Inter-reader variability when applying the 2013 Fleischner guidelines for potential solitary subsolid lung nodules.

BACKGROUND: In 2013, the Fleischner Society published recommendations for managing subsolid pulmonary nodules. Inter-reader variability has not yet been defined and has potential implications for the ease and reproducibility of applying the guidelines to clinical practice. PURPOSE: To evaluate inter-reader variability when applying the 2013 Fleischner guidelines for potential solitary subsolid lung nodules. MATERIAL AND METHODS: Potential nodules were identified through a systematic retrospective review of CT studies that reported a ground-glass lesion. Three radiologists decided whether these lesions fit criteria of a subsolid nodule and thus merit application of the Fleischner Society guidelines, determined if a solid component was present, and measured each component in two dimensions. Final management recommendations were based on these intermediate decisions. Inter-reader variability for management was calculated and Fleiss' kappa was used to determine significance. Logistic regression and Fisher's exact test determined whether management was contingent on each intermediate decision. RESULTS: Forty-four nodules with mean diameter of 9.4 mm were evaluated by three radiologists. Final management recommendations were in agreement for 93 out of 132 cases (70.4%, kappa = 0.56). Inter-reader variability in management recommendation was contingent on disagreement over whether a pulmonary lesion fit criteria of a subsolid nodule for 24 cases (P < 0.01), whether there was a solid component for 10 cases (P = 0.01), and whether the measurement met the threshold of 5 mm for five cases (P = 0.12). CONCLUSION: There is moderate inter-reader variability when applying the 2013 Fleischner Society management recommendations. Significant contributors of variability include whether the potential lesions fit subsolid nodule criteria and whether a solid component is present. Measurement variability does not significantly affect the final management decisions.

Use of DNA microarray and small animal positron emission tomography in preclinical drug evaluation of RAF265, a novel B-Raf/VEGFR-2 inhibitor.

Positron emission tomography (PET) imaging has become a useful tool for assessing early biologic response to cancer therapy and may be particularly useful in the development of new cancer therapeutics. RAF265, a novel B-Raf/vascular endothelial growth factor receptor-2 inhibitor, was evaluated in the preclinical setting for its ability to inhibit the uptake of PET tracers in the A375M(B-Raf(V600E)) human melanoma cell line. RAF265 inhibited 2-deoxy-2-[(18)F]fluoro-d-glucose (FDG) accumulation in cell culture at 28 hours in a dose-dependent manner. RAF265 also inhibited FDG accumulation in tumor xenografts after 1 day of drug treatment. This decrease persisted for the remaining 2 weeks of treatment. DNA microarray analysis of treated tumor xenografts revealed significantly decreased expression of genes regulating glucose and thymidine metabolism and revealed changes in apoptotic genes, suggesting that the imaging tracers FDG, 3-deoxy-3-[(18)F]fluorothymidine, and annexin V could serve as potential imaging biomarkers for RAF265 therapy monitoring. We concluded that RAF265 is highly efficacious in this xenograft model of human melanoma and decreases glucose metabolism as measured by DNA microarray analysis, cell culture assays, and small animal FDG PET scans as early as 1 day after treatment. Our results support the use of FDG PET in clinical trials with RAF265 to assess early tumor response. DNA microarray analysis and small animal PET studies may be used as complementary technologies in drug development. DNA microarray analysis allows for analysis of drug effects on multiple pathways linked to cancer and can suggest corresponding imaging tracers for further analysis as biomarkers of tumor response.

Pattern of 18F-FDG uptake in the spinal cord in patients with non-central nervous system malignancy.

