Kinetic quantification of protein polymer nanoparticles using non-invasive imaging.

Protein polymers are repetitive amino acid sequences that can assemble monodisperse nanoparticles with potential applications as cancer nanomedicines. Of the currently available molecular imaging methods, positron emission tomography (PET) is the most sensitive and quantitative; therefore, this work explores microPET imaging to track protein polymer nanoparticles over several days. To achieve reliable imaging, the polypeptides were modified by site-specific conjugation using a heterobifunctional sarcophagine chelator, AmBaSar, which was subsequently complexed with (64)Cu. AmBaSar/(64)Cu was selected because it can label particles in vivo over periods of days, which is consistent with the timescales required to follow long-circulating nanotherapeutics. Using an orthotopic model of breast cancer, we observed four elastin-like polypeptides (ELPs)-based protein polymers of varying molecular weight, amino acid sequence, and nanostructure. To analyze this data, we developed a six-compartment image-driven pharmacokinetic model capable of describing their distribution within individual subjects. Surprisingly, the assembly of an ELP block copolymer (78 kD) into nanoparticles (R(h) = 37.5 nm) minimally influences pharmacokinetics or tumor accumulation compared to a free ELP of similar length (74 kD). Instead, ELP molecular weight is the most important factor controlling the fate of these polymers, whereby long ELPs (74 kD) have a heart activity half-life of 8.7 hours and short ELPs (37 kD) have a half-life of 2.1 hours. These results suggest that ELP-based protein polymers may be a viable platform for the development of multifunctional therapeutic nanoparticles that can be imaged using clinical PET scanners.

Changes in regional brain perfusion during functional brain activation: comparison of [(64)Cu]-PTSM with [(14)C]-Iodoantipyrine.

A dilemma in behavioral brain mapping is that conventional techniques immobilize the subject, extinguishing all but the simplest behaviors. This is avoided if brain activation is imaged after completion of the behavior and tissue capture of the tracer. A single-pass flow tracer proposed for positron emission tomography (PET) is a radiolabeled copper(II) complex of pyruvaldehyde bis(N(4)-methylthiosemicarbazone), [Cu(64)]-PTSM. [Cu(64)]-PTSM reaches steady-state cerebral distribution more rapidly than the metabolic tracer [(18)F]-fluorodeoxyglucose, allowing imaging with substantially greater temporal resolution. Using dual-label autoradiography, this study compares the relative regional cerebral blood flow tracer distribution (CBF-TR) of [(64)Cu]-PTSM to that of the classic perfusion tracer [(14)C]-iodoantipyrine in a rat model during treadmill walking. Rats were exposed to continuous walking on a treadmill and compared to quiescent controls. [(64)Cu]-PTSM was bolus injected (iv) after 1 min, followed by a 5-minute uptake and subsequent bolus injection of [(14)C]-iodoantipyrine. CBF-TR was quantified by autoradiography and analyzed in the three-dimensionally reconstructed brain by statistical parametric mapping, as well as by region-of-interest analysis. A high homology was found between the [(64)Cu]-PTSM and [(14)C]-iodoantipyrine patterns of cerebral activation in cortical and subcortical regions. For white matter, however, [(64)Cu]-PTSM showed lower perfusion than [(14)Cu]-iodoantipyrine. [(64)Cu]-PTSM is a useful tracer for functional brain mapping in freely-moving subjects. Its application in conjunction with PET promises to increase our understanding of the neural circuitry of behaviors dependent on locomotion.

Diagnostic role of [F-18]-FDG positron emission tomography in restaging renal cell carcinoma.

AIMS: To retrospectively assess the diagnostic utility of positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) in restaging renal cell carcinoma. MATERIALS AND METHODS: We performed whole-body PET scans (45 minutes after intravenous injection of 10 - 15 mCi FDG) for restaging 25 patients (18 male, 7 female, 42 - 81 years old) with known or suspected metastatic renal cell carcinoma. Prior treatments included immunotherapy (n = 1), nephrectomy (n = 16), nephrectomy followed by chemotherapy (n = 3), by radiation therapy (n = 1), and by combined chemoradiation therapy (n = 4). Contrast-enhanced chest, abdomen and pelvis CT studies were available for all patients. Diagnostic validation was by histological sampling (n = 2) and clinical and imaging follow-up for up to 1 year (n = 23). RESULTS: PET was concordant with the findings of CT in 18 patients (3 TN, 15 TP). PET was discordant with CT in 7 patients (28% of total). PET was falsely negative in 6 of these patients and did not demonstrate hypermetabolism in pulmonary (n = 4), mediastinal (n = 2), adrenal (n = 1) and lytic osseous (n = 2) metastatic lesions. PET was falsely positive in the remaining 1 patient in the discordant group with lumbar facet arthropathy. The diagnostic performance of PET in detection of recurrent and metastatic renal cell carcinoma revealed a sensitivity of 71%, specificity of 75%, accuracy of 72%, negative predictive value of 33% and positive predictive value of 94%. CONCLUSIONS: FDG PET demonstrates modest accuracy in the diagnostic imaging evaluation of patients with suspected or known metastatic renal cell carcinoma. A negative study may not exclude disease while a positive study is suspicious for malignancy.

