Michele Lamuraglia

Clinical relevance of contrast-enhanced ultrasound in monitoring anti-angiogenic therapy of cancer: Current status and perspectives

Angiogenesis regulation is one of the newest fronts in the fight against cancer. Anti-angiogenic therapy is based on inhibiting factors required to solicit vessel formation thus cutting-off the tumors supply of nutriments and oxygen. Initial vascular response is followed by formation of necrosis. Volumetric regression occurs more tardively. Effective monitoring of this new therapeutic approach thus requires imaging techniques that can detect early microvascular changes. A number of clinical studies provide evidence that contrast-enhanced ultrasound (CEUS) can provide early indication of tumor response to anti-angiogenic therapy. More sophisticated imaging and analysis techniques for CEUS and contrast agents targeted for adhesion to anti-angiogenic markers have also demonstrated promise in animal model studies. This review underlines the relevance of CEUS for anti-angiogenic therapy monitoring by summarizing the current clinical results, emerging CEUS techniques and preclinical data.

VEGFR2-TARGETED CONTRAST-ENHANCED ULTRASOUND TO DISTINGUISH BETWEEN TWO ANTI-ANGIOGENIC TREATMENTS

The aim of this study was to evaluate the capacity of BR55, an ultrasound contrast agent specifically targeting vascular endothelial growth factor receptor 2 (VEGFR2), to distinguish the specific anti-VEGFR2 therapy effect of sunitinib from other anti-angiogenic effects of a therapy (imatinib) that does not directly inhibit VEGFR2. Sunitinib, imatinib and placebo were administered daily for 11 d (264 h) to 45 BalbC mice bearing ectopic CT26 murine colorectal carcinomas. During the course of therapy, B-mode ultrasound, contrast-enhanced ultrasound and VEGFR2-targeted contrast-enhanced ultrasound were performed to assess tumor morphology, vascularization and VEGFR2 expression, respectively. The angiogenic effects on these three aspects were characterized using tumor volume, contrast-enhanced area and differential targeted enhancement. Necrosis, microvasculature and expression of VEGFR2 were also determined by histology and immunostaining. B-Mode imaging revealed that tumor growth was significantly decreased in sunitinib-treated mice at day 11 (p < 0.05), whereas imatinib did not affect growth. Functional evaluation revealed that the contrast-enhanced area decreased significantly (p < 0.02) and by similar amounts under both anti-angiogenic treatments by day 8 (192 h): -23% for imatinib and -21% for sunitinib. No significant decrease was observed in the placebo group. Targeted contrast-enhanced imaging revealed lower differential targeted enhancement, that is, lower levels of VEGFR2 expression, in sunitinib-treated mice relative to placebo-treated mice from 24 h (p < 0.05) and relative to both placebo- and imatinib-treated mice from 48 h (p < 0.05). Histologic assessment of tumors after the final imaging indicated that necrotic area was significantly higher for the sunitinib group (21%) than for the placebo (8%, p < 0.001) and imatinib (11%, p < 0.05) groups. VEGFR2-targeted ultrasound was able to sensitively differentiate the anti-VEGFR2 effect from the reduced area of tumor with functional flow produced by both anti-angiogenic agents. BR55 molecular imaging was, thus, able both to detect early therapeutic response to sunitinib in CT26 tumors as soon as 24 h after the beginning of the treatment and to provide early discrimination (48 h) between tumor response during anti-angiogenic therapy targeting VEGFR2 expression and response during anti-angiogenic therapy not directly acting on this receptor.

