Applications of PET imaging with the proliferation marker [18F]-FLT.

[18F]-3'-fluoro-3'-deoxythymidine (FLT) is a nucleoside-analog imaging agent for quantifying cellular proliferation that was first reported in 1998. It accumulates during the S-phase of the cell cycle through the action of cytosolic thymidine kinase, TK1. Since TK1 is primarily expressed in dividing cells, FLT uptake is essentially limited to dividing cells. Thus FLT is an effective measure of cell proliferation. FLT uptake has been shown to correlate with the more classic proliferation marker, the monoclonal antibody to Ki-67. Increased cellular proliferation is known to correlate with worse outcome in many cancers. However, the Ki-67 binding assay is performed on a sampled preparation, ex vivo, whereas FLT can be quantitatively measured in vivo using positron emission tomography (PET). FLT is an effective and quantitative marker of cell proliferation, and therefore a useful prognostic predictor in the setting of neoplastic disease. This review summarizes clinical studies from 2011 forward that used FLT-PET to assess tumor response to therapy. The paper focuses on our recommendations for a standardized clinical trial protocol and components of a report so multi center studies can be effectively conducted, and different studies can be compared. For example, since FLT is glucuronidated by the liver, and the metabolite is not transported into the cell, the plasma fraction of FLT can be significantly changed by treatment with particular drugs that deplete this enzyme, including some chemotherapy agents and pain medications. Therefore, the plasma level of metabolites should be measured to assure FLT uptake kinetics can be accurately calculated. This is important because the flux constant (KFLT) is a more accurate measure of proliferation and, by inference, a better discriminator of tumor recurrence than standardized uptake value (SUVFLT). This will allow FLT imaging to be a specific and clinically relevant prognostic predictor in the treatment of neoplastic disease.

Kinetic quantitation of cerebral PET-FDG studies without concurrent blood sampling: statistical recovery of the arterial input function.

Kinetic quantitation of dynamic positron emission tomography (PET) studies via compartmental modeling usually requires the time-course of the radio-tracer concentration in the arterial blood as an arterial input function (AIF). For human and animal imaging applications, significant practical difficulties are associated with direct arterial sampling and as a result there is substantial interest in alternative methods that require no blood sampling at the time of the study. A fixed population template input function derived from prior experience with directly sampled arterial curves is one possibility. Image-based extraction, including requisite adjustment for spillover and recovery, is another approach. The present work considers a hybrid statistical approach based on a penalty formulation in which the information derived from a priori studies is combined in a Bayesian manner with information contained in the sampled image data in order to obtain an input function estimate. The absolute scaling of the input is achieved by an empirical calibration equation involving the injected dose together with the subject's weight, height and gender. The technique is illustrated in the context of (18)F -Fluorodeoxyglucose (FDG) PET studies in humans. A collection of 79 arterially sampled FDG blood curves are used as a basis for a priori characterization of input function variability, including scaling characteristics. Data from a series of 12 dynamic cerebral FDG PET studies in normal subjects are used to evaluate the performance of the penalty-based AIF estimation technique. The focus of evaluations is on quantitation of FDG kinetics over a set of 10 regional brain structures. As well as the new method, a fixed population template AIF and a direct AIF estimate based on segmentation are also considered. Kinetics analyses resulting from these three AIFs are compared with those resulting from radially sampled AIFs. The proposed penalty-based AIF extraction method is found to achieve significant improvements over the fixed template and the segmentation methods. As well as achieving acceptable kinetic parameter accuracy, the quality of fit of the region of interest (ROI) time-course data based on the extracted AIF, matches results based on arterially sampled AIFs. In comparison, significant deviation in the estimation of FDG flux and degradation in ROI data fit are found with the template and segmentation methods. The proposed AIF extraction method is recommended for practical use.

Hypoxia imaging-directed radiation treatment planning.

