Masayuki Inubushi

Positron Emission Tomography Imaging of Cardiac Reporter Gene Expression in Living Rats

Background—Imaging reporter gene expression is useful for noninvasive monitoring of gene therapy. In this study, we imaged cardiac reporter gene expression in living rats using micro positron emission tomography (microPET). Methods and Results—Rats (n=10) underwent intramyocardial injection with 1×109 pfu of adenovirus carrying cytomegalovirus promoter-driving herpes simplex virus type 1 mutant thymidine kinase (Ad-CMV-HSV1-sr39tk) as PET reporter gene. Control rats (n=4) received 1×109 pfu of adenovirus carrying cytomegalovirus promoter-driving firefly luciferase (Ad-CMV-Fluc). On days 2 to 4, microPET images were obtained after a tail vein injection of nitrogen-13 ammonia ([13N]-NH3) as myocardial perfusion tracer, followed by 9-(4-[18F]-fluoro-3 hydroxymethylbutyl) guanine ([18F]-FHBG) to assess HSV1-sr39tk expression. After imaging, hearts were removed for ex vivo [18F] gamma counting and thymidine kinase enzyme assay. Results show homogenous myocardial distribution of [13N]-NH3 on all microPET images. Rats injected with Ad-CMV-HSV1-sr39tk have significant [18F]-FHBG uptake in the anterolateral wall compared with background signal in controls. Gamma counting shows 20.0±4.4-fold increase of radioactivity, whereas enzyme assay shows 22.1±6.1-fold increase of thymidine kinase activity in Ad-CMV-HSV1-sr39tk injected rats (P <0.05). Conclusions—Successful imaging of cardiac HSV1-sr39tk expression was performed in living rats with microPET. The presence of [18F]-FHBG uptake is confirmed by gamma counting and the presence of HSV1-sr39TK protein by thymidine kinase enzyme assay. Cardiac reporter gene imaging by PET may eventually be applied toward human gene therapy studies.

Optical Imaging of Cardiac Reporter Gene Expression in Living Rats

Background—Studies of cardiac gene transfer rely on postmortem analysis using histologic staining or enzyme assays. Noninvasive imaging of the temporal and spatial characteristics of cardiac gene expression in the same subject offers significant advantages. Methods and Results—Rats underwent direct myocardial injection via left thoracotomy with adenovirus-expressing firefly luciferase (Ad-CMV-Fluc; n=30). The reporter substrate d-luciferin was injected intraperitoneally. Serial images were acquired by use of a cooled charged couple detector (CCD) camera. Results are expressed as relative light unit per minute (RLU/min). Rats transduced with 1×109 plaque-forming units show decremental cardiac luciferase activity over time: 152 070±21 170 (day 2), 195 806±62 630 (day 5), 7250±2941 (day 8), and 2040±971 RLU/min (day 14). To assess the detection sensitivity, serially diluted titers of Ad-CMV-Fluc were injected: 1×109 (195 393±14 896), 1×108 (33 777±18 179), 1×107 (417±91), 1×106 (185±64), 1×105 (53±1), and control (54±1) (P <0.05 for 1×109, 1×108, and 1×107 plaque-forming units versus control adenovirus-expressing mutant thymidine kinase [Ad-CMV-HSV1-sr39tk]; n=3). Finally, rats were euthanized, and in vitro luciferase activity correlated with in vivo CCD signals (r2=0.92). Conclusions—This study demonstrates for the first time the feasibility of imaging the location, magnitude, and time course of cardiac reporter gene expression in living rats. Cardiac gene therapy studies could be aided with wider application of this approach.

Assessment of regional and global left ventricular function by reinjection Tl-201 and rest Tc-99m sestamibi ECG-gated SPECT: Comparison with three-dimensional magnetic resonance imaging