Evaluation of recurrent gastric malignancy with [F-18]-FDG positron emission tomography.

AIM: We retrospectively assessed the use of [(18)F] fluorodeoxyglucose positron emission tomography (FDG PET) in the evaluation of recurrent disease in patients with history of gastric malignancy. MATERIALS AND METHODS: Eighteen patients were referred for FDG PET for evaluation of recurrent gastric cancer. Prior treatments included total (n = 4) or partial gastrectomy (n = 14) followed by chemotherapy alone (n = 7) or combined chemoradiation therapy (n = 2). The interval between the most recent treatment and PET ranged from 3 months to 2 years. Correlative diagnostic data were available in 16 patients and were all obtained within 3 months of the PET study. Validation was by clinical or imaging follow-up (2-45 months) in 16 patients and histology in two patients. RESULTS: PET was concordant with computed tomography (CT) in 12 patients (5 TP, 6 TN, 1 FN). In one patient with negative imaging studies, an incidental finding of left obstructive uropathy was determined to be due to metastatic ureteral stricture. Discordant imaging findings were present in four patients (22% of total). PET-detected diffuse metastatic lesions in three of these patients with rising serum tumour markers while other imaging studies were negative. Additional chemotherapy was initiated in these three patients (17% of total) based on PET localization of disease. PET and a gastric anastomosis biopsy were negative in another patient with positive CT. The remaining two patients without correlative imaging studies died shortly after positive PET studies with presumed recurrent cancer. CONCLUSION: FDG PET may be useful in the evaluation of recurrent gastric cancer, and can localize the disease when CT is non-diagnostic. Imaging evaluation with PET may also impact on the clinical management of patients with recurrent gastric cancer.

Preclinical evaluation of the penciclovir analog 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine for in vivo measurement of suicide gene expression with PET.

UNLABELLED: The gene for herpes simplex virus thymidine kinase (HSV-tk) is widely used as a suicide gene in experimental gene therapy of cancer. 9-(4-Fluoro-3-hydroxymethylbutyl)guanine (FHBG) is an antiviral nucleoside analog that is rapidly phosphorylated by viral thymidine kinase but is a poor substrate for mammalian thymidine kinase. Recently, FHBG labeled in the 4-fluoro position with (18)F has shown promise relative to other similar compounds for imaging in vivo expression of HSV-tk using PET. In this study, we evaluated the uptake of [(18)F]FHBG in vitro and in vivo using transduced and wild-type human colon cancer cells (HT-29). We also imaged [(18)F]FHBG and measured the radioactivity concentrations of circulating [(18)F]FHBG and its metabolites in monkeys. METHODS: Sterile, pyrogen-free [(18)F]FHBG was produced routinely in good yields. Cells were transduced with the retroviral vector G1Tk1SvNa containing HSV-tk gene. In vitro uptake studies were performed by incubating cells with [(18)F]FHBG at 37 degrees C for 1 and 5 h. Biodistribution studies were performed at 2 and 5 h after injection in nude mice bearing tumors grown from wild-type or transduced cells. Sequential, whole-body PET scans of cynomolgus monkeys were obtained over a period of >2 h after intravenous injection of [(18)F]FHBG. Arterial plasma samples obtained from monkeys 15-120 min after intravenous injection were subjected to acid extraction, and the acid-soluble fractions were analyzed by high-performance liquid chromatography. RESULTS: In vitro studies showed 31 and 71 (P < 0.001) times higher uptake of the probe at 1 and 5 h, respectively, in transduced cells compared with nontransduced cells. In vivo studies in mice showed that tumor uptake of the radiotracer was 4-fold (P < 0.05) and 13-fold (P < 0.001) higher at 2 and 5 h, respectively, in tumors grown from transduced cells compared with control cells. Transduced tumor-to-normal tissue ratios ranged from 2 to 25 at 2 h and from 2 to 22 at 5 h. Recirculating labeled metabolites had only a minor effect on the biodistribution of radiolabel from [(18)F]FHBG in monkeys. CONCLUSION: These results indicate that [(18)F]FHBG may yield high-contrast PET images of HSV-tk expression in tumors and, therefore, it is a very promising radiotracer for monitoring of gene therapy of cancer with PET.

