Changes In Tumor Vasculature Research Articles

Monitoring Combretastatin A4-induced tumor hypoxia and hemodynamic changes using endogenous MR contrast and DCE-MRI.

To benchmark MOBILE (Mapping of Oxygen By Imaging Lipid relaxation Enhancement), a recent noninvasive MR method of mapping changes in tumor hypoxia, electron paramagnetic resonance (EPR) oximetry, and dynamic contrast-enhanced MRI (DCE-MRI) as biomarkers of changes in tumor hemodynamics induced by the antivascular agent combretastatin A4 (CA4). NT2 and MDA-MB-231 mammary tumors were implanted subcutaneously in FVB/N and nude NMRI mice. Mice received 100 mg/kg of CA4 intraperitoneally 3 hr before imaging. The MOBILE sequence (assessing R1 of lipids) and the DCE sequence (assessing K(trans) hemodynamic parameter), were assessed on different cohorts. pO2 changes were confirmed on matching tumors using EPR oximetry consecutive to the MOBILE sequence. Changes in tumor vasculature were assessed using immunohistology consecutive to DCE-MRI studies. Administration of CA4 induced a significant decrease in lipids R1 (P = 0.0273) on pooled tumor models and a reduction in tumor pO2 measured by EPR oximetry. DCE-MRI also exhibited a significant drop of K(trans) (P < 0.01) that was confirmed by immunohistology. MOBILE was identified as a marker to follow a decrease in oxygenation induced by CA4. However, DCE-MRI showed a higher dynamic range to follow changes in tumor hemodynamics induced by CA4.

Pigment epithelium-derived factor enhances tumor response to radiation through vasculature normalization in allografted lung cancer in mice.

This study aimed to explore the potential therapeutic effects of the combination of pigment epithelium-derived factor (PEDF) and radiation on lung cancer. The Lewis lung cancer (LLC) allografts in nude mice were treated with radiation, PEDF and PEDF combined with radiation. The morphologic changes of tumor vasculature and the hypoxic fraction of tumor tissues were evaluated. Significant inhibition of tumor growth was observed when radiation was applied between the 3rd and 7th day (the vasculature normalization window) after the initiation of PEDF treatment. During the vasculature normalization window, the tumor blood vessels in PEDF-treated mice were less tortuous and more uniform than those in the LLC allograft tumor treated with phosphate-buffered saline. Meanwhile, the thickness of the basement membrane was remarkably reduced and pericyte coverage was significantly increased with the PEDF treatment. We also found that tumor hypoxic fraction decreased during the 3rd to the 7th day after PEDF treatment, suggesting improved intratumoral oxygenation. Taken together, our results show that PEDF improved the effects of radiation therapy on LLC allografts by inducing a vascular normalization window from the 3rd to the 7th day after PEDF treatment. Our findings provide a basis for treating lung cancer with the combination of PEDF and radiation.

Abstract 771: TLR4 activation by paclitaxel promotes breast cancer recurrence and metastasis

Abstract Background: Paclitaxel (PXL) is a widely-used drug for breast cancer therapy. Resistance, however, occurs frequently and the evasion mechanisms remain unclear. PXL is known to activate Toll-like Receptor-4 (TLR4), a major signaling receptor expressed on immune cells responding to lipopolysaccharide (LPS). LPS activation of TLR4 in the immune cells substantially enhances their migratory, invasive, proliferative, and pro-survival functions as necessary for the body defense. Since PXL is a functional LPS mimetic, we hypothesized that acquisition of such phenotype by TLR4-expressing cancer cells promotes tumor growth, metastasis, and resistance to therapy. Methods: We tested this hypothesis in two breast cancer luciferase-tagged models genetically modified to either suppress TLR4 expression in a positive line, MDA-MB-231, or overexpress it in a negative line, HCC1806. Modified cell lines and their controls were orthotopically implanted in mice followed by measuring tumor growth prior and post-PXL treatment. Incidence and burden of lymph node (LN) and lung metastases were quantified by measuring luciferase activity in respective organs. TLR4-induced inflammation was assessed by measuring cytokines in blood, tumor lysates, and metastatic organs using RT-qPCR and ELISA. Cell composition of spleens and bone marrow from control and treated mice were analyzed by FACS. TLR4-induced changes in tumor vasculature were determined by immunostaining for blood and lymphatic vessel markers. Results: TLR4 expressed in tumor cells significantly increased rate of recurrence and metastasis, an event augmented by PXL treatment. Local inflammation was also enhanced by the PXL•TLR4 axis as illustrated by a 276-fold increase in IL-6 in lysates of TLR4+ tumors from treated mice as compared with samples of isogenic tumors with depleted TLR4. Activation of this pathway also increased cytokine levels in the blood and distant organs as indicted by 3-6 fold increase in IL-4 and IL-10 in LNs and lungs. These pro-inflammatory systemic changes promoted generation of myeloid progenitors in bone marrow and spleen evident by 4-5 fold increase in Ly6C+ cell population in TLR4-overexpressing tumors compared with those lacking TLR4. Importantly, activation of the PXL•TLR4 axis induced intratumoral formation of lymphatic vessels in HCC1806 tumors absent in all other experimental groups. Conclusion: These data imply that PXL therapy activates TLR4 often overexpressed in breast cancer. Activation of this pathway substantially increases local tumor and systemic inflammation leading to generation of myeloid progenitors that can promote metastasis by inducing angiogenesis and lymphatic vessel formation, particularly inside the tumor. These findings suggest that tumor expression of TLR4 may indicate poor prognosis and response to therapy, and that blocking the TLR4 pathway may improve current anti-cancer treatments. Citation Format: Sandeep Rajput, Lisa Volk-Draper, Kelly Hall, Sophia Ran. TLR4 activation by paclitaxel promotes breast cancer recurrence and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 771. doi:10.1158/1538-7445.AM2014-771