OBJECTIVES The purpose of this study was to test the ability of reinjection thallium-201 and rest technetium-99m sestamibi ECG (electrocardiographic)-gated SPECT (i.e., reinjection-g-SPECT [single-photon emission computed tomography] and MIBI-g-SPECT) to determine regional and global functional parameters. BACKGROUND The ECG-gated perfusion SPECT was reported to provide accurate left ventricular ejection fraction (LVEF) using an automated algorithm. We hypothesized that other various functional data may be obtained using reinjection-g-SPECT and MIBI-g-SPECT. METHODS Reinjection-g-SPECT, MIBI-g-SPECT, and three-dimensional magnetic resonance imaging (3DMRI) were conducted in 20 patients with coronary artery disease. Regional wall motion (RWM) and wall thickening (RWT) were analyzed using semiquantitative visual scoring by each g-SPECT and 3DMRI. The left ventricular end-systolic and end-diastolic volumes (EDV, ESV) and LVEF estimated by reinjection- and MIBI-g-SPECT were compared with the results of 3DMRI. RESULTS A high degree of agreement in RWM and RWT assessment was observed between each g-SPECT and 3DMRI (kappa >.70, p < .001). The LVEF values by reinjection- and MIBI-g-SPECT correlated and agreed well with those by 3DMRI (reinjection: r = .92, SEE = 5.9%, SD of differences = 5.7%; sestamibi: r = .94, SEE = 4.4%, SD of differences = 5.1%). The same also pertained to EDV (reinjection: r = .85, SEE = 18.7 ml, SD of differences = 18.4 ml; sestamibi: r = .92, SEE = 13.1 ml, SD of differences = 13.0 ml) and ESV (reinjection: r = .94, SEE = 10.3 ml, SD of differences = 10.3 ml; sestamibi: r = .97, SEE = 6.7 ml [p < .05 vs. reinjection by F test], SD of differences = 6.6 ml [p < .05 vs. reinjection by F test]). CONCLUSIONS Reinjection- and MIBI-g-SPECT provide clinically satisfactory various functional data. These functional data in combination with the perfusion information will improve diagnostic and prognostic accuracy without an increase in cost or the radiation dose to the patients.

Positron-Emission Tomography Reporter Gene Expression Imaging in Rat Myocardium

Background—This study examines the quantitative accuracy, detection sensitivity, and time course of imaging the expression of a mutant herpes simplex type-1 virus thymidine kinase (HSV1-sr39tk) PET reporter gene in rat myocardium by using the PET reporter probe 9-(4-[18F]-Fluoro-3-Hydroxymethylbutyl)-Guanine ([18F]-FHBG) and a small-animal PET (microPET). Methods and Results—In 40 rats, adenovirus expressing HSV1-sr39tk driven by a cytomegalovirus promoter (Ad-CMV-HSV1-sr39tk, 1×106 to 1×109 pfu) was injected through a thoracotomy directly into the left ventricular myocardium. After 3 days, myocardial perfusion was imaged with [13N]-ammonia for delineating the left ventricular myocardium, followed by imaging the expression of the reporter gene with intravenous [18F]-FHBG. The total myocardial [18F]-FHBG accumulation was quantified in percent of injected dose (%ID). Immunohistochemistry and autoradiography demonstrated HSV1-sr39tk enzyme (HSV1-sr39TK) and accumulation of [18F]-FHBG in the inoculated myocardium in 3 rats each. In 24 rats with various viral titers, the %ID was correlated with ex vivo well counting (r2=0.981, P <0.0001) and myocardial HSV1-sr39TK activity by tissue enzyme activity assay (r2=0.790, P <0.0001). Myocardial [18F]-FHBG accumulation was identified at viral titers down to 1×107 pfu. In 6 rats serially imaged up to day 17, myocardial [18F]-FHBG accumulation on microPET peaked on days 3 to 5 and was no longer identified on days 10 to 17. Conclusions—HSV1-sr39tk reporter gene expression can be monitored with [18F]-FHBG and microPET in rat myocardium quantitatively and serially with high detection sensitivity. Cardiac PET reporter gene imaging offers the potential of monitoring the expression of therapeutic genes in cardiac gene therapy.

Assessment of cervical lymph node metastases using FDG-PET in patients with head and neck cancer

ObjectiveTo evaluate the diagnostic accuracy of fluorodeoxyglucose positron emission tomography (FDG-PET) relative to computed tomography (CT) for detecting metastatic cervical lymph nodes in patients with squamous cell carcinoma of the head and neck (HNSCC), and to ascertain the factors that affect this accuracy.MethodsA total of 1076 lymph nodes obtained from 35 neck dissections in 26 HNSCC patients who preoperatively underwent both FDG-PET and CT were retrospectively analyzed. For pathological metastatic lymph nodes, the lymph node size (short-axis diameter), the ratio of intranodal tumor deposits, and the size of intranodal tumor deposits (maximum diameter of metastatic foci in each lymph node) were histologically recorded.ResultsForty-six lymph nodes from 23 neck sides were pathologically diagnosed metastases. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of FDG-PET evaluated individually per neck side were 74%, 92%, 80%, 94%, and 65%, respectively, whereas those of CT were 78%, 58%, 71%, 78%, and 58%, respectively. FDG-PET detected 100% of metastatic lymph nodes ≥10 mm, intranodal tumor deposits ≥9 mm, and intranodal tumor deposits with a ratio >75%, whereas no nodes or tumor deposits smaller than 5 mm were detected. The spatial resolution limitations of FDG-PET were responsible for 16 of 20 (80%) false-negative PET results in lymph nodes.ConclusionsFDG-PET is a useful tool for preoperative evaluation of the neck because it accurately detects metastatic lymph nodes ≥10 mm and has fewer false-positive cases than CT. The high specificity of FDG-PET for lymph node metastases may play an important role in avoiding unnecessary neck dissection.