A cost analysis of positron emission tomography.

OBJECTIVE: Changes in regulations and improvements in reimbursement have propelled positron emission tomography (PET) into clinical use, making it increasingly important to understand the costs of this emerging service. Cost analyses are important tools to do this. Data published previously on these topics reflect assumptions that are no longer valid. The aim of this study was to determine the cost of developing and operating a PET facility and to evaluate whether a regional cyclotron serving several scanners reduces costs. MATERIALS AND METHODS: Financial data were collected on capital expense and global operating costs through interviews with industry experts, evaluation of prior studies, and review of expenses incurred at the University of Southern California PET center. A data model and cost templates were developed. Expenses were allocated either to the production or purchase of radiopharmaceuticals or to the provision of the PET scan, and the cost per procedure was determined. A sensitivity analysis was performed on the net present value for key parameters. RESULTS: A cyclotron serving a single scanner is not financially viable. The radiopharmaceutical distribution configurations were financially sound. In these cases, the cost of the radiopharmaceutical was approximately $700 per dose with modest levels of production (12 doses per day). In addition, the average cost of PET scans (technical scan and professional charges) ranged from approximately $900 to $1400. The critical factor for profitability was shown to be throughput. CONCLUSION: This analysis provides significant insight into the cost of PET and the comparative costs of offering PET through four operating configurations. Reductions in equipment prices, increased availability of radiopharmaceuticals, growth in demand, and improvements in reimbursement have all contributed to the financial viability of this imaging technique.

Blocking catabolism with eniluracil enhances PET studies of 5-[18F]fluorouracil pharmacokinetics.

UNLABELLED: Noninvasive methods for measuring the pharmacokinetics of chemotherapeutic drugs such as 5-fluorouracil (FU) are needed for individualized optimization of treatment regimens. PET imaging of [18F]FU (PET/[18F]FU) is potentially useful in this context, but PET/[18F]FU is severely hampered by low tumor uptake of radiolabel and rapid catabolism of FU in vivo. Pretreatment with eniluracil (5-ethynyluracil) prevents catabolism of FU. Hypothesizing that suppression of catabolism would enhance PET/[18F]FU, we examined the effects of eniluracil on the short-term pharmacokinetics of the radiotracer. METHODS: Anesthetized rats bearing a subcutaneous rat colorectal tumor were given eniluracil or placebo and injected intravenously 1 h later with [18F]FU or [3H]FU. In the 18F studies, dynamic PET image sequences were obtained 0-2 h after injection. Tumors were excised and frozen at 2 h and then analyzed for labeled metabolites by high-performance liquid chromatography. Biodistribution of radiolabel was determined by direct tissue assay. RESULTS: Eniluracil improved tumor visualization in PET images. With eniluracil, tumor standardized uptake values ([activity/g]/[injected activity/g body weight]) increased from 0.72 +/- 0.06 (mean +/- SEM; n = 6) to 1.57 +/- 0.20 (n = 12; P < 0.01), and tumor uptake increased by factors of 2 or more relative to plasma (P < 0.05) and bone, liver, and kidney (P < 0.01). Without eniluracil (n = 5), 57% +/- 4% of recovered radiolabel in tumor at 2 h was on catabolites, with the rest divided among FU (2% +/- 1%), anabolites of FU (38% +/- 7%), and unidentified peaks (4% +/- 2%). With eniluracil (n = 8), catabolites, FU, and anabolites comprised 2% +/- 1%, 41% +/- 5%, and 57% +/- 4%, respectively, of the recovered radiolabel in tumors. CONCLUSION: Eniluracil increased tumor accumulation of 18F relative to host tissues and fundamentally changed the biochemical significance of that accumulation. With catabolism suppressed, tumor radioactivity reflected the therapeutically relevant aspect of FU pharmacokinetics--namely, uptake and anabolic activation of the drug. With this approach, it may be feasible to measure the transport and anabolism of [18F]FU in tumors by kinetic modeling and PET. Such information may be useful in predicting and increasing tumor response to FU.

