Ultrasound Imaging of Cancer Therapy

C H A P T E R

8 Ultrasound Imaging of Cancer Therapy Dorde Komljenovic and Tobias Ba¨uerle,† 

Department of Medical Physics in Radiology, German Cancer Research Center, Heidelberg, Germany University Hospital Erlangen, Institute of Radiology, Maximiliansplatz 1, 91054 Erlangen, Germany

†

O U T L I N E Introduction Ultrasound Physics Ultrasound in Cancer Research and Treatment

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Ultrasound Imaging Concepts Technical Considerations Microbubble-Based Contrast Agents Nonmicrobubble-Based Contrast Agents Contrast Agent Applications

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Assessment of Cancer Therapy Experimental Applications Nontargeted Microbubble-Based Ultrasound Targeted Microbubble-Based Molecular Ultrasound

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Conclusion

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Acknowledgments

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Abbreviations

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Glossary

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References

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INTRODUCTION Biomedical imaging is pivotal for the assessment of treatment response in the clinical setting. Besides the assessment of clinical presentation of the patients and acquisition of serum markers, imaging modalities including conventional X-rays, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), and ultrasound (US) are most frequently used to determine response to therapies. Among these, ultrasound has many advantages over other modalities including its low costs and broad availability, portability, not exposing patients to ionizing radiation, and its minimally invasive, highresolution real-time view. Ultrasound scans are, on the other hand, highly dependent on the expertise of the observer. Furthermore, there are tissues and organs

X. Chen and S. Wong (Eds): Cancer Theranostics. DOI: http://dx.doi.org/10.1016/B978-0-12-407722-5.00008-6

Clinical Applications Nontargeted Microbubble-Based Ultrasound Targeted Microbubble-Based Molecular Ultrasound

that are characterized by poor ultrasound propagation (e.g., air-containing organs such as lungs and bowel) that disqualifies them for ultrasound examination. Sonography is an established method in cancer imaging, in particular for screening examinations, for indications in the urogenital system, and for detection of asymptomatic tumors. Together with X-ray examinations of the thoracic cavity, ultrasonography is the most common imaging modality to assess the tumor burden and its response to therapy [1]. Cancer therapy includes local and systemic approaches such as surgery and radiation as well as chemotherapy, hormonal treatments, and targeted therapy. According to the standardized systems to assess response to treatment in oncological patients by the World Health Organization (WHO) [2] and the International Union Against Cancer (UICC) [3] as well as the recently updated Response Evaluation Criteria

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in Solid Tumors (RECIST) [4,5], the respective lesions are classified based on morphology only. Obviously, the lesion size is the most important criterion to classify response as “complete response,” “partial response,” “stable disease,” and “progressive disease.” However, functional and molecular imaging data that give insight into pathophysiologic structures and processes in tumors such as parameters of vascularization or the expression of tumor-specific epitopes are currently not included in these classification systems. Sonography covers a broad imaging spectrum that ranges from morphological to functional and molecular imaging in high spatial and temporal resolution. Besides acquiring tumor morphology with high softtissue contrast, unenhanced and contrast-enhanced methods are applicable for quantification of functional parameters of vascularization. This chapter describes some basic principles of ultrasound techniques for treatment response assessment in tumors and focuses on novel methods exploiting the potential of ultrasound for acquiring information on the morphological, functional, and molecular level for this purpose. Ultrasound-mediated delivery of therapeutics to the tumor tissue is in the scope of Chapter 16 “Ultrasound for Gene/Drug Delivery” and will not be covered here.