Non-invasive quantification of anti-angiogenic therapy by contrast-enhanced MRI in experimental pancreatic cancer

Currently, early changes of tumor vasculature after angiogenesis inhibition can only be evaluated by histopathology, a method not suitable in a clinical setting. To quantify effects of different angiogenesis inhibitors on the microvasculature of orthotopically implanted pancreatic cancers by contrast-enhanced magnetic resonance imaging (MRI) in order to establish a non-invasive technique for monitoring antiangiogenic cancer treatment. DSL-6A/C1 pancreatic cancers were implanted in the pancreas of 109 Lewis rats. Three weeks later, antiangiogenic treatment was initiated by administration of Bevacizumab (n = 38) or Suramin (n = 27) while the control group (n = 44) remained untreated. Dynamic MRI was performed 24 h, 1 week, and 4 weeks after treatment initiation. Fractional tumor plasma volume (fPV, %) and vascular permeability (K(PS), mL/min/100 cc) were calculated based on the MRI data by using a pharmacokinetic model. Twenty-four hours after the initial dose, a significant decline in K(PS) was observed in the Bevacizumab group compared to the control and Suramin group (0.002 ± 0.008; 0.057 ± 0.046 and 0.064 ± 0.062 (mean ± SD); P < 0.05). At 1 week, fPV was significantly smaller in Bevacizumab and Suramin treated tumors compared to control tumors (6.25 ± 2.74, 7.47 ± 3.44, and 15.10 ± 9.97, respectively; P < 0.05). Differences in tumor volumes were first observed after 4 weeks of treatment with significantly larger control tumors (4380.3 ± 1590.6 vs. 869.6 ± 717.2 and 1676.5 ± 2524.1 mm(3); P < 0.05). Dynamic MRI can quantify antiangiogenic effects on tumor microvasculature before changes in tumor volumes are detectable. Thus, this technique is a reasonable addition to morphological MRI and may be applied as an alternative to histopathology.

Comparison of dynamic contrast-enhanced MR, ultrasound and optical imaging modalities to evaluate the antiangiogenic effect of PF-03084014 and sunitinib.

Noninvasive imaging has been widely applied for monitoring antiangiogenesis therapy in cancer drug discovery. In this report, we used different imaging modalities including high-frequency ultrasound (HFUS), dynamic contrast enhanced-MR (DCE-MR), and fluorescence molecular tomography (FMT) imaging systems to monitor the changes in the tumor vascular properties after treatment with γ-secretase inhibitor PF-03084014. Sunitinib was tested in parallel for comparison. In the MDA-MB-231Luc model, we demonstrated that antiangiogenesis was one of the contributing mechanisms for the therapeutic effect of PF-03084014. By immunohistochemistry and FITC-lectin perfusion assays, we showed that the vascular defects upon treatment with PF-03084014 were associated with Notch pathway modulation, evidenced by a decrease in the HES1 protein and by the changes in VEGFR2 and HIF1α levels, which indicates down-stream effects. Using a 3D power Doppler scanning method, ultrasound imaging showed that the% vascularity in the MDA-MB-231Luc tumor decreased significantly at 4 and 7 days after the treatment with PF-03084014. A decrease in the tumor vessel function was also observed through contrast-enhanced ultrasound imaging with microbubble injection. These findings were consistent with the PF-03084014-induced functional vessel changes measured by suppressing the Ktrans values using DCE-MRI. In contrast, the FMT imaging with the AngioSence 680EX failed to detect any treatment-associated tumor vascular changes. Sunitinib demonstrated an outcome similar to PF-03084014 in the tested imaging modalities. In summary, ultrasound and DCE-MR imaging successfully provided longitudinal measurement of the phenotypic and functional changes in tumor vasculature after treatment with PF-03084014 and sunitinib.