STUDY DESIGN: Retrospective review. OBJECTIVE: To (1) propose a standard method to quantitate 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG) uptake in the spinal cord and (2) use this methodology to retrospectively characterize the pattern of uptake within the entire spinal cord using whole-body positron emission tomography/computed tomography (PET/CT) imaging. SUMMARY OF BACKGROUND DATA: A physiologic understanding of glucose metabolism within the spinal cord may provide insight regarding infectious, inflammatory, vascular, and neoplastic spinal cord diseases. METHODS: Institutional review board approval was obtained. A total of 131 consecutive whole-body PET/CT studies from July to August 2004 were reviewed, and using exclusionary criteria of: (1) severe spinal arthropathy or curvature, (2) motion artifact, (3) canal hardware, (4) spinal tumor, and (5) marrow hyperplasia, 92 studies of neurologically intact patients (49 men and 43 women) were selected for a retrospective review of spinal cord 18F-FDG activity. The transaxial CT was used to define the canal and circular regions of interests were placed within the canal at the level of the vertebral body midpoint from C1 to L3. Region of interest total count, area, and maximum standardized uptake value (SUVmax) were recorded. Measurements at L5 served as an internal control. For comparative analysis, the cord-to-background (CTB) ratio was defined as spinal cord SUVmax to L5 SUVmax. RESULTS: Mean CTB decreased along each spinal level from cranial to caudal (P < 0.001). Significant relative increases were observed at the T11-T12 vertebral body levels (P < 0.001). Although insignificant, a relative increase was observed at C4. No significant interactions of age or sex on CTB were observed. CONCLUSION: The pattern of 18F-FDG uptake within the spinal cord, observed in patients with non-central nervous system malignancy, may be helpful in understanding glucose physiology of spinal cord diseases and warrants further research.

A strategy for blood biomarker amplification and localization using ultrasound.

Blood biomarkers have significant potential applications in early detection and management of various diseases, including cancer. Most biomarkers are present in low concentrations in blood and are difficult to discriminate from noise. Furthermore, blood measurements of a biomarker do not provide information about the location(s) where it is produced. We hypothesize a previously undescribed strategy to increase the concentration of biomarkers in blood as well as localize the source of biomarker signal using ultrasound energy directly applied to tumor cells. We test and validate our hypothesis in cell culture experiments and mouse tumor xenograft models using the human colon cancer cell line LS174T, while measuring the biomarker carcinoembryonic antigen (CEA) before and after the use of ultrasound to liberate the biomarker from the tumor cells. The results demonstrate that the application of low-frequency ultrasound to tumor cells causes a significant release of tumor biomarker, which can be measured in the blood. Furthermore, we establish that this release is specific to the direct application of the ultrasound to the tumor, enabling a method for localization of biomarker production. This work shows that it is possible to use ultrasound to amplify and localize the source of CEA levels in blood of tumor-bearing mice and will allow for a previously undescribed way to determine the presence and localization of disease more accurately using a relatively simple and noninvasive strategy.

(18)F and (18)FDG PET imaging of osteosarcoma to non-invasively monitor in situ changes in cellular proliferation and bone differentiation upon MYC inactivation.

Osteosarcoma is one of the most common pediatric cancers. Accurate imaging of osteosarcoma is important for proper clinical staging of the disease and monitoring of the tumor's response to therapy. The MYC oncogene has been commonly implicated in the pathogenesis of human osteosarcoma. Previously, we have described a conditional transgenic mouse model of MYC-induced osteosarcoma. These tumors are highly invasive and are frequently associated with pulmonary metastases. In our model, upon MYC inactivation osteosarcomas lose their neoplastic properties, undergo proliferative arrest and differentiate into mature bone. We reasoned that we could use our model system to develop noninvasive imaging modalities to interrogate the consequences of MYC inactivation on tumor cell biology in situ. We performed positron emission tomography (PET) combining the use of both (18)F-fluorodeoxyglucose ((18)FDG) and (18)F-flouride ((18)F) to detect metabolic activity and bone mineralization/remodeling. We found that upon MYC inactivation, tumors exhibited a slight reduction in uptake of (18)FDG and a significant increase in the uptake of (18)F along with associated histological changes. Thus, these cells have apparently lost their neoplastic properties based upon both examination of their histology and biologic activity. However, these tumors continue to accumulate (18)FDG at levels significantly elevated compared to normal bone. Therefore, PET can be used to distinguish normal bone cells from tumors that have undergone differentiation upon oncogene inactivation. In addition, we found that (18)F is a highly sensitive tracer for detection of pulmonary metastasis. Collectively, we conclude that combined modality PET/CT imaging incorporating both (18)FDG and (18)F is a highly sensitive means to non-invasively measure osteosarcoma growth and the therapeutic response, as well as to detect tumor cells that have undergone differentiation upon oncogene inactivation.

The synthesis of 18F-FDS and its potential application in molecular imaging.