Increasing evidence supports the role of the tumor microenvironment in modulating cancer behavior. Tissue hypoxia, an important and common condition affecting the tumor microenvironment, is well established as a resistance factor in radiotherapy. Increasing evidence points to the ability of hypoxia to induce the expression of gene products, which confer aggressive tumor behavior and promote broad resistance to therapy. These factors suggest that determining the presence or absence of tumor hypoxia is important in planning cancer therapy. Recent advances in PET hypoxia imaging, conformal radiotherapy, and imaging-directed radiotherapy treatment planning now make it possible to perform hypoxia-directed radiotherapy. We review the biological aspects of tumor hypoxia and PET imaging approaches for measuring tumor hypoxia, along with methods for conformal radiotherapy and image-guided treatment, all of which provide the underpinnings for hypoxia-directed therapy. As a case example, we review emerging data on PET imaging of hypoxia to direct radiotherapy.

[(18)F]FMISO and [(18)F]FDG PET imaging in soft tissue sarcomas: correlation of hypoxia, metabolism and VEGF expression.

Hypoxia imparts resistance to radiotherapy and chemotherapy and also promotes a variety of changes in tumor biology through inducible promoters. The purpose of this study was to evaluate the use of positron emission tomography (PET) imaging with fluorine-18 fluoromisonidazole (FMISO) in soft tissue sarcomas (STS) as a measure of hypoxia and to compare the results with those obtained using [(18)F]fluorodeoxyglucose (FDG) and other known biologic correlates. FDG evaluates energy metabolism in tumors while FMISO uptake is proportional to tissue hypoxia. FMISO uptake was compared with FDG uptake. Vascular endothelial growth factor (VEGF) expression was also compared with FMISO uptake. Nineteen patients with STS underwent PET scanning with quantitative determination of FMISO and FDG uptake prior to therapy (neo-adjuvant chemotherapy or surgery alone). Ten patients receiving neo-adjuvant chemotherapy were also imaged after chemotherapy but prior to surgical resection. Standardized uptake value (SUV) was used to describe FDG uptake; regional tissue to blood ratio (>or=1.2 was considered significant) was used for FMISO uptake. Significant hypoxia was found in 76% of tumors imaged prior to therapy. No correlation was identified between pretherapy hypoxic volume (HV) and tumor grade ( r=0.15) or tumor volume ( r=0.03). The correlation of HV with VEGF expression was 0.39. Individual tumors showed marked heterogeneity in regional VEGF expression. The mean pixel-by-pixel correlation between FMISO and FDG uptake was 0.49 (range 0.09-0.79) pretreatment and 0.32 (range -0.46-0.72) after treatment. Most tumors showed evidence of reduced uptake of both FMISO and FDG following chemotherapy. FMISO PET demonstrates areas of significant and heterogeneous hypoxia in soft tissue sarcomas. The significant discrepancy between FDG and FMISO uptake seen in this study indicates that regional hypoxia and glucose metabolism do not always correlate. Similarly, we did not find any relationship between the hypoxic volume and the tumor volume or VEGF expression. Identification of hypoxia and development of a more complete biologic profile of STS will serve to guide more rational, individualized cancer treatment approaches.

Multinuclear NMR studies of an actively dividing artificial tumor.

Growth of the A549 cell line in a perfusion system suitable for use in a magnetic resonance study has been characterized and shown to be stable physiologically and hence appropriate for serial observations. Several methods of monitoring cell growth were compared to assess the behavior of the cells in this system. Comparison between NMR metabolite data and cell growth via cell counting showed that 31P NMR signals accurately reported cell doubling time. In contrast to most NMR cell culture systems, viable cells can be recovered from the perfusion system after the NMR measurements for further biochemical studies. These data further suggest that this system will be useful for studying the physiology and biochemistry of exponentially growing cells for at least two days in NMR tube culture.

Molecular imaging of regional brain tumor biology.

Energy metabolism measurements in gliomas in vivo are now performed widely with positron emission tomography (PET). This capability has developed from a large number of basic and clinical science investigations that have cross fertilized one another. This article presents several areas that exemplify questions that have been explored over the last two decades. While the application of PET with [(18)F]-2-fluoro-2-deoxyglucose (FDG-PET) has proven useful for grading and prognosis assessments, this approach is less clinically suitable for assessing response to therapy, even though results to date raise very intriguing biological questions. Integration of metabolic imaging results into glioma therapy protocols is a recent and only preliminarily tapped method that may prove useful in additional trials that target DNA or membrane biosynthesis, or resistance mechanisms such as hypoxia. There are exciting future directions for molecular imaging that will undoubtedly be fruitful to explore, especially apoptosis, angiogenesis and expression of mutations of genes, e.g., epidermal growth factor receptor, that promote or suppress cellular malignant behavior.