Monitoring Tumor Response to Linifanib Therapy with SPECT/CT Using the Integrinαvβ3–Targeted Radiotracer99mTc-3P-RGD2

The objective of this study was to determine the utility of (99m)Tc-3P-Arg-Gly-Asp (RGD2) single photon emission computed tomography (SPECT)/computed tomography (CT) for noninvasive monitoring of integrin αvβ3-expression response to antiangiogenic treatment with linifanib. Linifanib or vehicle therapy was carried out in female athymic nu/nu mice bearing U87MG glioma (high αvβ3 expression) or PC-3 prostate (low αvβ3 expression) tumors at 12.5 mg/kg twice daily. The average tumor volume was 180 ± 90 mm(3) the day prior to baseline SPECT/CT. Longitudinal (99m)Tc-3P-RGD2 SPECT/CT imaging was performed at baseline (-1 day) and days 1, 4, 11, and 18. Tumors were harvested at all imaging time points for histopathological analysis with H&E and immunohistochemistry. A significant difference in tumor volumes between vehicle- and linifanib-treated groups was observed after 4 days of linifanib therapy in the U87MG model. The percent injected dose (%ID) tumor uptake of (99m)Tc-3P-RGD2 peaked in the vehicle-treated group at day 11, while the %ID/cm(3) tumor uptake decreased slowly over the whole study period. During the first 2 days of linifanib treatment, a rapid decrease in both %ID/cm(3) tumor uptake and tumor/muscle ratios of (99m)Tc-3P-RGD2 was observed, followed by a slow decrease until day 18. No decrease in tumor uptake of (99m)Tc-3P-RGD2 or tumor volume was observed for either treatment group in the PC-3 model. Changes in tumor vasculature were confirmed by histopathological H&E analysis and immunohistochemistry. Longitudinal imaging using (99m)Tc-3P-RGD2 SPECT/CT may be a useful tool for monitoring the downstream biologic effects of linifanib therapy.

Dose-dependent response of tumor vasculature to radiation therapy in combination with Sunitinib depicted by three-dimensional high-frequency power Doppler ultrasound

Large doses of radiation (8-20 Gy) preferentially target tumor vasculature. This vascular response is suggested to regulate tumor response to radiotherapy. Here, we investigate the relative contributions of direct cell killing by radiation versus tumor cell death due to radiation effects on the vasculature. We also examine Sunitinib's mechanism of action as a tumor radiosensitizer. MDA-MB-231 xenografts were treated with radiation doses of 2-16 Gy alone, or in combination with bFGF (endothelial radio-protector) or Sunitinib as pharmacological modulators of the vasculature. Sunitinib was orally administered for 2 weeks at 30 mg/kg before radiotherapy; bFGF was intravenously injected 1 h prior to irradiation. Three-dimensional high-frequency power Doppler ultrasound was used to assess relative changes in tumor vasculature. Immunohistochemistry, clonogenic and tumor growth assays were used to quantify tumor response. Significant reductions in power Doppler signal of up to 50 % were observed for 8 and 16 Gy treatments, along with a dose-dependent increase in cell death. No significant change in power Doppler signal and minimal tumor cell death were noted for tumors treated with radiation and bFGF. Treatments where Sunitinib was combined with radiation demonstrated a significant increase in flow signal at doses equal or greater than 8 Gy. This was accompanied with a significant increase in cell death when compared to radiation or Sunitinib alone. We confirm that tumor response to high doses of radiation is regulated by its vasculature. We also posit that the response observed when radiation is combined with Sunitinib is linked to a vascular "normalization" effect.

Integrin αvβ3-Targeted Radiotracer 99mTc-3P-RGD2 Useful for Noninvasive Monitoring of Breast Tumor Response to Antiangiogenic Linifanib Therapy but not Anti-Integrin αvβ3 RGD2 Therapy