Abstract 1497: In vivo discrimination of tumor modifications during antiangiogenic and cytotoxic therapy using ultrasonography modalities: Shear Wave Elastography (SWE), Contrast Enhanced Ultrasound (CEUS) and Quantitative Ultrasound (QUS):

Robust, novel image-based information on the tumor microenvironment would improve therapeutic follow-up in oncology. SWE evaluates tissue stiffness, CEUS assesses microvascular flow using intravascular microbubbles and QUS measures tissue microstructure. Our goal was to evaluate sensitivity and complementarity of these techniques during cytotoxic and antiangiogenic therapies in ectopic, murine tumors. A multi-targeted receptor tyrosine kinase antiangiogenic drugs inhibiting vascularization (sunitinib, N = 24, 40mg/kg/day), an alkylating cytotoxic agent that adds an alkyl group to DNA to interfere with DNA replication (cyclophosphamide, N = 26, 150mg/kg for 3 days) and placebo (N = 26) were administered in murine Lewis Lung Carcinomas bearing C57BL6 mice. Six days after implantation, tumors were imaged (SWE, CEUS and QUS) prior to therapy (Day 0) and on Days 1, 3, 7, 9 and 13. Tumors were harvested at Day 9 (N = 2 per group) and Day 13 (N = 10 per group) and stained to evaluate% necrosis and fibrosis. Table. 1 summarizes the% variation of each measured parameter from the start to the end of therapy. Imaging parameters for microvascular function and stiffness were most strongly modified for tumors treated with sunitinib compared to cytotoxic and placebo therapies. At Day 13, tumors treated with sunitinib presented a heterogeneous structure with more fibrosis and necrosis than for other treatment groups. This heterogeneous structure may explain why the standard deviation of stiffness was highest for the antiangiogenic group. The sensitivity of CEUS to microvascular modifications and SWE to biomarkers such as necrosis and fibrosis, in this model, underline potential for monitoring complementarity aspects of the tumor microenvironment during therapy. Table 1: Variation between Day 0 and Day 13 of mean values of each parameter for each group. Citation Format: Alexandre Dizeux, Thomas Payen, Guillaume Barrois, Michele Lamuraglia, Capucine Baldini, Delphine Le Guillou, Eva Comperat, Jean-Luc Gennisson, Mickael Tanter, Michael Oelze, S. Lori Bridal. In vivo discrimination of tumor modifications during antiangiogenic and cytotoxic therapy using ultrasonography modalities: Shear Wave Elastography (SWE), Contrast Enhanced Ultrasound (CEUS) and Quantitative Ultrasound (QUS). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1497. doi:10.1158/1538-7445.AM2015-1497

Abstract 2665: Dynamic contrast-enhanced ultrasound monitoring of tumor perfusion in a murine pancreatic tumor model to assess the effect of (ziv)-aflibercept and sorafenib, two anti-angiogenic drugs.

Background: anti-angiogenic drug combinations provide a promising new therapeutic approach. Noninvasive monitoring techniques would help to choose drug based on patient response. The effects on tumor perfusion by combining Ziv-aflibercept (VEGF-Trap is being developed as a part of a collaboration between Sanofi and Regeneron Pharmaceuticals, Inc.) and Sorafenib (Nexavar TM , Bayer Schering Pharma) was tested. Methods: pancreatic adenocarcinomas were induced by subcutaneous injection of 100 μl containing 1 x 10 6 MIA PaCa2 cells in the left flank of 50 female, 2-4 week-old, nude mice (Naval Medical Research Institute [NMRI] Elevage Janvier, Le Genest-St-Isle, France). Dynamic contrast-enhanced ultrasound (DCE-US) was performed using an Aplio 50 ultrasound imaging system with a 12-MHz probe (PLT 1202 S, TOSHIBA, Tokyo, Japan). DCE-US data sequences were acquired after intravenous injection of a 100 μl bolus of Luminity contrast agent. Data were acquired from each mouse on days 15, 16 and 24 after tumor cell injection. Treatment was started on day 15 just after DCE-US baseline scan. Fifty mice were included in this study: 15 controls received placebo, 11 mice were treated with sorafenib (60 mg/kg/day by gavage), 12 mice were treated with ziv-aflibercept (40 mg/kg 2 twice a week by injection) and 12 mice were treated with the combination of the two anti-angiogenic drugs. Image sequences were recorded in raw data format and extracted with TOSHIBA software (CHIQ) allowing calculation of the linear time-intensity curve in regions of interest within the tumor. Using in-house software, the time-intensity bolus curve was fit to a lognormal model from which the Area Under the Curve (AUC) was calculated to assess microvascular perfusion in the tumor. Statistical analysis was performed using Wilcoxon Signed-Rank tests. Results: the AUC decreased significantly from day 15 to day 16 for the group receiving ziv-aflibercept (p=0,020) and for the group receiving the combination of ziv-aflibercept and sorafenib (p=0,005), while no significant differences were found for the placebo or sorafenib monotherapy groups. From day 15 to day 24, the AUC obtained from the fit to the bolus curve decreased significantly for the group receiving the combination of ziv-aflibercept and sorafenib (p=0,031), while no significant differences were found for the three other groups. Conclusions: the DCE-US functional evaluation used in this study demonstrated that the association of the combination of ziv-aflibercept and sorafenib and ziv-aflibercept alone reduced functional perfusion in the tumor 24 hours after therapy onset. The long term (9 days) perfusion reduction was maintained for combined therapy but was not significant for placebo, ziv-aflibercept or sorafenib mono-therapy. Citation Format: Michele Lamuraglia, Guillaume Barrois, Mathieu Santin, Delphine Le Guillou-Buffello, Lori S. Bridal, Olivier Lucidarme. Dynamic contrast-enhanced ultrasound monitoring of tumor perfusion in a murine pancreatic tumor model to assess the effect of (ziv)-aflibercept and sorafenib, two anti-angiogenic drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2665. doi:10.1158/1538-7445.AM2013-2665