Ultrasound Physics First attempts to implement ultrasound into clinics date back to the first half of the last century by using hyperphonography to visualize brain ventricles [6]. Modern ultrasound devices recognize echoes from the tissue and/or medium after the emission of an ultrasound pulse characterized by a specific frequency. Approximately 1% of the ultrasound waves emitted to the tissue are reflected directly back to the transducer. Sound waves travel effectively through soft body tissues and water since they require a support medium. On the other hand, ultrasound cannot penetrate airfilled organs, due the total reflected of the sound waves at tissue-air interfaces. Besides reflection, physical processes that affect ultrasound imaging include scattering, refraction, attenuation, and absorption. Clinical ultrasound imaging is enabled by the fact that each tissue in the human body has a specific resistance to sound, a phenomenon commonly referred to as acoustic impedance, which is, for a plane wave, a product of the speed of sound propagation and the density of the medium it is traveling through. Frequency emission of common ultrasound devices used in clinics and preclinical research is typically in the megahertz (MHz) range (human ear recognizes sounds between 20 Hz and 20 kHz). It is the interface between tissues of different acoustic impedances

(rather than the tissue itself) that gives contrast on an ultrasound image. While some ultrasound beams are reflected at the acoustic interface, some are refracted, meaning that the waves change the direction after passing through the interface. When the acoustic interface is in perpendicular position to the beam of the ultrasound, a larger part of the echo pulses are reflected back to the transducer, which results in the bright signal on the image, contrary to less bright signal obtained when the interface conforms to a less perpendicular position to the sound beam. However, interface between tissues of different acoustic impedances is only rarely perpendicular to the sound beam in clinical applications, but rather irregular or rough [6], which induces backscattering of echoes rather than reflection. As a consequence, a proportion of the energy backscattered to the transducer and used to generate ultrasound image is smaller than the energy originally emitted to the tissue. It is due to scattering that parenchymal organs appear to have characteristic echo structure in ultrasound images [6,7]. Attenuation accounts for the decrease of the sound wave amplitude as it propagates through the tissue and/or medium, due to various mechanisms, including absorption (conversion of sound energy into heat) and scattering. In B-mode scanning (also known as brightness mode), a two-dimensional image is obtained, with echoes reflected from interfaces between tissues of different acoustic impedance registered as bright spots. The brightness of each spot represents the amplitude of the returned echo in a way that a given brightness value (gray scale) is attributed to a certain amplitude value (for example, amplitude value of 0 corresponds to black color whereas amplitude value of 255 corresponds to white color). B-mode is used to visualize anatomical structures and to guide/visualize diagnostic and therapeutic procedures. In addition to morphologic imaging, Doppler sonography allows for the detection and quantification of motion, e.g. the blood flow. When the transmitted wave is reflected or backscattered by a moving object, e.g. a red blood cell, the reflected frequency is shifted compared to the insonated wave. This Doppler shift can be detected and the motion can be analyzed, e.g. for the assessment of blood flow velocity and vascularity of physiological or pathological elements.

Ultrasound in Cancer Research and Treatment Overall, most extracranial and extraosseous locations are accessible for ultrasound evaluation. Standard applications in oncological patients are the evaluation

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of suspected lymph nodes and parenchymatous organs of the abdomen and pelvis for detection and followup. Thereby, noncontrast-enhanced B-mode imaging allows the acquisition of soft-tissue contrast differences between organ stroma and tumor tissue or Doppler techniques facilitate evaluation of perfusion in these locations. Although current imaging diagnosis is based on primarily morphologic aspects of tumors, functional and molecular information in particular of tumor vasculature has been of increasing interest to derive quantitative parameters in cancerous tissue by ultrasound. Angiogenesis is one of the hallmarks of cancer as defined by Hanahan and Weinberg [8] and describes the formation of tumor-associated vasculature, which is a critical and rate-limiting determinant of tumor growth beyond a lesion size of approximately 0.2 mm [9]. The result of this angiogenic activity of tumors is a heterogeneous tumor vasculature consisting of tortuous and immature smaller vessels that might be leaky as well as more mature and larger vessels enabling the delivery of a considerable amount of blood in highly proliferative and fast-growing tumors. Obviously, such characteristics influence functional parameters (e.g., perfusion, permeability, blood volume, filling time, etc.) when compared to the regular architecture of nontumor vessels under physiologic conditions. Another difference between normal and angiogenic vessels is the difference of endothelial cell epitopes that are predominantly exposed by tumor vasculature. Thus, tumor-associated endothelial cells express characteristic factors such as the transmembrane proteins vascular endothelial growth factor receptor (VEGFR) or integrins, in particular αvβ3 and αvβ5 integrin subclasses. For treatment, angiogenesis represents an attractive target in cancer as compared to the ever-changing cancer cells concerning their expression profile, even in the same patient over time. Inhibitors of angiogenesis aim to block key players in the cascade such as VEGF (e.g., anti-VEGF antibody bevacizumab) and VEGFR (e.g., tyrosine kinase inhibitor sunitinib) resulting in vessel remodeling, vessel rarefaction, and finally in decreased tumor perfusion. Interestingly, not only antiangiogenic drugs reduce aspects of tumor vascularization, but also chemotherapy (e.g., cytotoxic paclitaxel) or antiresorptive agents (e.g., bisphosphonate zoledronic acid) [10,11]. Using noncontrast-enhanced (Doppler) and contrast-enhanced techniques, ultrasound enables quantitative and semiquantitative assessment of tumor vascularization including functional parameters (e.g., blood volume, filling time, and transit time) for detection and follow-up of cancer lesions. For the latter, changes in vascularization are relatively fast as compared to an increase or decrease