The physical chemistry of ligand-receptor binding identifies some limitations to the analysis of receptor images.

The biophysical chemistry of ligand-receptor interactions imposes some restrictions on the characteristics of a radioligand if it is to be a useful tracer for accurately measuring the in vivo concentration of a specific cellular membrane receptor. This review discusses thermodynamic and kinetic rate constant considerations in selecting a ligand for radiolabeling and imaging. When radioligands of only modest specific activity are injected, one is able to use kinetic analysis to calculate the rate constant for the bimolecular binding reaction as well as the receptor concentration. Images of regional receptor density can be constructed from analysis of emission imaging data when the binding occurs at a rate that is slower than the collision frequency. A tracer that reacts with each collision cannot distinguish receptor density from blood flow. The theory of diffusion-limited reactions is reviewed and individual ligand-receptor examples are presented to demonstrate conditions where, even for very fast forward reactions, the binding of radioligand to receptor is controlled by local biochemistry rather than by the purely physical process of diffusion.

Evaluation of alternative approaches for imaging cellular growth.

Uncontrolled growth is a characteristic of malignant tumors. Histochemical techniques to measure tumor growth rate in tissue specimens have proved useful, but are limited because of sampling and the difficulty of following response to therapy. PET imaging offers the opportunity to measure tumor growth non-invasively and repeatedly as an early assessment of response to therapy. Measuring cellular growth instead of energy metabolism offers significant advantages in evaluating therapy. The rationale is that a cell's biosynthetic machinery, rather than its fueling process, is more susceptible to cancer therapy. Cytostatic agents may not reduce the quantity of viable tumor; so imaging a change in cellular proliferation may be the only effective way to assess the response to therapy. Radiopharmaceuticals to image growth include labeled amino acids, lipid precursors, and nucleosides. The biochemical characteristic that most uniquely distinguishes successfully treated cancer cells is that they no longer synthesize DNA and no longer divide. Thus imaging with labeled thymidine, which is incorporated into DNA but not into RNA, provides definitive evidence of a cell that is proliferating and, therefore, whether it has responded to treatment.

Imaging cellular proliferation as a measure of response to therapy.

Cell proliferation imaging is based on extensive laboratory investigations of labeled thymidine being selectively incorporated into DNA. [11C]-Thymidine labeled in the ring-2 or the methyl position is the natural extension of earlier work using tritiated thymidine. Proliferation imaging using [11C]-thymidine requires correction for labeled metabolites; however, quantitative approaches can provide reliable estimates of cellular proliferation by measuring thymidine flux from the blood into DNA in tumors. 18F-labeled thymidine analogs that are resistant to catabolism in vivo, [18F]-FLT and [18F]-FMAU, may simplify quantitative analysis and may be more suitable for clinical studies but will require careful validation to determine how their uptake is quantitatively related to cell growth. Clinical studies using [11C]-thymidine have demonstrated the power of cellular proliferation imaging to characterize tumors and monitor response early in the course of therapy. Patient imaging using the PET thymidine analogs is at an earlier stage but appears promising as a clinically feasible approach to cellular proliferation imaging.

[18F]fluoroestradiol radiation dosimetry in human PET studies.

UNLABELLED: [18F]16alpha-fluoroestradiol (FES) is a PET imaging agent useful for the study of estrogen receptors in breast cancer. We estimated the radiation dosimetry for this tracer using data obtained in patient studies. METHODS: Time-dependent tissue concentrations of radioactivity were determined from blood samples and PET images in 49 patients (52 studies) after intravenous injection of FES. Radiation absorbed doses were calculated using the procedures of the MIRD committee, taking into account the variation in dose based on the distribution of activities observed in the individual patients. Effective dose equivalent was calculated using International Commission on Radiological Protection Publication 60 weights for the standard woman. RESULTS: The effective dose equivalent was 0.022 mSv/MBq (80 mrem/mCi). The organ that received the highest dose was the liver (0.13 mGy/MBq [470 mrad/mCi]), followed by the gallbladder (0.10 mGy/MBq [380 mrad/mCi]) and the urinary bladder (0.05 mGy/MBq [190 mrad/mCi]). CONCLUSION: The organ doses are comparable to those associated with other commonly performed nuclear medicine tests. FES is a useful estrogen receptor-imaging agent, and the potential radiation risks associated with this study are well within accepted limits.