Purpose: 99mTc-3P-RGD2 is a 99mTc-labeled dimeric cyclic RGD peptide that binds to integrin αvβ3 with high affinity and specificity. The purpose of this study was to demonstrate the utility of 99mTc-3P-RGD2 SPECT/CT (single photon emission computed tomography/computed tomography) as a molecular imaging tool for noninvasive monitoring breast tumor early response to antiangiogenesis therapy with linifanib, and to illustrate its limitations in monitoring the efficacy of anti-αvβ3 treatment.Methods: To support SPECT/CT imaging, biodistribution and therapy studies, the xenografted breast cancer model was established by subcutaneous injection of 5 × 106 MDA-MB-435 cells into the fat pad of each athymic nude mouse. Linifanib (ABT-869) was used as antiangiogenesis agent. The tumor volume was 180 ± 90 mm3 on the day (-1 day) before baseline SPECT/CT. Each animal was treated twice daily with vehicle or 12.5 mg/kg linifanib. Longitudinal 99mTc-3P-RGD2 SPECT/CT imaging was performed on days -1, 1, 4 and 11. Tumors were harvested at each time point for pathological analysis of hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Tumor uptake of 99mTc-3P-RGD2 was calculated from SPECT/CT quantification. When cyclic peptide E[c(RGDfK)]2 (RGD2) was used as the anti-αvβ3 agent, SPECT/CT images were obtained only at 7 and 21 days after last RGD2 dose.Results: The tumor uptake of 99mTc-3P-RGD2 from SPECT/CT quantification was almost identical to that from biodistribution. There was a dramatic reduction in both %ID and %ID/cm3 tumor uptake of 99mTc-3P-RGD2 during the first 24 hours of linifanib therapy. The therapeutic effect of linifanib was on both tumor cells and vasculature, as determined by IHC analysis of integrin αvβ3 and CD31. Changes in tumor vasculature were further confirmed by pathological H&E analysis of tumor tissues. While its %ID tumor uptake increased steadily in vehicle-treated group, the %ID tumor uptake of 99mTc-3P-RGD2 decreased in linifanib-treated group slowly over the 11-day study period. The degree of tumor response to linifanib therapy correlated well to the integrin αvβ3 expression levels before linifanib therapy.Conclusion: 99mTc-3P-RGD2 is an excellent radiotracer for monitoring integrin αvβ3 expression during and after linifanib therapy. 99mTc-3P-RGD2 SPECT/CT is an useful molecular imaging tool for patient selection before antiangiogenic and anti-αvβ3 therapy; but it would be difficult to use 99mTc-3P-RGD2 for accurate and noninvasive monitoring of early tumor response to anti-αvβ3 therapy.

Feasibility of volume perfusion CT (VPCT) imaging in antiangiogenic treatment of rabbit VX2 soft-tissue tumor

To explore the feasibility of volume perfusion CT imaging to dynamically monitor and evaluate the response of rabbit VX2 soft-tissue tumor to antiangiogenic treatment. To establish an experimental animal model of VX2 soft tissue tumor on 20 New Zealand white rabbits. Twenty rabbits were randomly divided into 2 groups. The therapy group was treated with recombinant human endostatin (3 mg·kg⁻¹·d⁻¹) for 7 days, and the control group received saline in the same dose only. Four times of CT volume perfusion scan were performed before treatment and on the second, forth, seventh days of treatment, respectively. The value of blood flow (BF), blood volume (BV), mean transit time (MTT), and permeability (PMB) in the VX2 tumors were measured after scanning. The microvessel density (MVD) and expression of vascular endothelial growth factor (VEGF) in the tumors were determined using immunohistochemical staining. The tumor volume of the therapy group was (1.36 ± 0.73) cm³ on the forth day of treatment and (1.69 ± 0.68) cm³ on the seventh day of the treatment. The tumor volume of the control group was (2.35 ± 0.62) cm³ on the fourth day of treatment and (3.87 ± 0.93) cm³ on the seventh day of the treatment (P < 0.05). On the seventh day of treatment, tumor necrosis ratio of the therapy group and the control group was (25.58 ± 5.51)% and (42.93 ± 4.34)%, respectively (P < 0.05). Comparing the perfusion parameters between the two groups on the same day, and the second, forth, seventh days of treatment, the value of PMB of the therapy group was (70.36 ± 23.46) ml·100 ml⁻¹·min⁻¹, (79.64 ± 13.68) ml·100 ml⁻¹·min⁻¹ and (84.76 ± 3.55) ml·100 ml⁻¹·min⁻¹, respectively, and that in the control group was (26.61 ± 6.47) ml·100 ml⁻¹·min⁻¹, (33.74 ± 16.47) ml·100 ml⁻¹·min⁻¹ and (30.47 ± 10.64) ml·100 ml⁻¹·min⁻¹, respectively (P < 0.05). The value of BF in the therapy group and control group was (71.19 ± 12.21) ml·100 ml⁻¹·min⁻¹ and (43.56 ± 12.21) ml·100 ml⁻¹·min⁻¹, respectively, on the seventh day of treatment (P < 0.05). The parameters on different days in the same group were compared. In the control group, the value of BF on the seventh day of treatment was significantly lower than that before and on the second and forth days of treatment (P < 0.05). However, in the therapy group, the value of PMB on the second, forth, and seventh days of treatment was significantly higher than that before treatment (P < 0.05). MVD of tumor in the control group was increased gradually, whereas increased on the first day and then decreased more in the therapy group. The VEGF expressions did not differ significantly between the experimental and control groups. Volume perfusion CT is helpful to quantify the tumor perfusion and evaluate the functional changes of tumor vasculature, and then evaluate the early therapeutic effect of antiangiogenic treatment.