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of lesion size that might be hampered by therapyinduced effects such as cell swelling, necrosis, or inflammation, which are often not visible on B-mode images only.

ULTRASOUND IMAGING CONCEPTS In recent years there has been extensive research on targeted, antiangiogenic antitumor therapy, resulting in considerable improvement of diagnostic modalities to assess the effects of such therapies. Use of ultrasound as a molecular imaging tool was enabled by the introduction of microbubble-based contrast agents, which can be functionalized by the use of ligands that specifically and with high affinity bind to tumor markers of interest expressed either directly on tumor cells or on structures in their environment. Introduction of gas-filled microbubbles significantly improves sensitivity and specificity of ultrasonography, in particular in abdominal pathology (e.g., focal liver lesions) where it serves as an imaging modality of choice [1]. A distinct advantage of microbubbles is their nonlinear response after insonation, which is used to suppress the background signal and improve the detection of specific signals. Due to their size, hemodynamic properties of microbubbles mimic those of erythrocytes, a feature that enables their extensive use in preclinical studies focused on investigation of tumor blood vessels. Microbubble-based ultrasonography not only serves as an optimal modality to assess the distribution volume of a vascular bed of the tissue of interest such as tumor but also to quantitatively assess molecular markers on the inner vascular wall after the specific molecular targeting of microbubbles [12]. Notably, when it comes to quantification of a signal within a voxel, in clinical settings the concentration of microbubbles may be underestimated, due to the fact that the slice thickness exceeds the size of one microbubble/voxel. This is of particular importance during quantification of signal from areas with locally increased microbubble accumulation. In an attempt to overcome this drawback, a sensitive particle acoustic quantification (SPAQ) concept was developed [13,14], which aims to detect single microbubbles by a volumetric scan of the object with thin slices, for a more precise quantification of microbubbles.

Technical Considerations According to their size, contrast agents used for ultrasound imaging may be grouped into microbubbleand nanobubble-based contrast agents.

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Microbubble-Based Contrast Agents Microbubbles are gas-liquid emulsions, consisting of a gaseous core (e.g., perfluorocarbon, sulfur hexafluoride, or nitrogen) surrounded by a shell of biocompatible properties (e.g., made of albumin, galactose, lipids, or polymers) and usually of a diameter between 1 and 4 μm (Figure 8.1). The safety profile of such gas-filled, encapsulated microbubbles is, according to current knowledge, not fully understood, yet the incidence of side effects remains low with minor and major adverse event rates of 0.13% and 0.0086%, respectively (minor adverse events include dizziness, erythematous rash, itching, nausea, and vomiting; major adverse events include dyspnea, bronchospasm, hypotension, bradycardia, cutaneous rash, dorsolumbar pain, and clouding of consciousness) [15,16]. Microbubble-based agents are injectable intravenously and, due to their size, pass the pulmonary capillaries. Microbubblebased ultrasound contrast agents are isotonic to human plasma [17] and dissolve after intravenous injection, producing remnants that are readily metabolized and cleared, with minimized danger to form emboli. The half-life of microbubbles in the vascular compartment is typically 3 to 5 minutes after intravenous injection. It is due to compressibility of microbubbles and elastic properties of their shell that they oscillate in a nonlinear fashion after insonation. A physical phenomenon of particular importance for the acquisition of signal obtained using ultrasound contrast agents is scattering, as previously mentioned [18]. One of the main reasons for scattering of ultrasound after insonation of microbubble-containing tissues is indeed the large difference of acoustic impedance of gas in the core of a microbubble on one side and the surrounding