Abstract 2428: Echo-contrast monitoring of sorafenib early efficacy: animal model for Ewing's sarcoma tumors

Background: Anti-angiogenic drugs are a pillar of the therapeutic approach against cancer. Contrast ultrasound (CUS) can detect perfusion modifications during anti-angiogenic therapy preceding tumor size regression. However, contrast imaging would benefit from better standardization of the technique. This work compares discrimination of tumors in Sorafenib-treated and placebo mice using CUS evaluation of relative blood volume (rBV), mean transit time (mTT) and wash in rate (WiR). Methods: Ewing9s sarcoma tumors were grafted orthotopically by sub-capsular injection of 100 µL of SK-NEP-1 cells (5 x 10 6 cells/mL) in the left kidney of nude fifteen mice (Elevage Janvier, Le Genest-St-Isle, France). Contrast enhanced ultrasonography was performed using a clinical ultrasound system Acuson Sequoia 512 with a 14 MHz probe (Siemens Medical Solutions, Mountain View). The tumor area (A) was calculated according to A = (≥ x L x w)/2. CUS (Sonovue, Bracco SA, Milan, Italy) with destruction-reperfusion was performed using cadence pulse-sequence (CPS). Data were acquired from each mouse at 14 days after tumor-cell injection. The treatment was started at day 14 just after the CUS baseline. At day 14 (day of start therapy) the 12 mice were randomized into two groups. One group received (6 mice) the anti-angiogenic treatment Sorafenib daily (10mg/kg/jr) per os and the other group (6 mice) received corresponding doses of placebo by the same route for 8 days. Image sequences were recorded in DICOM format and analyzed with prototype software from Bracco Suisse SA (Geneva, Switzerland) allowing selection of regions of interest, approximate linearization of the data and fitting of flow models to time-intensity data to estimate functional parameters within the tumor. All parameters were normalized with respect to normal cortex perfusion. Statistical analysis was performed using Wilcoxon Signed-Rank tests. Results: Ultrasound estimates of the tumor size increased with time for both groups, but tumor size was never significantly different between the two groups. Before the normalization with the adjacent renal cortex perfusion, the functional parameters did not present a significant difference between the two groups. Therefore after normalization: the rBV shows a significant difference Sorafenib vs placebo (p=0.008) at 8 days, the mTT also becomes significant after 8 days of treatment (p=0.02). The WiR shows a significant difference between groups at 8 days (p=0.005), but also at 6 days (p=0.04). Conclusions: functional CUS was an effective technique for monitoring the early response of anti-angiogenic treatment. The parameters obtained by fitting flow models of destruction-reperfusion demonstrated the capacity to standardize this angiogenesis analysis. Also the normalization with respect to adjacent normal tissue appears to reduce estimate variability and improve functional contrast assessment. 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 2428. doi:1538-7445.AM2012-2428