Magnetic resonance-imaging of the effect of targeted antiangiogenic gene delivery in a melanoma tumour model.

OBJECTIVES: We investigated the effect of targeted gene therapy to melanoma tumours (M21) by MR-imaging. METHODS: M21 and M21-L tumours were grown to a size of 850 mm(3). M21 and M21-L tumours were intravenously treated with an αvß3-integrin-ligand-coupled nanoparticle (RGDNP)/RAF(-) complex five times every 72 hours. MRI was performed at set time intervals 24h and 72h after the i.v. injection of the complex. The MRI protocol was T1-wt-SE±CM, T2-wt-FSE, DCE-MRI, Diffusion-wt-STEAM-sequence, T2-time obtained on a 1.5-T-GE-MRI device. RESULTS: The size of the treated M21 tumours kept nearly constant during the treatment phase (847.8±31.4 mm(3) versus 904.8±44.4 mm(3)). The SNR value (T2-weighted images) of the tumours was 36.7±0.6 and dropped down to 30.6±1.9 (p=0.004). At the beginning the SNR value (T1-weighted images) of the tumours after contrast medium application was 42.3±1.9 and dropped down to 28.5±3.0 (p<0.001). In the treatment group the diffusion coefficient increased significantly under therapy (0.54±0.01x10(-3) mm(2)/s versus 0.67±0.04x10(-3) mm(2)/s). The DCE-MRI showed a reduction of the slope and of the Akep of 67.8±4.3 % respectively 64.8±3.3 % compared to baseline. CONCLUSIONS: Targeted gene delivery therapy induces significant changes in MR-imaging. MRI showed a significant reduction of contrast medium uptake parameters and increase of the diffusion coefficient of the tumours. KEY POINT: • Treatment with targeted gene-delivery therapy can be monitored by MR imaging • DCE and diffusion-weighted imaging are appropriate methods for monitoring this therapy • Functional changes are significant prior to any morphological changes.

Imaging of firefly luciferase activity under antiangiogenic therapy in a heat shock protein 70-transfected melanoma tumor model.

We investigated the effect of targeted gene therapy on heat shock protein 70 (Hsp70) expression in a melanoma tumor model (M21). M21 cells transfected with a plasmid containing the firefly luciferase reporter gene (ffluc), whose expression is driven by the hsp70 (hspa1b) or the cytomegalovirus (CMV) promoter, were grown to a size of 600 mm3. Five animals in each group were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein [RGD-NP/RAF(-)] complex. Bioluminescence imaging (BLI) (IVIS, Xenogen, Alameda, CA) was performed at set time intervals. Western blot analysis of the HSP70 protein was simultaneously performed. The size of the treated M21 tumors was nearly constant (637.8 ± 33.4 mm3 vs 674.8 ± 34.4 mm3). BLI showed that if transcription was controlled by the CMV promoter, firefly luciferase activity decreased to 51.1% ± 8.3%. When transcription was controlled by the hsp70 promoter, the highest firefly luciferase activity (4.4 ± 0.3-fold) was observed after 24 hours. In accordance with BLI, Western blot analysis showed an increase in the level of HSP70, with the maximum detection 24 hours after the injection of the RGD-NP/RAF(-) complex. Targeted antiangiogenic therapy can induce luciferase activity where transcription is controlled by an hsp70 promoter and HSP70 protein in melanoma tumors.

Comparison of contrast-enhanced multi-station MR angiography and digital subtraction angiography of the lower extremity arterial disease.