PURPOSE: 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) is the most commonly used positron emission tomography (PET) tracer for oncological and neurological imaging, but it has limitations on detecting tumor or inflammation in brain gray matter. In this study, we describe the development of 2-deoxy-2-[(18)F]fluorosorbitol ((18)F-FDS) and its possible application in lesion detection around brain area. PROCEDURES: (18)F-FDS was obtained by reduction of FDG using NaBH(4) (81 +/- 4% yield in 30 min). Cell uptake/efflux experiments in cell culture and small animal PET imaging on tumor and inflammation models were performed. RESULTS: Despite the low accumulation in cell culture, (18)F-FDS had good tumor uptake and contrast in the subcutaneous U87MG tumor model (4.54%ID/g at 30 min post-injection). Minimal uptake in the normal mouse brain facilitated good tumor contrast in both U87MG and GL-26 orthotopic tumor models. (18)F-FDS also had increased uptake in the inflamed foci of the TPA-induced acute inflammation model. CONCLUSIONS: Because of the ease of synthesis and favorable in vivo kinetics, (18)F-FDS may have potential applications in certain cases where FDG is inadequate (e.g., brain tumor).

Preclinical efficacy of the c-Met inhibitor CE-355621 in a U87 MG mouse xenograft model evaluated by 18F-FDG small-animal PET.

UNLABELLED: The purpose of this study was to evaluate the efficacy of CE-355621, a novel antibody against c-Met, in a subcutaneous U87 MG xenograft mouse model using (18)F-FDG small-animal PET. METHODS: CE-355621 or control vehicle was administered intraperitoneally into nude mice (drug-treated group, n = 12; control group, n = 14) with U87 MG subcutaneous tumor xenografts. Drug efficacy was evaluated over 2 wk using (18)F-FDG small-animal PET and compared with tumor volume growth curves. RESULTS: The maximum %ID/g (percentage injected dose per gram of tissue) of (18)F-FDG accumulation in mice treated with CE-355621 remained essentially unchanged over 2 wk, whereas the %ID/g of the control tumors increased 66% compared with the baseline. Significant inhibition of (18)F-FDG accumulation was seen 3 d after drug treatment, which was earlier than the inhibition of tumor volume growth seen at 7 d after drug treatment. CONCLUSION: CE-355621 is an efficacious novel antineoplastic chemotherapeutic agent that inhibits (18)F-FDG accumulation earlier than tumor volume changes in a mouse xenograft model. These results support the use of (18)F-FDG PET to assess early tumor response for CE-355621.

Reproducibility of 18F-FDG microPET studies in mouse tumor xenografts.

UNLABELLED: (18)F-FDG has been used to image mouse xenograft models with small-animal PET for therapy response. However, the reproducibility of serial scans has not been determined. The purpose of this study was to determine the reproducibility of (18)F-FDG small-animal PET studies. METHODS: Mouse tumor xenografts were formed with B16F10 murine melanoma cells. A 7-min small-animal PET scan was performed 1 h after a 3.7- to 7.4-MBq (18)F-FDG injection via the tail vein. A second small-animal PET scan was performed 6 h later after reinjection of (18)F-FDG. Twenty-five sets of studies were performed. Mean injected dose per gram (%ID/g) values were calculated from tumor regions of interest. The coefficient of variation (COV) from studies performed on the same day was calculated to determine the reproducibility. Activity from the second scans performed after 6 h were adjusted by subtracting the estimated residual activity from the first (18)F-FDG injection. For 7 datasets, an additional scan immediately before the second injection was performed, and residual activity from this additional delayed scan was subtracted from the activity of the second injection. COVs of both subtraction methods were compared. Blood glucose values were measured at the time of injection and used to correct the %ID/g values. RESULTS: The COV for the mean %ID/g between (18)F-FDG small-animal PET scans performed on the same day 6 h apart was 15.4% +/- 12.6%. The delayed scan subtraction method did not produce any significant change in the COV. Blood glucose correction increased the COV. The injected dose, tumor size, and body weight did not appear to contribute to the variability of the scans. CONCLUSION: (18)F-FDG small-animal PET mouse xenograft studies were reproducible with moderately low variability. Therefore, serial small-animal PET studies may be performed with reasonable accuracy to measure tumor response to therapy.

Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging.

Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3-8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843+/-28) to passage 20 (250+/-10) to passage 40 (44+/-3) to passage 60 (3+/-1 RLU/microg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation.