Kinetic characterization of hexokinase isoenzymes from glioma cells: implications for FDG imaging of human brain tumors.

Quantitative imaging of glucose metabolism of human brain tumors with PET utilizes 2-[(18)F]-fluorodeoxy-D-glucose (FDG) and a conversion factor called the lumped constant (LC), which relates the metabolic rate of FDG to glucose. Since tumors have greater uptake of FDG than would be predicted by the metabolism of native glucose, the characteristic of tumors that governs the uptake of FDG must be part of the LC. The LC is chiefly determined by the phosphorylation ratio (PR), which is comprised of the kinetic parameters (Km and Vmax) of hexokinase (HK) for glucose as well as for FDG (LC proportional to (Km(glc) x Vmax(FDG))/(Km(FDG) x Vmax(glc)). The value of the LC has been estimated from imaging studies, but not validated in vitro from HK kinetic parameters. In this study we measured the kinetic constants of bovine and 36B-10 rat glioma HK I (predominant in normal brain) and 36B-10 glioma HK II (increased in brain tumors) for the hexose substrates glucose, 2-deoxy-D-glucose (2DG) and FDG. Our principal results show that the KmGlc < KmFDG << Km2DG and that PR2DG < PRFDG. The FDG LC calculated from our kinetic parameters for normal brain, possessing predominantly HK I, would be higher than the normal brain LC predicted from animal studies using 2DG or human PET studies using FDG or 2DG. These results also suggest that a shift from HK I to HK II, which has been observed to increase in brain tumors, would have little effect on the value of the tumor LC.

Prothrombin G20210A mutation in a child with spinal cord infarction.

Prothrombin G20210A is a newly described common mutation that is associated with an increased risk of arterial and venous thrombosis. We describe a healthy child heterozygous for this prothrombin mutation who had a spinal cord infarct with no other prothrombotic risk factors.

Kinetic analysis of 2-[11C]thymidine PET imaging studies: validation studies.

UNLABELLED: 2-[11C]thymidine has been tested as a PET tracer of cellular proliferation. We have previously described a model of thymidine and labeled metabolite kinetics for use in quantifying the flux of thymidine into DNA as a measure of tumor proliferation. We describe here the results of studies to validate some of the model's assumptions and to test the model's ability to predict the time course of tracer incorporation into DNA in tumors. METHODS: Three sets of studies were conducted: (a) The uptake of tracers in proliferative tissues of normal mice was measured early after injection to assess the relative delivery of thymidine and metabolites of thymidine catabolism (thymine and CO2) and calculate relative blood-tissue transfer rates (relative K1s). (b) By using sequential injections of [11C]thymidine and [11C]thymine in normal human volunteers, the kinetics of the first labeled metabolite were measured to determine whether it was trapped in proliferating tissue such as the bone marrow. (c) In a multitumor rat model, 2-[14C]thymidine injection, tumor sampling and quantitative DNA extraction were performed to measure the time course of label uptake into DNA for comparison with model predictions. RESULTS: Studies in mice showed consistent relative delivery of thymidine and metabolites in somatic tissue but, as expected, showed reduced delivery of thymidine and thymine in the normal brain compared to CO2. Thymine studies in volunteers showed only minimal trapping of label in bone marrow in comparison to thymidine. This quantity of trapping could be explained by a small amount of fixation of labeled CO2 in tissue, a process that is included as part of the model. Uptake experiments in rats showed early incorporation of label into DNA, and the model was able to fit the time course of uptake. CONCLUSION: These initial studies support the assumptions of the compartmental model and demonstrate its ability to quantify thymidine flux into DNA by using 2-[11C]thymidine and PET. Results suggest that further work will be necessary to investigate the effects of tumor heterogeneity and to compare PET measures of tumor proliferation to in vitro measures of proliferation and to clinical tumor behavior in patients undergoing therapy.

2-[C-11]thymidine imaging of malignant brain tumors.