PURPOSE: To compare diagnostic accuracy of multi-station, high-spatial resolution contrast-enhanced MR angiography (CE-MRA) of the lower extremities with digital subtraction angiography (DSA) as the reference standard in patients with symptomatic peripheral arterial occlusive disease. MATERIALS AND METHODS: Of 485 consecutive patients undergoing a run-off CE-MRA, 152 patients (86 male, 66 female; mean age, 71.6 years) with suspected peripheral arterial occlusive disease were included into our Institutional Review Board approved study. All patients underwent MRA and DSA of the lower extremities within 30 days. MRA was performed at 1.5 Tesla with a single bolus of 0.1 mmol/kg body weight of gadobutrol administered at a rate of 2.0 mL/s at three stations. Two readers evaluated the MRA images independently for stenosis grade and image quality. Sensitivity and specificity were derived. RESULTS: Sensitivity and specificity ranged from 73% to 93% and 64% to 89% and were highest in the thigh area. Both readers showed comparable results. Evaluation of good and better quality MRAs resulted in a considerable improvement in diagnostic accuracy. CONCLUSION: Contrast-enhanced MRA demonstrates good sensitivity and specificity in the investigation of the vasculature of the lower extremities. While a minor investigator experience dependence remains, it is standardizable and shows good inter-observer agreement. Our results confirm that the administration of Gadobutrol at a standard dose of 0.1 mmol/kg for contrast-enhanced runoff MRA is able to detect hemodynamically relevant stenoses. Use of contrast-enhanced MRA as an alternative to intra-arterial DSA in the evaluation and therapeutic planning of patients with suspected peripheral arterial occlusive disease is well justified.

Use of in vivo bioluminescence and MRI to determine hyperthermia-induced changes in luciferase activity under the control of an hsp70 promoter.

We investigated the in vivo effect of hyperthermia on the expression of heat shock proteins and MRI changes in three tumor cell lines. Three tumor cell lines (SCCVII, NIH3T3, M21) were transfected with a plasmid containing the heat shock protein 70 gene (hsp70) promoter fragment and the luciferase reporter gene, and injected into mice. Tumors of 1100 mm³ in size were exposed to five different temperatures (38, 40, 42, 44 and 46 °C) in a water bath. Bioluminescence and MRI were performed at set time intervals. The MRI scan protocol was as follows: T1-weighted spin echo ± contrast medium, T2-weighted fast spin echo, dynamic contrast-enhanced MRI, diffusion-weighted stimulated echo acquisition mode sequence, T2 time obtained on a 1.5T General Electric MRI scanner. Immunoblotting was also performed. hsp70 transcription was strongly induced at 42 and 44 °C, reaching values as high as 8531.5 ± 432.1-fold above baseline in NIH3T3 tumors. At these temperatures, significant increases in the uptake of contrast medium, slope of initial enhancement, Ak(ep) values and apparent diffusion coefficient (ADC) were observed in the 8-h scan of the NIH3T3 cell line. In SCCVII tumors, ADC increased by about 23% (p = 0.010) in the scans performed at 8, 24, 48 and 96 h. At 46 °C, luciferase activity was reduced significantly in the three cell lines. In all tumor types, a significant increase in ADC was observed, which was highest in SCCVII tumors (33.8%; p < 0.01). In accordance with the bioluminescence results, significant Hsp70 protein production was shown by immunoblot analysis. The best correlation coefficient between luciferase activity and immunoblotting results was found for M21 tumors (r = 0.93, p < 0.0001). Different tissue types display distinct patterns of hsp70 transcription. MRI can be used, in combination with optical imaging, to provide information on hsp70 transcription and protein production. The major finding of the present study was that heat-related biochemical changes in tumor tissue can be determined by MRI.

Gene expression analysis of SCC tumor cells in muscle tissue.

The purpose of this study was to evaluate microarray technology of HNSCC cells in muscle tissue. 200 SCCVII tumor cells were injected intramuscularly into the right flank of ten C3H/Km mice each. One week later the animals were killed and the tissue taken out. Histology (H&E staining) and microarray of the tissue were performed. Histology showed a few tumor cells between the muscle fibers. Microarray technology showed different gene expression pattern of the muscle tissue with SCCVII cells in comparison with normal muscle tissue. Only those genes showing a fold change difference of 5 or higher were considered. Gene expression analysis revealed changes in the expression levels of SCCVII cells in muscle tissue in 220 genes. Significant gene expression differences between SCCVII cells in muscle tissue and pure muscle tissue could be seen.