Reproducibility of 3'-deoxy-3'-(18)F-fluorothymidine microPET studies in tumor xenografts in mice.

UNLABELLED: 3'-Deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) has been used to image tumor proliferation in preclinical and clinical studies. Serial microPET studies may be useful for monitoring therapy response or for drug screening; however, the reproducibility of serial scans has not been determined. The purpose of this study was to determine the reproducibility of (18)F-FLT microPET studies. METHODS: C6 rat glioma xenografts were implanted into nude mice (n = 9) and grown to mean diameters of 5-17 mm for approximately 2 wk. A 10-min acquisition was performed on a microPET scanner approximately 1 h after (18)F-FLT (1.9-7.4 MBq [50-200 muCi]) was injected via the tail vein. A second microPET scan was performed approximately 6 h later on the same day after reinjection of (18)F-FLT to assess for reproducibility. Most of the mice were studied twice within the same week (for a total of 17 studies). Images were analyzed by drawing an ellipsoidal region of interest (ROI) around the tumor xenograft activity. Percentage injected dose per gram (%ID/g) values were calculated from the mean activity in the ROIs. Coefficients of variation and differences in %ID/g values between studies from the same day were calculated to determine the reproducibility after subtraction of the estimated residual tumor activity from the first (18)F-FLT injection. RESULTS: The coefficient of variation (mean +/- SD) for %ID/g values between (18)F-FLT microPET scans performed 6 h apart on the same day was 14% +/- 10%. The difference in %ID/g values between scans was -0.06% +/- 1.3%. Serum thymidine levels were mildly correlated with %ID/g values (R(2) = 0.40). Tumor size, mouse body weight, injected dose, and fasting state did not contribute to the variability of the scans; however, consistent scanning parameters were necessary to ensure accurate studies, in particular, controlling body temperature, the time of imaging after injection, and the ROI size. CONCLUSION: (18)F-FLT microPET mouse tumor xenograft studies are reproducible with moderately low variability. Serial studies may be performed to assess for significant changes in therapy response or for preclinical drug development.

Molecular imaging of the kinetics of vascular endothelial growth factor gene expression in ischemic myocardium.

BACKGROUND: Angiogenic gene therapy is a promising treatment paradigm for patients with ischemic heart disease. In this study, we used micro-positron emission tomography (microPET) to monitor the transgene expression, function, and effects in a whole-body system. METHODS AND RESULTS: Adenovirus with cytomegalovirus promoter driving an angiogenic gene (vascular endothelial growth factor [VEGF]) linked to a PET reporter gene (herpes simplex virus type 1 mutant thymidine kinase; Ad-CMV-VEGF121-CMV-HSV1-sr39tk) was used to transfect rat embryonic cardiomyoblasts in vitro. Expression of both genes correlated strongly (r=0.98; P<0.001). Afterward, rats underwent ligation of the left anterior descending artery followed by injection of 1x10(10) pfu of Ad-CMV-VEGF121-CMV-HSV1-sr39tk (study; n=35) or Ad-null (control; n=15) at the peri-infarct region. Noninvasive microPET imaging was used to assess the uptake of 9-(4-[18F]-fluoro-hydroxymethylbutyl)guanine ([18F]-FHBG) PET reporter probe by cells expressing the HSV1-sr39tk PET reporter gene. Cardiac transgene expression peaked at day 1 and declined over the next 2 weeks. Repeat adenoviral injections at day 60 yielded no detectable signal. The in vivo reporter gene expression (% injected dose/g of [18F]-FHBG) correlated well with ex vivo gamma counting (r=0.92), myocardial tissue HSV1-sr39TK enzyme activity (r=0.95), and myocardial tissue VEGF level (r=0.94; P<0.001 for all). The VEGF121 isoform induced significant increases in capillaries and small blood vessels. However, the level of neovasculature did not translate into significant improvements in functional parameters such as myocardial contractility by echocardiography, perfusion by nitrogen-13 ammonia imaging, and metabolism by [18F]-fluorodeoxyglucose imaging. CONCLUSIONS: Taken together, these findings establish the feasibility of molecular imaging for monitoring angiogenic gene expression with a PET reporter gene and probe noninvasively, quantitatively, and repetitively. The principles demonstrated here can be used to evaluate other therapeutic genes of interest in animal models before future clinical trials are initiated.