Malignant brain tumors pose diagnostic and therapeutic problems. Despite the advent of new brain imaging modalities, including magnetic resonance imaging (MRI) and [F-18]fluorodeoxyglucose (FDG) positron emission tomography (PET), determination of tumor viability and response to treatment is often difficult. Blood-brain barrier disruption can be caused by tumor or nonspecific reactions to treatment, making MRI interpretation ambiguous. The high metabolic background of the normal brain and its regional variability makes it difficult to identify small or less active tumors by FDG imaging of cellular energetics. We have investigated 2-[C-11]thymidine (dThd) and PET to image the rate of brain tumor cellular proliferation. A series of 13 patients underwent closely spaced dThd PET, FDG PET, and MRI procedures, and the image results were compared by standardized visual analysis. The resulting dThd scans were qualitatively different from the other two scans in approximately 50% of the cases, which suggests that dThd provided information distinct from FDG PET and MRI. In two cases, recurrent tumor was more apparent on the dThd study than on FDG; in two other patients, tumor dThd uptake was less than FDG uptake, and these patients had slower tumor progression than the three patients with both high dThd and FDG uptake. To better characterize tumor proliferation, kinetic modeling was applied to dynamic dThd PET uptake data and metabolite-analyzed blood data in a subset of patients. Kinetic analysis was able to remove the confounding influence of [C-11]CO2, the principal labeled metabolite of 2-[C-11]dThd, and to estimate the flux of dThd incorporation into DNA. Sequential, same-day [C-11]CO2 and [C-11]dThd imaging demonstrated the ability of kinetic analysis to model both dThd and CO2 simultaneously. Images of dThd flux obtained using the model along with the mixture analysis method for pixel-by-pixel parametric imaging significantly enhanced the contrast of tumor compared with normal brain. Comparison of model estimates of dThd transport versus dThd flux was able to discern increased dThd uptake simply on the basis of blood-brain barrier disruption retention on the basis of increased cellular proliferation. This preliminary study demonstrates the potential for imaging brain tumor cellular proliferation to provide unique information for guiding patient treatment.

Carbon-11-thymidine and FDG to measure therapy response.

UNLABELLED: This study was performed to determine if PET imaging with 11C-thymidine could measure tumor response to chemotherapy early after the initiation of treatment. Imaging of deoxyriboneucleic acid biosynthesis, quantitated with 11C-thymidine, was compared with measurements of tumor energetics, obtained by imaging with 18F-fluorodeoxyglucose (FDG). METHODS: We imaged four patients with small cell lung cancer and two with high-grade sarcoma both before and approximately 1 wk after the start of chemotherapy. Thymidine and FDG studies were done on the same day. Tumor uptake was quantified by standardized uptake values (SUVs) for both tracers by the metabolic rate of FDG and thymidine flux constant (K(TdR)) using regions of interest placed on the most active part of the tumor. RESULTS: In the four patients with clinical response to treatment, both thymidine and FDG uptake markedly declined 1 wk after therapy. Thymidine measurements of SUV and K(TdR) declined by 64% +/- 15% and 84% +/- 33%, respectively. FDG SUV and the metabolic rate of FDG declined by 51% +/- 9% and 63% +/- 23%, respectively. In the patient with metastatic small cell lung cancer who had disease progression, the thymidine SUV decreased by only 8% (FDG not done). In a patient with abdominal sarcoma and progressive disease, thymidine SUV was essentially unchanged (declined by 3%), whereas FDG SUV increased by 69%. CONCLUSION: Images show a decline in both cellular energetics and proliferative rate after successful chemotherapy. In the two patients with progressive disease, thymidine uptake was unchanged 1 wk after therapy. In our limited series, K(TdR) measurements showed a complete shutdown in tumor proliferation in patients in whom FDG showed a more limited decrease in glucose metabolism.

1-[Carbon-11]-glucose radiation dosimetry and distribution in human imaging studies.