Prospective comparison of image quality and diagnostic accuracy of 0.5 molar gadobenate dimeglumine and 1.0 molar gadobutrol in contrast-enhanced run-off magnetic resonance angiography of the lower extremities.

PURPOSE: To compare image quality and diagnostic accuracy of 0.5 molar gadobenate dimeglumine and 1.0 molar gadobutrol in contrast-enhanced (CE) magnetic resonance angiography (MRA) of the lower extremities interindividually. MATERIALS AND METHODS: The study was approved by our Institutional Review Board. Written informed consent was obtained from all patients before enrollment in the study. We prospectively included 74 patients (21 women, 53 men; mean age ± SD: 67.9 ± 11.0 years) with suspected peripheral occlusive vascular disease. All patients underwent a contrast-enhanced MRA of both lower extremities with either 0.1 mL/kg body weight gadobutrol or gadobenate dimeglumine. Image quality, stenosis grade, and artifacts were assessed by two blinded, independent investigators. Signal intensity (SI), signal-to-noise ratio (SNR), and contrast-to-noise ratio (CNR) were measured by a third investigator. Contrast agent groups were compared to each other using a two-sided Student's t-test. RESULTS: The results did not show significant differences for SI, SNR, or CNR. Both investigators were in significant accordance (P < 0.05) with regard to stenosis detection. CONCLUSION: We conclude that application of standard clinical doses (0.1 mL/kg body weight) of both contrast agents provides similar diagnostic results and gadolinium dose could be reduced by the application of a single dose of gadobenate dimeglumine for CE run-off MRA.

Detection of micrometastases of squamous cell carcinoma tumor cells in muscle tissue.

The aim of this study was to evaluate microarray technology in the detection of micrometastases of head and neck squamous cell carcinoma (HNSCC) in muscle tissue. Three hundred SCCVII tumor cells were injected intramuscularly into the right flank of ten C3H/Km mice. One week later, the animals were euthanized and the muscle tissue was taken out. Histology (H&E staining), microarray and reverse transcriptase polymerase chain reaction analysis (RT-PCR) of the tissue was performed. Histology showed a few tumor cells between the muscle fibers. Microarray technology showed the different gene expression pattern of the muscle tissue with micrometastases in comparison to normal muscle tissue. Only genes with a fold change difference of 10 or greater were considered. Gene expression analysis revealed changes in the expression levels of 91 genes of micrometastases in muscle tissue. RT-PCR confirmed gene up-regulation. Significant differences in gene expression between micrometastases in muscle tissue and pure muscle tissue were found. The genes found to be up-regulated could be used to detect micrometastases in muscle tissue.

Combined treatment of subacute and acute synthetic and venous bypass-graft occlusions with percutaneous mechanical thrombectomy and thrombolysis.

INTRODUCTION: Percutaneous mechanical thrombectomy (PMT) is a third choice of treatment for acute arterial occlusions, in addition to thrombolysis and surgical thrombectomy. The aim of this retrospective study was to compare the combined treatment of PMT and local thrombolysis with thrombolysis therapy alone. MATERIALS AND METHODS: Sixty-nine patients with acute (<14 days [n = 35]) or subacute (14-42 days [n = 34]) femoropopliteal bypass occlusions were treated with PMT combined with thrombolysis. Seventy-two patients with acute [n=40] or subacute [n = 32] femoropopliteal bypass occlusions were treated with thrombolysis alone. The thrombolysis in myocardial infarction (TIMI) classification was used to assess the bypass occlusion. Local thrombolysis time and dosage, reopening time, time in the intensive care unit, necessary surgical re-interventions, and clinical outcome were compared between the 2 groups. RESULTS: The TIMI scores were significantly higher in the PMT plus thrombolysis group than in the thrombolysis group (acute occlusions 1188 versus 935, p<0.001; subacute occlusions 935 versus 605, p<0.001). The total urokinase dosage, the total hours of thrombolysis, time in the intensive care unit, and total hospital stay in the acute PMT plus thrombolysis group were significantly lesser than those in the thrombolysis group. After 24h of treatment, the ankle-brachial index improved in all groups (p<0.001): in the acute and subacute PMT plus thrombolysis group to 0.63 ± 0.14 and 0.43 ± 0.08, respectively; and in the acute and subacute thrombolysis group to 0.51 ± 0.11 and 0.41 ± 0.04, respectively. CONCLUSIONS: PMT combined with thrombolysis is a safe and very effective therapy for acute and subacute femoropopliteal bypass occlusions compared to treatment with thrombolysis alone.