media on the other side. The possibility of engineering versatile shells and combining them with different gases as a core enables the production of various classes of microbubbles [15]. The advantage of such contrast agents is that the gaseous core induces a high echogenic response after insonation, which results in improved contrast-to-background tissue ratio [12,15]. Nonlinear scattering of sound waves results in strong signals at frequencies twice (second harmonic) or half (subharmonic) the input frequency [18]. This enables filtering of the frequencies of the nonlinear and linear oscillations to sensitively distinguish specific signals originating from the microbubbles and from the suppressed background tissue signal, respectively. Nowadays, ultrasound imaging techniques are engineered to be able to preferentially detect such microbubble-originating, nonlinear events. Formation of emboli after the intravenous injection of microbubbles is prevented by the properties of the shell itself, and additional protection may be achieved by the insertion of arms of polyethylene glycol (PEG) polymer onto the shell [12]. Chemical properties of the shell allow incorporation of molecules that may react with target structures on the endothelial cell (e.g., in tumor blood vessels). Nonmicrobubble-Based Contrast Agents Nonmicrobubble-based contrast agents, submicron or nanosized, are small enough to leave blood vessels and may be employed to assess structures in extravascular space and therefore image targets inaccessible to microbubbles. Nanobubbles remain, however, poorly detectable by the ultrasound for their low acoustic reflectivity due to their size and the fact that these

FIGURE 8.1

Different types of ultrasound contrast agents. a) Microbubbles are gas-liquid emulsions with a polyethylene gycol (PEG) polymer on the surface to prevent aggregation. b) Perfluorocarbon emulsion (PFC) nanodroplets are liquid-liquid emulsions that can be vaporized into echogenic gas-bubbles following administration of acoustic energy. c) Liposomes are phospholipid bilayers that can enclose air pockets for ultrasound imaging. d) Nanobubbles are gas-liquid emulsions that can fuse into echogenic microbubbles at the target site. e) Solid nanoparticles are solid amorphous substances with gas entrapped in their pores or fissures increasing echogenicity. Source: Reprinted from [15].

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compounds have relatively incompressible core. Different types of nanosized contrast agents are now synthesized (Figure 8.1), including perfluorocarbon (PFC) nanodroplets of a size of 200 to 400 nm and polylactic acid-stabilized nanobubbles of a size of 40 of 200 nm [15]. Efforts were made to test whether solid nanoparticles could be employed as ultrasound contrast agents. A study by Liu et al. [19] investigated the applicability of different nanoparticles including silica (100 nm) and polystyrene nanoparticles (500 3000 nm) for B-mode contrast enhancement, and reported correlation of both the size and the concentration of these particles with B-mode contrast enhancement.

Contrast Agent Applications Relatively simple use of ultrasound devices and a good safety profile of ultrasound contrast agents has enabled extensive application of the method in experimental conditions, focusing on disease processes that affect vascular compartment including angiogenesis, inflammation, and thrombus formation. Following encouraging preclinical results, microbubble-based diagnostics have also been seen in clinics in recent years. After the notion that the angiogenic switch [20] occurs in a variety of solid tumors as they grow in size, tumor blood vessels were set in the focus of anticancer research, resulting in the identification of various molecular markers over-expressed on tumor endothelium [21]. An approach based on targeted microbubbles enabled the monitoring of the expression of such markers on the activated endothelium and, as such, was extensively applied to assess the tumor angiogenesis and inflammation in preclinical models. BR55 (Bracco) is a VEGFR2-targeted ultrasound contrast agent for molecular imaging of angiogenesis that is currently in clinical study to test its efficacy in identification of prostate cancers based on their over-expression of VEGFR2 [21]. More detailed insight into experimental applications of microbubble-based contrast agents in cancer therapy is given in the next section. Inflammation is often found as a pathophysiological process in a variety of diseases. Molecular ultrasound may be beneficial for early diagnosis and assessment of treatment response in diseases characterized by the inflammation since one of the major features of inflammation is activation of leukocytes in blood and their transmigration in the extracellular space. This process is largely influenced by the interaction of adhesion molecules on leukocytes and different factors expressed by the endothelial cells. Targets identified to take part in transmigration of leukocytes include E- and P-selectin, and adhesion molecules ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and