Pharmacokinetics of the thymidine analog 2'-fluoro-5-[(14)C]-methyl-1-beta-D-arabinofuranosyluracil ([(14)C]FMAU) in rat prostate tumor cells.

2'-Fluoro-5-[(14)C]-methyl-1-beta-D-arabinofuranosyluracil (FMAU) is an analog of thymidine (TdR) that is resistant to catabolism, is incorporated into DNA, and has been labeled with (11)C for use with positron emission tomography. We compared the uptake and metabolism of [(14)C]FMAU with that of [(3)H]TdR in fast- and slow-growing cell lines of a rat prostate tumor. Although FMAU was incorporated much less rapidly than TdR, FMAU behaved very similarly to TdR with respect to correlation between uptake velocity and cell growth rate, saturability of cellular incorporation, and intracellular metabolite pools. Thus, FMAU warrants further evaluation as an in vivo indicator of tumor cell division.

Imaging of locally recurrent and metastatic thyroid cancer with positron emission tomography.

Serum thyroglobulin and imaging have been routinely used in the evaluation of thyroid cancer patients suspected of having metastatic or recurrent disease. A more sensitive technique capable of identifying the sites of disease not detected by current imaging methods might improve overall management. The objective in this study was to demonstrate the feasibility of using positron emission tomography (PET) for the detection of recurrent thyroid cancer. Thirty patients with a history of either papillary/follicular or medullary thyroid cancer suspected of having locally recurrent or metastatic cancer on the basis of elevated or rising blood markers were evaluated with PET. Imaging studies were performed with the radiotracer [F-18] fluorodeoxyglucose (FDG). A retrospective review of other imaging results was performed and compared to the PET results. PET was able to identify locally recurrent or metastatic papillary/follicular disease in all 24 patients studied with elevated or rising thyroglobulin. Similar results were obtained in 6 patients with medullary cancer recurrences in the presence of elevated calcitonin. In cases where follow-up data was obtainable (17/24 papillary/follicular cancers and 4/6 medullary cancers), disease was confirmed either directly by surgery and/or indirectly through changes or persistence of laboratory findings. The results support the hypothesis that in the presence of elevated blood markers indicative of recurrent thyroid cancer, PET may prove valuable as an adjunctive imaging test for identifying disease and influencing management in cases where conventional imaging fails to detect suspected disease.

Evaluation of 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]-FHPG) in vitro and in vivo as a probe for PET imaging of gene incorporation and expression in tumors.

Preparation of 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl]-guanine ([18F]-FHPG) for clinical use, and its evaluation as a positron emission tomography (PET) imaging agent for gene incorporation and expression in tumors are reported. In vitro studies in human colon cancer cells, HT-29, transduced with the retroviral vector G1Tk1SvNa and nontransduced (wild type) showed 4, 8, 12, and 15 times higher uptake of the probe in 1, 3, 5, and 7 h, respectively, in transduced cells compared with the controls. In vivo studies in tumor-bearing nude mice demonstrated that the tumor uptake of the radiotracer is three and six-fold higher in 2 and 5 h, respectively, in transduced cells compared with the control cells. These results suggest that [18F]-FHPG is a potential in vivo PET imaging agent for monitoring gene incorporation and expression in gene therapy of cancer.

Cerebral vasculopathy in sickle cell anemia: diagnostic contribution of positron emission tomography.