Evaluation of contrast medium enhancement and [(18)F]-FDG uptake of liver metastasis in PET/CT prior to therapy.

OBJECTIVE: To evaluate the contrast medium enhancement and [(18)F]-FDG uptake of liver metastases in patients suffering from colon or breast carcinoma prior to therapy. MATERIAL AND METHODS: PET/CT (Philips Gemini) with 200MBq [(18)F]-FDG and contrast medium was performed in 50 patients with colon and 39 patients with breast carcinoma. Lesions were characterized with the presence or the absence of a rim enhancement. The area size, the HU(mean), HU(max), SUV(mean), SUV(max) of the lesion and of the liver were determined. The standard uptake values (SUVs) were correlated with the tumor markers CEA and CA 15-3. RESULTS: The lesions of colon carcinoma had HU(mean)-values of 70.7±19.2 and of breast carcinoma 88.1±21.7 (p<0.0001). In breast cancer the SUV(mean) was 3.9±1.3 versus 4.4±1.9 in colon carcinoma (p=0.0182). Lesion of colon carcinoma with rim enhancement had a significantly higher SUV(mean) (4.4±1.5 versus 3.6±1.2; p=0.001) and SUV(max) (6.7±2.6 versus 5.1±2.1; p=0.000) than lesions without a rim enhancement. A good correlation between tumor markers and SUVs(max) could be found in both tumor groups; r=0.83 (p<0.01) for colon carcinoma and r=0.82 (p<0.01) for breast carcinoma. CONCLUSIONS: The rim enhancement of the lesions in colon carcinoma indicate a significantly higher SUV.

Effect of antiangiogenic therapy on luciferase activity in a cytomegalovirus- or HSP70-promoter-transfected M21 tumor model.

We investigated the effect of targeted gene therapy on heat shock protein 70 expression (Hsp70) and protein production (HSP70) in a melanoma tumor model (M21; M21-L). M21 and M21-L cells transfected with a plasmid containing the Hsp70 (Hspa1b) or the cytomegalovirus (CMV) promoter and the luciferase reporter gene were injected into mice; the resulting tumors grew to a size of 650 mm(3). Mice (five per group) were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein complex [RGD-NP/RAF(-)] or with a nanoparticle control. Bioluminescence imaging (IVIS®, Xenogen, USA) was performed at 12, 24, 48, and 72 h after the treatment cycle. Western blot analysis of HSP70 protein was performed to monitor protein expression. The size of the treated M21 tumors remained fairly constant (647.8 ± 103.4 mm(2) at the beginning versus 704.8 ± 94.4 mm(3) at the end of the experiment). The size of the M21-L tumors increased, similar to the untreated control tumors. Bioluminescent imaging demonstrated that when transcription was controlled by the CMV promoter, luciferase activity decreased to 17.9% ± 4.3% of baseline values in the treated M21 tumors. When transcription was controlled by the Hsp70 promoter, the highest luciferase activity (4.5 ± 0.7-fold increase over base-line values) was seen 24 h after injection in the M21 tumors; however, no luciferase activity was seen in the M21-L tumors. In accordance with bioluminescent imaging, western blot analysis showed a peak in HSP70 production at 24 h after the injection of the RGD-NP/RAF(-) complex in the M21 tumors; however, no HSP70 protein induction was seen in the M21-L tumors. Thus, targeted antiangiogenic therapy can induce Hsp70 expression and HSP70 protein in melanoma tumors.