Noninvasive Measurement of Myocardial Activity Concentrations and Perfusion Defect Sizes in Rats With a New Small-Animal Positron Emission Tomograph

Background—We explored the feasibility of measuring regional tracer activity concentrations and flow defects in myocardium of rats with a high spatial resolution small-animal PET system (microPET). Methods and Results—Myocardial images were obtained after intravenous 18F-fluorodeoxyglucose (18FDG) in 11 normal rats (group 1) and assembled into polar maps. Regional 18F activity concentrations were measured in 9 regions of interest and compared with tissue activity concentrations measured by well counting. In another 9 rats (group 2), myocardial perfusion images were acquired with 13N-ammonia at baseline and during coronary occlusion. On the polar maps recorded during coronary occlusion, the size of perfusion defects was measured as the myocardium with <50% of maximum activity and expressed as percent total myocardium and was correlated with the area at risk defined by postmortem staining. The diagnostic quality of 18FDG and 13N-ammonia microPET images was good to excellent; the images were easily assembled into polar maps. In group 1, regional 18F concentrations by microPET and postmortem were correlated linearly (r =0.99;P <0.01 for average and r =0.97;P <0.01 for regional concentrations). In group 2, perfusion defect sizes by microPET and postmortem were correlated linearly (P <0.01;r =0.93). Conclusions—The findings indicate the feasibility of noninvasive studies of the myocardium in rats with a dedicated small-animal PET-imaging device.

Comparison of 99mTc-annexin A5 with 18F-FDG for the detection of atherosclerosis in ApoE−/− mice

Purpose99mTc-annexin A5, a marker of ongoing apoptosis, and 18F-FDG, a marker of the increased metabolism of inflammatory cells, are supposed to be useful in the detection of metabolically active atheroma. This study reports a comparison of the intralesional distribution of these tracers in relation to lesion development in ApoE−/− mice.MethodsMale ApoE−/− mice (n = 12–14/group) were maintained on a Western-type diet after the age of 5 weeks. At 25 weeks, 99mTc-annexin A5 or 18F-FDG was injected and the aortas were harvested for autoradiography (ARG) and Oil Red O staining. Regional radioactivity accumulation was compared in relation to the Oil Red O staining score (ranging from 0 to 3, a semiquantitative parameter for evaluating lesion development).ResultsBoth 99mTc-annexin A5 and 18F-FDG showed preferential uptake into atherosclerotic lesions, with higher uptake levels for 18F-FDG (mean, 56.07 %ID×kg/m2) than for 99mTc-annexin A5 (mean, 10.38 %ID×kg/m2). The regional uptake levels of each tracer correlated with the Oil Red O staining score (r = 0.65, p < 0.05 for 99mTc-annexin A5; r = 0.56, p < 0.05 for 18F-FDG). The uptake ratios of advanced lesions (score >0.5) to early lesions (score <0.5) were significantly higher for 99mTc-annexin A5 than for 18F-FDG (f = 4.73, p = 0.03).ConclusionBoth 99mTc-annexin A5 and 18F-FDG accumulate in atherosclerotic lesions and correlate with the severity of each lesion. The higher absolute uptake levels of 18F-FDG may be advantageous for lesion detection, whereas the preferential uptake of 99mTc-annexin A5 in advanced lesions may be a useful indicator of late-stage lesions or vulnerable plaque transformation.

Decreased Myocardial β-Adrenergic Receptor Density in Relation to Increased Sympathetic Tone in Patients with Nonischemic Cardiomyopathy