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vascular cell adhesion molecule-1, respectively). These inflammatory markers have been targeted by microbubbles enabling the quantification of inflammation in different organs and organ systems. In a study employing P-selectin-targeted contrast echocardiography on ischemia-reperfusion rat model, recent ischemic areas in the myocardium without infarction could be identified [22]. This finding may be an opportunity to rapidly detect ischemia in patients presenting with atypical chest pain. The same group reported the feasibility of contrast-enhanced molecular imaging of VCAM-1 to quantify vascular inflammatory changes during atherosclerosis [23]. Molecular ultrasound has a great potential in the imaging of thrombus formation, although this approach is still at its early stage. Schumann et al. [24] reported targeted-microbubble binding to GPIIb/IIIa receptors (also known as integrin αIIbβ3) of platelet thrombi. Noninvasive imaging of intravascular thrombi by employing molecular ultrasound using microbubbles targeted to components of blood coagulation may provide information valuable for treatment of patients with stroke, but also of those considered to be at high risk for cerebral embolic disease [15,24].

ASSESSMENT OF CANCER THERAPY Relying predominantly on the observation of morphological changes of a tumor during treatment is insufficient to assess complex effects that antitumor therapies often have, in particular during earlier stages of the treatment. Some reasons for inadequacy of the therapy assessment based only on morphology are that anatomical changes very often occur rather slowly and that volumetric morphologic measurements in clinical settings are often observer-dependent and affected by irregular shape and heterogeneous appearance of tumors. Currently, extensive research is undertaken to identify parameters that may predict an outcome of the antitumor therapy, especially its early effects, when it may be possible to identify nonresponders and subject them, if available, to the other therapeutic options. As mentioned in the introduction to this chapter, sonography is a valuable tool to accomplish these tasks as it enables acquisition of morphological as well as functional data in high resolution; from the volumetric quantification of a tumor as a whole, to the use of nontargeted as well as targeted microbubbles that are used for the assessment of specific molecular markers. Furthermore, ultrasound enables assessment of tissue mechanical properties by ultrasound elastography as well as blood flow velocities by Doppler techniques.

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Experimental Applications Ultrasound is one of the imaging methods well suited to obtaining basic information about experimentally induced tumors such as their size or volume. In a majority of cases, unenhanced, B-mode ultrasound imaging would suffice to accomplish this task. By using nontargeted microbubbles in a dynamic contrast mode, ultrasound allows for quantification of functional vascular parameters. Finally, use of targeted ultrasound enables direct or indirect monitoring of the spatiotemporal distribution of molecular or cellular processes for diagnostic applications [25]. Nontargeted Microbubble-Based Ultrasound Application of nontargeted contrast agents may help to further characterize tumors, for example, to assess functional parameters of tumor vasculature as it responds to a therapy. Dynamic contrast-enhanced (DCE) ultrasound enables quantitative assessment of the perfusion in solid tumors and acquisition of functional parameters. A recent study [26] aimed to investigate the feasibility of the contrast-enhanced ultrasound in assessing effects of the antiangiogenic tyrosine kinase inhibitor sunitinib on the vasculature of the soft tissue compartment of experimental breast cancer bone metastases model in nude rats. Nontargeted ultrasound contrast agent was used to perform descriptive analysis of the exported time intensity curves, which enabled assessment of the following