Novel approach to complex pulmonary arteriovenous malformation embolization using detachable coils and Amplatzer vascular plugs.

INTRODUCTION: We evaluated the feasibility of a modified embolization technique of pulmonary arteriovenous malformations (PAVM) using venous sac embolization with detachable coils combined with the feeding artery embolization with the Amplatzer vascular plug (AVP). MATERIALS AND METHODS: We retrospectively studied technical and clinical success in the treatment of 11 complexe PAVMs. We recorded number and size of feeding arteries and draining vein, the last prior and post treatment in the follow up CT, size of PAVMs; and the number of devices needed to occlude each PAVM. RESULTS: 11 complexe PAVM were treated with detachable coils to venous sac embolization followed by AVP to embolize feeding arteries. In all but one case a complete occlusion of the PAVM was angiographically achieved. The mean number of feeding vessel was 2.64 ± 0.92 (2-5). The mean number of coils was 7.82 ± 5.09 (3-20 coils). CT-follow-up, that was possible in 8 patients, showed a significant reduction of the draining vein size. The mean diameter reduction of the draining vein was 62 ± 18% varying between 29% and 77%. In all but one case with the complexe angioarchitecture the reduction of draining vein size close to 70% was achieved. CONCLUSIONS: Our study implies that the venous sac embolization using the detachable coils followed by occlusion of the large feeding arteries using the AVP is a highly efficient method for the treatment of the complex PAVMs with large out-flow vessels and short feeding arteries.

In vivo monitoring of antiangiogenic therapy by magnetic resonance and bioluminescence imaging in an M21 tumor model through activation of an hsp70 promoter-luciferase reporter construct.

We have investigated the effect of targeted gene therapy on the melanoma cell line M21, using a combination of bioluminescence imaging (BLI) and magnetic resonance imaging (MRI). M21 cells transfected with a plasmid containing either an hsp70 (Hspa1b) or a CMV promoter fragment, along with the luciferase reporter gene, were grown to a tumor size of 900 mm(3) . Five mice in each group were intravenously treated every 72 h with a complex consisting of a nanoparticle, an Arg-Gly-Asp-peptide, and a dominant negative mutant protein kinase inhibitor gene. BLI and MRI were performed at specific time intervals. The MRI scan protocol included T(1) -weighted-spin-echo ± contrast medium, T(2) -weighted-fast-spin-echo, dynamic contrast-enhanced MRI (DCE-MRI), and diffusion-weighted-stimulated-echo-acquisition-mode-sequence. The T(2) times were obtained using a 1.5 T GE MRI scanner. The size of the treated M21 tumors remained almost constant during the treatment phase (837.8 ± 133.4 vs 914.8 ± 134.4 mm(3) ). BLI showed that, if transcription was controlled by the CMV promoter, the luciferase activity decreased to 51.1 ± 8.3%. After transcription was controlled by the hsp70 promoter, the highest luciferase activity (4.4 ± 0.3 fold) was seen after 24 h. The signal-to-noise ratio (SNR; T(2) -weighted images) of the tumors was 36.7 ± 0.6 and subsequently dropped to 31.2 ± 4.4 (p=0.004). DCE-MRI showed a reduction of the slope and the Ak(ep) of 67.8% ± 4.3 and 64.8% ± 3.3%, respectively, compared with the baseline. The SNR value (T(1) -weighted images) of the tumors was 42.3 ± 1.9 immediately following contrast medium application and subsequently dropped to 28.5 ± 3.0 (p<0.001). In the treatment group, the diffusion coefficient increased significantly under therapy (0.66 ± 0.05 vs the pretreatment value of 0.54 ± 0.009 p<0.01). Thus, we observed that targeted antiangiogenic therapy can induce activation of the hsp70 promoter through a heat shock/luciferase reporter system. Moreover, MRI showed a significant reduction of the contrast medium uptake parameters and an increase in the diffusion coefficient of the tumors.