Children with sickle cell anemia (SS) have an increased risk for cerebral vasculopathy with stroke (CVA) and cognitive impairment. The present study examines the extent to which adding positron emission tomography (PET) to magnetic resonance imaging (MRI) can improve the detection of cerebral vasculopathy. Whereas MRI has been the prime modality for showing anatomical lesions, PET excels at assessing the functional metabolic state through glucose utilization 2-deoxy-2 [18F] fluoro-D-glucose (FDG) and microvascular blood flow ([15O]H2O). Forty-nine SS children were studied. Among them, 19 had clinically overt CVA, 20 had life-threatening hypoxic episodes or soft neurologic signs, and 10 were normal based on neurological history and examination. For the entire sample of 49 subjects, 30 (61%) had abnormal MRI findings, 36 (73%) had abnormal PET findings, and 44 (90%) showed abnormalities on either the MRI or the PET or both. Of the 19 subjects with overt CVA, 17 had abnormal MRI (89%), 17 had abnormal PET (89%), and 19 (100%) had either abnormal MRI or PET or both. Among the 20 subjects with soft neurologic signs, 10 (50%) had abnormal MRI, 13 (65%) had abnormal PET, and 17 (85%) had abnormal MRI and/or PET. Six (60%) of the 10 neurologically normal subjects had abnormal PET. Among the 30 subjects with no overt CVA, 25 (83%) demonstrated imaging abnormalities based on either MRI or PET or both, thus, silent ischemia. Lower than average full-scale intelligence quotient (FSIQ) was associated with either overt CVA or silent ischemic lesions. Four subjects who received chronic red blood cell transfusion showed improved metabolic and perfusion status on repeat PET scans. In conclusion, (1) the addition of PET to MRI identified a much greater proportion of SS children with neuroimaging abnormalities, particularly in those who had no history of overt neurologic events. (2) PET lesions are more extensive, often bihemispheric, as compared with MRI abnormalities. (3) PET may be useful in management as a tool to evaluate metabolic improvement after therapeutic interventions, and (4) the correlation of PET abnormalities to subsequent stroke or progressive neurologic dysfunction requires further study.

Synthesis and preliminary evaluation of 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG): a new potential imaging agent for viral infection and gene therapy using PET.

Synthesis and preliminary biological evaluation of 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)-guanine ([18F]FHBG) is reported. 9-(4-Hydroxy-3-hydroxymethylbutyl)-guanine (penciclovir) 4 was converted to 9-[N2, O-bis-(methoxytrityl)-3-(tosylmethybutyl)]guanine 7 by treatment with methoxytrityl chloride followed by tosylation. The tosylate 7 was reacted with either tetrabutylammonium fluoride or KF in the presence of kryptofix 2.2.2. to produce the 4-fluoro-N2-O-bis-(methoxytrityl) derivative 8. Removal of the methoxytrityl groups by acidic hydrolysis produced FHBG 5. Radiolabeled product [18F]FHBG was prepared by fluorination of the tosylate 7 with [18F]KF and kryptofix 2.2.2. The labeled product was isolated by HPLC purification on a reverse-phase C18 column, and eluted at 12 min with 15% acetonitrile in water at a flow rate of 2.25 mL/min. Radiochemical yield was 8.0-22.3% with an average of 12% in 7 runs (corrected for decay). Synthesis time was 90 to 100 min including HPLC purification with radiochemical purity >99%, and average specific activity of 320 mCi/micromol. In vitro studies of the compound in HT-29 colon cancer cells revealed 18.2-fold higher uptake into transduced cells compared to control in 3 h. The agent may be useful for imaging viral infection or transfected cells in gene therapy.

Second-look laparotomy for stage III epithelial ovarian cancer: rationale and current issues.

During the past two decades, the initial treatment of an advanced ovarian malignancy has been generally uniform: it begins with an exploratory laparotomy surgically to remove as much tumor as possible (1) and to stage the cancer (2). For the 70% of patients classified as stages III and IV, surgery is then followed by combination chemotherapy. Although opinions differ as to the optimal regimen, the standard involves a platinum-based program (3), recently also including paclitaxel (4). A second-look laparotomy is often performed in all patients who achieve a clinical complete remission, that is the inability to detect disease by physical examination and non-invasive laboratory tests. This surgical procedure is able to detect clinical disease not apparent on computerized axial tomography (CT scan), ultrasound, magnetic resonance imaging (MRI), serum CA-125 levels or physical examination (5-7). Major questions, however, have arisen around the need for such a procedure, and whether one can justify it in terms of an improved outcome or merely as an assessment of prognosis (8-14). We shall review: (i) the technique; (ii) the rationale; (iii) the results that have been reported from its routine application; and (iv) controversial issues, particularly as they relate to the evolution of therapeutic strategies.

PET and [18F]-FDG in oncology: a clinical update.

Positron emission tomography (PET) has become a very useful adjunct to anatomic imaging techniques, adding unique information to the characterization of disease. The whole-body PET FDG technique developed over the last few years has surpassed most expectations with respect to its utility in clinical oncology. The large spectrum of neoplasms that now can be studied with this approach makes it an essential clinical imaging tool in diagnosis and management for many patients with cancer. The metabolic information provided by this technique is complementary to results from standard clinical and morphological examinations. It may be anticipated that through application of the multi-modality imaging approach, significant advances in medical care will come.