WE-E-BRA-01: Introduction to Treatment Assessment of Radiation Therapy Using MR Functional Imaging and Its Application to Intracranial Stereotactic Radiosurgery.

1. Understand the principles of MR functional imaging techniques 2. Understand the issues related to MR functional imaging 3. Understand the clinical application of MR functional imaging in assessing treatment response of SRS.

Abstract 2438: Bevacizumab treatment hampers tumor uptake of antibodies in human tumor bearing mice

Abstract Background: Extensive angiogenesis, defective vascular architecture, impaired lymphatic drainage and increased vascular permeability are characteristics unique for solid tumors. These characteristics result in enhanced tumor permeability and retention (EPR) effect. The EPR effect has been recognized as an important factor for intratumoral delivery of macromolecular drugs, like antibodies. Rapid changes in tumor vasculature and permeability due to antiangiogenic treatment could possibly restrain the EPR effect and thereby lead to a decreased uptake of antibodies. This would have implications for combining antiangiogenic therapy with monoclonal antibodies. We therefore investigated the effect of bevacizumab treatment on the tumor uptake of antibodies by means of immunoPET imaging. Methods: The effect of bevacizumab treatment on the antibody tumor uptake was studied with in vivo PET imaging of 89Zr-trastuzumab, 89Zr-bevacizumab and 89Zr-IgG in a SKOV-3 human xenograft tumor model. PET images were made before and during bevacizumab (5mg/kg; 2qw) or placebo (normal saline) treatment. All animals also received trastuzumab, bevacizumab or IgG labeled with the near-infrared fluorescence dye IRDye 800CW as a co-injection. Immunohistochemistry was performed to determine the mean vascular density (MVD). Immunofluorescence was used to visualize trastuzumab-800CW, bevacizumab-800CW and IgG-800CW and to determine vessel normalization by vessel pericyte coverage. Results: Bevacizumab treatment decreased 89Zr-trastuzumab, 89Zr-bevacizumab and 89Zr-IgG tumor uptake with 41 ± 5% (P&amp;lt;0.0001), 43 ± 4% (P&amp;lt;0.0001) and 28 ± 6% (P=0.0004) respectively. Placebo treatment did not affect tumor uptake of 89Zr-IgG. Immunofluorescence showed less accumulation of IgG-800CW in bevacizumab treated tumor sections compared to placebo treated tumor sections. In all groups, bevacizumab treatment decreased the MVD (P&amp;lt;0.0003). Remaining vessels in bevacizumab treated tumor sections showed an increase in the amount and degree of vessel pericyte coverage compared to placebo. Conclusion: Bevacizumab treatment reduces tumor uptake of trastuzumab, bevacizumab and IgG possibly by restraining the EPR effect through vessel normalization. This deserves attention in the design of clinical trials, when combining antiangiogenic treatment with antibodies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2438. doi:1538-7445.AM2012-2438

Hyperpolarized 13C Spectroscopy Detects Early Changes in Tumor Vasculature and Metabolism after VEGF Neutralization

No clinically validated biomarkers exist to image tumor responses to antiangiogenic therapy. Here, we report the utility of hyperpolarized (13)C magnetic resonance spectroscopy (MRS) to detect the early effects of anti-VEGF therapy. In two colorectal cancer xenograft models, displaying differential sensitivity to VEGF blockade, we compared hyperpolarized MRS with measurements of tumor perfusion using dynamic contrast agent-enhanced (DCE)-MRI and tumor cellularity using diffusion-weighted MRI of the apparent diffusion coefficient (ADC) of tissue water. In tumors sensitive to anti-VEGF therapy, (13)C flux between hyperpolarized [1-(13)C]pyruvate and [1-(13)C]lactate decreased after anti-VEGF therapy and correlated with reduced perfusion. Production of [1,4-(13)C(2)]malate from hyperpolarized [1,4-(13)C(2)]fumarate increased in parallel with tumor cell necrosis, preceding any change in tumor ADC. In contrast, tumors that were less sensitive to anti-VEGF therapy showed an increase in (13)C flux from hyperpolarized [1-(13)C]pyruvate and an increase in uptake of a gadolinium contrast agent, whereas tumor ADC decreased. Increased label flux could be explained by vascular normalization after VEGF blockade, increasing delivery of hyperpolarized [1-(13)C]pyruvate as observed. Despite the minimal response of these tumors to treatment, with only a minor increase in necrosis observed histologically, production of [1,4-(13)C(2)]malate from hyperpolarized [1,4-(13)C(2)]fumarate in therapy-resistant tumors also increased. Together, our findings show that hyperpolarized (13)C MRS detects early responses to anti-VEGF therapy, including vascular normalization or vascular destruction and cell death.