UNLABELLED: 1-[Carbon-11]-D-glucose ([11C]-glucose) is an important imaging agent for PET studies that have been used to study the normal brain, encephalitis, epilepsy, manic-depressive disorder, schizophrenia and brain tumors. METHODS: Dosimetry estimates were calculated in subjects undergoing imaging studies to help define the radiation risk of [11C]-glucose PET imaging. Time-dependent radioactivity concentrations in normal tissues in 33 subjects after intravenous injection of [11C]-glucose were obtained by PET imaging. Radiation absorbed doses were calculated according to the procedures of the Medical Internal Radiation Dose (MIRD) committee along with the variation in dose based on the calculated standard deviation of activity distribution seen in the individual patients. RESULTS: Total body exposure was a median of 3.0 microGy/MBq in men and 3.8 microGy/MBq in women. The effective dose equivalent was 3.8 microGy/ MBq in men and 4.8 microGy/MBq in women. The critical organs were those that typically take up the most glucose (brain, heart wall and liver). CONCLUSION: The organ doses reported here are small and comparable to those associated with other commonly performed nuclear medicine tests and indicate that potential radiation risks associated with this radiotracer are within generally accepted limits.

Quantitation of presynaptic cardiac sympathetic function with carbon-11-meta-hydroxyephedrine.

UNLABELLED: The purpose of this study was to validate an axially distributed blood-tissue exchange model for the quantitation of cardiac presynaptic sympathetic nervous system function that could be applied to PET images. The model accounts for heterogeneity in myocardial blood flow, differences in transport rates of 11C-meta-hydroxyephedrine (mHED) across the capillary endothelium and/or neuronal membranes, the virtual volumes of distribution in the interstitial space and neuron and retention of mHED in the neuronal vesicles. METHODS: Multiple indicator outflow dilution and residue detection methods were used to measure the kinetics of radiolabeled intravascular space and interstitial space markers and 11C-mHED in isolated perfused rat heart at baseline and during norepinephrine neuronal transporter blockade with desipramine (DMI). The outflow dilution and residue detection data were modeled with a multiple pathway, four-region, axially distributed model of blood-tissue exchange describing flow in the capillary and exchange between regions using permeability-surface area products with units of clearance of milliliters per minute per gram. Meta-hydroxyephedrine may enter the nerve terminal via membrane transport, where it may be sequestered by first-order unidirectional uptake within vesicles. Release of mHED from the vesicles is modeled via exchange with the interstitial space. RESULTS: After intracoronary injection, mHED transport across the capillary endothelium and in the interstitial space closely followed that of sucrose. Subsequently, mHED was retained in the heart, whereas sucrose washed out rapidly. With DMI the outflow dilution curves more closely resembled those of sucrose. Model parameters reflecting capillary-interstitial kinetics and volumes of distribution were unchanged by DMI, whereas parameters reflecting the neuronal transporter process and volumes of distribution in the nerve terminal and vesicular sequestration were markedly decreased by DMI. Application of the model to a pilot set of canine PET images of mHED suggests the feasibility of this approach. CONCLUSION: Meta-hydroxyephedrine kinetics in the heart can be quantitated using an axially distributed, blood-tissue exchange model that accounts for heterogeneity of flow, reflects changes in neuronal function and is applicable to PET images.

PET radiopharmaceuticals: state-of-the-art and future prospects.

In this review we provide a conceptual overview of radiopharmaceuticals containing positron-emitting isotopes, not a catalog of radiopharmaceuticals or details of syntheses. We hope to provide an integrated framework for understanding the radiopharmaceuticals that are available at this time, describing both their strengths and weaknesses, and to look forward to some of the improvements that might be anticipated in the next decade. The range of biology that can be studied with positron emission tomography (PET) radiopharmaceuticals has greatly expanded, involving more sophisticated tracers and more sophisticated data analysis. PET measurements now encompass increasingly more specific aspects of human biochemistry and physiology as described in this review. As the biology being studied becomes more complex, the demands on the radiopharmaceutical and the methods of data analysis also become more complex. New synthetic chemistry and data analysis must develop in tandem. Radiopharmaceuticals must be designed to ensure that the rate determining step that is of interest is the one reflected in the data from the radiopharmaceutical. The challenge to the PET community of chemists, biologists, and physicians is to apply new knowledge of human biochemistry for developing and validating useful PET radiopharmaceuticals that will, in turn, produce useful nuclear medicine procedures. Initially the synthesis of a compound containing a short-lived radionuclide was a triumph in itself. However as the science advances the radiochemical synthesis becomes just the first step in a long trail that terminates in the compound being used to provide data on biological processes via a well-designed PET experiment. The resulting list of compounds and experiments should be as diverse as all of human biology and pathophysiology.