with radiolabelled peptide analogues in patients with slowgrowing tumours. In patients with poorly differentiated tumours chemotherapy may be considered [3]. However, the management options are manifold, and there are only a few evidence-based controlled studies available. Furthermore, formal guidelines are required. One should be aware that not all centres have the same treatment approach. For example, some believe that the long-acting SST analogues exert a beneficial effect even when the patient does not have symptoms. Positive effects of long-acting octreotide on tumour progression were assessed in the PROMID study recently [4]. In addition, there is not a general agreement as to the scope and timing of tumour debulking surgery and/or chemoembolisation [1]. The assessment of the location and extent of NETs is crucial for clinical management. Although being a heterogeneous group of neoplasms, NETs are characterised by their ability to over-express SST receptors (SSTR) at the cell surface. For localisation, conventional SSTR scintigraphy/positron emission tomography (PET), computed tomograhpy (CT)/magnetic resonance imaging (MRI), fused PET/CT or PET/MRI imaging, endoscopic ultrasound, arterial stimulation and venous sampling are used [5–8]. Other nuclear medicine techniques are also available for rare NETs. Here, we give an overview of the clinical value of radiopharmaceuticals used for diagnosis and treatment of NET patients.