A novel multi-targeted tyrosine kinase inhibitor, linifanib (ABT-869), produces functional and structural changes in tumor vasculature in an orthotopic rat glioma model

Tyrosine kinase inhibitors represent a class of targeted therapy that has proven to be successful for cancer treatment. Linifanib is a novel, orally active multi-targeted receptor tyrosine kinase (RTK) inhibitor that exhibits potent antitumor and antiangiogenic activities against a broad spectrum of experimental tumors and malignancies in patients. The compound is currently being evaluated in phase 2 and 3 clinical trials. To investigate the effectiveness of linifinib against gliomas and the mechanism of drug action, we characterized treatment-induced antitumor and antiangiogenic responses to linifanib in an orthotopic rat glioma model. The effect of linifanib treatment on tumor growth was determined by tumor volume assessment using anatomical magnetic resonance imaging (MRI). Changes in tumor microvessel function were evaluated with dynamic contrast-enhanced MRI (DCE-MRI). Immunohistochemistry (IHC) was applied to excised tumor samples to examine underlying changes in vascular structures and target receptor expression. Linifanib (10 mg/kg) given twice daily inhibited tumor growth following treatment for 7 days with tumor volumes being 149 ± 30 and 66 ± 7 mm(3) for vehicle-and linifanib-treated groups, respectively. A significant reduction of 37 ± 13% in tumor perfusion and microvessel permeability (measured by K (trans)) was observed as early as 2 h after administration compared with vehicle treatment. Continuous linifanib administration further reduced K (trans) at later time points until the end of the study (7 days post-treatment). At day 7, K (trans) was reduced by 75 ± 32% for linifanib treatment compared with vehicle treatment. Significant reduction in total blood vessel density and improved vessel wall integrity were observed, and staining for target receptor expression confirmed inhibition of phospho VEGFR-2 and PDGFR-β by linifanib treatment. These results demonstrate significant antitumor and antiangiogenic activity against gliomas by linifanib, a property that may result from the inhibition of VEGFR-2 and PDGFR-β-mediated vascular changes. DCE-MRI measured K (trans) changes at early treatment stages may be a useful pharmacodynamic marker for linifanib activity in clinical trials, and basal K (trans) may provide predictive value for tumor progression.

Phase I Dose-Finding Study of Pazopanib in Hepatocellular Carcinoma: Evaluation of Early Efficacy, Pharmacokinetics, and Pharmacodynamics

A phase I dose-escalating study of pazopanib was conducted to determine the maximum tolerated dose (MTD), pharmacokinetic/pharmacodynamic relationships, and clinical activity in patients with advanced hepatocellular carcinoma (HCC). Asian patients (N = 28) were dose escalated on pazopanib (200-800 mg) once daily (QD) on 21-day cycles, with MTD as the primary endpoint using a modified 3 + 3 design. Changes in tumor vasculature were evaluated by dynamic contrast-enhanced MRI (DCE-MRI). Two of five patients at the 800-mg dose level experienced dose-limiting toxicities [grade 3 aspartate aminotransferase (AST)/alanine aminotransferase (ALT) elevations and grade 3 malaise]. The MTD in patients with HCC (Child-Pugh class A) was 600 mg QD. Diarrhea, skin hypopigmentation, and AST elevation were the most commonly reported adverse events at the MTD. Mean C(max) and area under the concentration-time curve (AUC(0-6)) of pazopanib and its metabolites did not increase dose proportionally across the 200 to 800 mg range. Reductions in IAUGC and K(trans) were shown at all pazopanib doses evaluated, with the greatest reductions at 600 and 800 mg. Although larger DCE-MRI parameter decreases were associated with larger C(24) and C(max) values, there was no constant relationship between tumor perfusion decreases measured by DCE-MRI and plasma pazopanib pharmacokinetic parameters. Overall, 19 patients (73%) had either partial response or stable disease. Pazopanib has a manageable safety profile in patients with advanced HCC, and 600 mg was chosen for further development of pazopanib in advanced HCCs. Moreover, pazopanib reduced tumor vessel leakage, as shown by DCE-MRI, indicating a direct effect on HCC vasculature that might be associated with its antitumor activity.