Cardiac sympathetic function plays an important role in the regulation of left ventricular (LV) function and the pathophysiology of LV dysfunction. 11C-CGP-12177 (11C-CGP) has been used to assess myocardial β-adrenergic receptor (β-AR) density in vivo using PET. The aim of this study is to measure myocardial β-AR density in patients with nonischemic cardiomyopathy and to compare the measurements with various standard parameters of heart failure (HF), particularly with presynaptic function assessed by 123I- metaiodobenzylguanidine (123I-MIBG) imaging. Methods: 11C-CGP PET was performed on 16 patients with nonischemic cardiomyopathy and 8 age-matched healthy volunteers using a double injection method. A 11C-CGP dynamic scan for 75 min was performed after the injection of 11C-CGP with a high specific activity. After 30 min, 11C-CGP with a low specific activity was injected. The β-AR density of the whole LV was calculated on the basis of the graphical analysis method. Additionally, β-AR density was compared with LV ejection fraction (LVEF), sympathetic presynaptic function assessed using 123I-MIBG kinetics, and neurohormonal parameters. Results: The β-AR density of patients was significantly lower than that of healthy volunteers (3.80 ± 0.96 vs. 7.70 ± 1.92 pmol/mL; P < 0.0001). In the patients, β-AR density correlated significantly with LVEF (r = 0.62, P < 0.05). Furthermore, β-AR density correlated significantly with the 123I-MIBG washout rate (r = −0.68, P < 0.01) and delayed heart-to-mediastinum ratio (H/M ratio) (r = 0.61, P < 0.05). On the other hand, the correlation between β-AR density and early H/M ratio was not significant (r = 0.40, P = 0.13). The β-AR density of patients with severe HF (New York Heart Association functional [NYHA] class III) was significantly lower than that of those with NYHA functional class I or class II HF (3.24 ± 0.96 vs. 4.24 ± 0.73 pmol/mL; P < 0.05). Conclusion: A reduction in β-AR density measured by 11C-CGP PET was observed in patients with nonischemic cardiomyopathy. This downregulation may be due to the increased presynaptic sympathetic tone as assessed by 123I-MIBG imaging.

Prospective Evaluation of Whole-Body Cancer Screening With Multiple Modalities Including [18F]Fluorodeoxyglucose Positron Emission Tomography in a Healthy Population: A Preliminary Report

PURPOSE To prospectively evaluate the utility of whole-body cancer screening with multiple modalities including [(18)F]fluorodeoxyglucose positron emission tomography (FDG-PET) in a healthy population. This report summarizes the results of the first three annual screenings. PARTICIPANTS AND METHODS A total of 1,197 healthy volunteers > or = 35 years old were enrolled between August 2003 and July 2004 and offered annual cancer screening for 5 years with subsequent long-term follow-up. Screening modalities included were whole-body FDG-PET, chest and abdominal computed tomography (CT), brain and pelvic magnetic resonance imaging, several tumor markers, and fecal occult blood testing. RESULTS As of the end of 2006, 22 primary cancers were pathologically confirmed. Nineteen of 22 were detected by the screening; 18 in the initial, one in the second, and none in the third. Three were diagnosed after development of symptoms. Of the 18 detected in the initial screening (six thyroid, four lung, three prostate, three breast, one endometrial, and one thymic), 12 were at stage I and 11 were PET positive. PET-negative cancers were detected by CT or the prostate-specific antigen (PSA) test. Sensitivity and specificity were 50.0% (11 of 22) and 93.2% (1,095 of 1,175), respectively, for FDG-PET alone and 81.8% (18 of 22) and 82.0% (963 of 1,175), respectively, for the combination of imaging modalities and PSA. CONCLUSION While FDG-PET alone is insufficient, whole-body cancer screening with selected modalities including FDG-PET has initial performance supporting possible utility by detecting a wide variety of early-stage cancers with reasonable sensitivity. However, the detection of many indolent cancers and false positives necessitate continuing study for appropriate evaluation.

Myocardial flow reserve is influenced by both coronary artery stenosis severity and coronary risk factors in patients with suspected coronary artery disease

PurposeMyocardial flow reserve (MFR) measurement has an important role in assessing the functional severity of coronary artery stenosis. However, a discrepancy between the anatomical severity of coronary artery stenosis and MFR is often observed. Such a discrepancy may be explained by coronary risk factors. In this study, we aimed to investigate the influence of coronary artery stenosis severity and risk factors on MFR.MethodsSeventy-four patients suspected to have coronary artery disease and seven age-matched healthy volunteers were enrolled. Myocardial blood flow (MBF) and MFR were measured using 15O-labelled water PET. Regional MFR was calculated in regions with significant coronary artery stenosis (stenotic regions) and in regions without significant stenosis (remote regions). The contributions of coronary artery stenosis severity and coronary risk factors were assessed using univariate and multivariate analyses.ResultsIn stenotic regions, MFR correlated inversely with coronary artery stenosis severity (r=−0.50, p<0.01). Univariate analysis did not show any significant difference in MFR between the patients with and the patients without each risk factor. In remote regions, however, MFR was significantly decreased in the diabetes and smoking groups (each p<0.05). By multivariate analysis, diabetes and smoking were independent predictors of MFR (each p<0.05). In the group with more than one risk factor, MFR was significantly lower (2.78±0.79) than in the other group (3.40±1.22, p<0.05).ConclusionMFR is influenced not only by coronary stenosis severity but also by coronary risk factors. In particular, the influence of risk factors should be considered in regions without severe coronary stenosis.