Hemorrhagic onset of cerebellar pilocytic astrocytoma in an adult: a case report and review of the literature implying a possible relation of degenerative vascular changes to the massive intratumoral hemorrhage

Pilocytic astrocytoma (PA) is a low-grade astrocytic tumor arising predominantly during the first two decades of life. Hemorrhagic onset of PAs is uncommon, and the etiology of hemorrhage remains unclear. Here we report a case of hemorrhagic onset of cerebellar PA in a 29-year-old man who presented with a week-long history of headache and gait instability. Computed tomography and magnetic resonance imaging revealed a hemorrhagic tumor located in the right cerebellar hemisphere, and total resection was performed. Histological examination showed bipolar glial cell proliferation in a biphasic pattern in a compact area and a loose microcystic area with Rosenthal fibers and eosinophilic granular bodies, indicating PA. Prominent changes in tumor vasculature, including aggregation of sclerotic thick-walled and ectatic thin-walled vessels, was observed, and nodules of thrombi containing complex vascular proliferation suggesting recanalized thrombi formed in partially ruptured vessels were also found. Thus, rupture of these abnormal vessels appeared to be the cause of hemorrhage. Review of the literature revealed that age distribution of patients with hemorrhagic PAs tends to be older than that of patients with general PAs. These findings imply a possibility that degenerative changes in blood vessels in long-standing PAs might be related to the mechanisms of spontaneous intratumoral hemorrhage.

ABT-869, a Multitargeted Receptor Tyrosine Kinase Inhibitor, Reduces Tumor Microvascularity and Improves Vascular Wall Integrity in Preclinical Tumor Models

N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N1-(2-fluoro-5-methyl phenyl)-urea (ABT-869) is a novel multitargeted receptor tyrosine kinase inhibitor that demonstrates single-agent activity in preclinical studies and has undergone phase I and II clinical trials. We characterized the mechanism of action of ABT-869 by examining vascular changes after treatment (25 mg/kg per day) in HT1080 fibrosarcoma and SW620 colon carcinoma cells, using immunohistochemistry, dynamic contrast enhanced-magnetic resonance imaging (DCE-MRI), and hypoxic protein detection. We observed the inhibition of vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor β phosphorylation in both tumors and changes in tumor vasculature. Reductions in microvessel density and diameter were observed. Vascular-wall integrity was assessed by colocalization of pericytes and basement membrane. Although both microvessel density and total number of pericytes decreased with treatment, the percentage of pericyte coverage on remaining vessels significantly increased. These data suggest the selective ablation of microvessels lacking pericyte coverage. Functional vascular measures DCE-MRI and hypoxia formation were also tested. After 2 days of treatment on the HT1080 model, vascular permeability, K(trans), was reduced by >60% and hypoxic tumor fraction was significantly decreased, which was also seen in the SW620 tumors after 4 days of treatment. Taken together, decreases in vascular permeability and changes in vascular integrity observed in these studies define the mode of action of ABT-869 and may aid in optimizing the timing of therapeutic window for combination therapies.

A phase II study of neoadjuvant bevacizumab and radiation therapy for resectable soft tissue sarcomas.

10023 Background: Numerous preclinical studies demonstrate that anti-angiogenic agents increase the efficacy of radiation therapy (RT) against solid tumors. However, there have been no clinical studies of bevacizumab (BV) and RT (without chemotherapy) examining efficacy and biological effects. Methods: 20 pts with >5 cm, intermediate- or high-grade soft tissue sarcomas at increased risk of local recurrence (LR) received neoadjuvant BV and RT. Blood and tumor tissue were collected before treatment, after BV alone (for 2 wks), and after BV plus RT (for 6 wks). Serial perfusion CT scans assessed changes in tumor vasculature. Tumors were resected 6-7 wks following BV plus RT. Results: 13 extremity, 1 trunk, and 6 retroperitoneal tumors of varied histological subtypes were treated, and median tumor size was 8.25 cm. During treatment, there were no grade 4 toxicities, and 3 pts (15%) had grade 3 toxicities (hypertension, LFT elevation). Following surgery, 4 pts (20%) had major wound complications and one had a pulmonary embolism. Neoadjuvant BV plus RT resulted in 9 of 20 tumors (45%) with ≥80% necrosis, which is over double the historical rate with radiation alone. 3 pts had no viable tumor remaining. Microvessel density decreased from a mean of 20.7 to 9.7 (p=0.01) after BV alone and remained decreased following combination therapy (7.4). Following BV plus RT, tumor cell proliferation by PCNA immunohistochemistry decreased 53% (p=0.01), apoptosis by TUNEL immunohistochemistry increased 3.7 fold (p=0.02), and blood volume and permeability by perfusion CT decreased 63-64% (p<0.05). Oligonucleotide microarray analysis of untreated tumors revealed global differences in gene expression between responders and non- responders as well as an 8-gene signature that highly correlated with treatment response. Analysis of circulating proteins and cells is pending. After a median follow-up of 20 months, there was one local recurrence (5%). All pts declined adjuvant chemotherapy, and 7 pts (35%) developed metastatic disease. Conclusions: Bevacizumab appears to increase the efficacy of radiation therapy against soft tissue sarcomas and may reduce local recurrence. Gene signatures may be useful as biomarkers to predict response. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca, Dyax, Millennium, SynDevRx SynDevRx Pfizer AstraZeneca, Dyax

Sonographic Assessment of Tumor Response

The advent of antiangiogenic drugs in cancer therapy necessitates an imaging modality that can longitudinally assess posttreatment changes in tumor vasculature. In this regard, microbubble contrast-enhanced ultrasonography (CEUS) offers several advantages over conventional imaging modalities. The small size of microbubbles (approximately 2-3 mum) permits their retention in the intravascular compartment and travel through the tortuous tumor vasculature. Mathematical models applied to signal intensity versus time depicting the kinetics of microbubble flow through the tumor are used to characterize tumor vascular density, blood flow velocity, and perfusion. In vivo studies using CEUS have demonstrated its comparability to dynamic contrast-enhanced magnetic resonance imaging and fluorodeoxyglucose positron emission tomography in distinguishing between diseased or malignant tissue and normal tissue. Moreover, CEUS has great potential for other novel clinical applications such as improved cancer diagnosis, enhanced medication delivery, and early antiangiogenic cancer treatment response evaluation. This review discusses the principles and potential clinical applications of CEUS in determining tumor response and its promising role in enhancing medication delivery in certain tumors.

Assessment of Vasculature of Meningiomas and the Effects of Embolization with Intra-arterial MR Perfusion Imaging: A Feasibility Study

Embolization of meningiomas has emerged as a preoperative adjuvant therapy that has proved effective in mitigating blood loss during surgical resection. Arterial supply to these tumors is typically identified by diffuse areas of parenchymal staining after selective x-ray angiograms. We investigate the benefits that selective injection of MR contrast may have in identifying vascular territories and determining the effects of embolization therapy. Selective intra-arterial (IA) injection of dilute MR contrast media was used to assess the vascular distribution territories of meningeal tumors before and after embolization therapy. Regions of the tumor that experienced loss of signal intensity after localized contrast injections into the external and common carotid as well as vertebral arteries were used to quantify the specific vessel's volume of distribution. Assessments were made before and after embolization to reveal changes in the vascular supply of the tumor. MR findings were compared with radiographic evaluation of tumor vascular supply on the basis of conventional x-ray angiography. MR proved to be an excellent means to assess tissue fed by selected arteries and clearly demonstrated the treated and untreated portions of the neoplasm after therapy. In some instances, MR revealed postembolization residual enhancement of the tumor that was difficult to appreciate on x-ray angiograms. Very low contrast dose was necessary, which made repeated assessment during therapy practical. MR perfusion imaging with selective IA injection of dilute contrast can reveal the distribution territory of vessels. Changes in tumor vasculature could be detected after embolization, which reveal the volumetric fraction of the tumor affected by the therapy.

Assessment of the morphological and functional effects of the anti‐angiogenic agent SU11657 on 9L gliosarcoma vasculature using dynamic susceptibility contrast MRI

To investigate the influence of anti-angiogenic agents on tumor perfusion, we employed a dynamic susceptibility contrast (DSC)-MRI method that utilizes a simultaneous gradient-echo (GE) and spin-echo (SE) imaging sequence to derive perfusion parameters (blood flow, blood volume, and mean transit time (MTT)). These parameters are sensitive to both the total vasculature (from the GE data) and the microvasculature (from the SE data), and can also provide a measure of the mean vessel diameter (mVD). This approach was used to evaluate the response of a 9L rat brain tumor model to 20 mg/kg and 40 mg/kg of the anti-angiogenic agent SU11657. The 20-mg/kg dose significantly decreased mVD by 29.9% (P = 0.02). The 40-mg/kg dose significantly decreased mVD by 30.4% (P = 0.0007), SE blood volume by 31.8% (P = 0.03), GE and SE MTT by 46.9% (P = 0.03) and 62.0% (P = 0.0005), and increased GE and SE blood flow by 36.6% (P = 0.04) and 52.6% (P = 0.02). These findings demonstrate that DSC-MRI perfusion methods can play a key role in the noninvasive evaluation of morphological and functional changes in tumor vasculature in response to therapy.