vascular parameters: wash-in rate, wash-out rate, peak enhancement, and area under the curve. A change in overall contrast agent uptake (lower peak enhancement and area-under-the-curve values compared to tumorbearing but untreated, control rats) as well as in washout of the contrast agent from the tumor (lower wash-out values in treated rats compared to the controls) was observed. Using a quantitative analysis software (Qontrast made by the Italian company Bracco), vascular parameters such as regional blood volume, regional blood flow, and filling time could be quantified. Study results suggest DCE ultrasound to be a feasible method for the assessment of effects of antiangiogenic therapy in experimental breast cancer bone metastases (Figure 8.2). In a study utilizing human hepatocellular carcinoma rat model, effects of sunitinib and FAK/Pyk2 tyrosine kinase inhibitor were effectively assessed using contrast-enhanced ultrasound applying MicroMarkers microbubbles (from the Canadian company VisualSonics) [27]. Targeted Microbubble-Based Molecular Ultrasound Increasing popularity of contrast-enhanced ultrasound in a variety of clinical settings is paralleled with preclinical studies that aim to test applicability of molecular ultrasound in diagnostics and treatment, in particular due to the possibility of molecularly targeting specific markers expressed on tumor microvessels. However, many experimental molecular US imaging studies employ streptavidin/biotin interaction for

FIGURE 8.2

Monitoring of sunitinib effects on experimental breast cancer bone metastases (B) compared to tumor-bearing, nontreated control (A) nude rats using ultrasound (metastases encircled white). Ultrasound imaging included B-mode (first row; for metastasis localization) and contrast mode (second row; Sonovues) and CPS (cadence contrast pulse sequence: transmission frequency, 7 MHz; mechanical index (MI), 0.18). Third to fifth row: color maps representing vascular parameters relative blood volume (RBV), relative blood flow (RBF), and filling time (FT). Values for given parameter range from high (h) in red to low (l) in blue. Days: time after the cancer cell injection (Day 30: no treatment; days 32 and 35: 2 and 5 days of treatment, respectively). Source: Reprinted from [26].

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binding the shell with an antibody. Since streptavidin is immunogenic, these microbubbles cannot be applied in humans. Advantages such as broad availability of antibodies and relatively simple conjugation chemistry with microbubbles enabled numerous proof-ofprinciple preclinical studies. Among these, antibody- or peptide-biotin-streptavidin-conjugated microbubbles were employed to image tumor angiogenic markers, including VEGFR2 and integrins [28 30]. Moleculartargeted microbubbles were used to image molecular adhesion molecules overexpressed in microvessels of inflamed tissues, such as mucosal addressin cellular adhesion molecule (MadCAM1) [31], VCAM-1, ICAM-1 [32], and P-selectin [33]. VEGFR2 and αvβ3 integrins emerged as the most studied molecular markers expressed on the endothelial cells of tumor vasculature both to investigate specificities of the activated endothelium and to assess how it responds to applied therapy. Lee and colleagues [34] reported the magnitude of the signal from VEGFR2targeted microbubbles to correlate well with the immunohistochemically quantified VEGFR2 expression level. When using dual-targeted microbubbles against VEGFR2 and αvβ3 integrin, and obtained signal intensity was noticeably higher compared to single-targeted as seen on the model employing angiogenic ovarian cancer xenografts [35]. After treatment with a matrix metalloproteinase (MMP) inhibitor, decreased binding of microbubbles targeted to these markers was noted in subcutaneous human squamous cell xenografts in mice when compared to untreated controls [21,36]. Here, authors reported a general decrease in vessel density and not a decrease in the expression level of a marker on tumor blood vessels, indicating necessity to combine functional and molecular ultrasound to distinguish whether the difference in signal intensity originates indeed from the alterations of a marker expression level. On the mouse model for pancreatic carcinoma, microbubbles targeting the VEGF VEGFR complex, VEGFR2, and endoglin were used to analyze angiogenic activity in xenografts and to monitor the response to antiangiogenic or cytotoxic agents [37].

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well-established indications for this kind of examination (Figure 8.3). A limited number of commercially available ultrasound contrast agents, different from each other with respect to either the gas employed as a core or the chemical properties of their shell, are being used in clinics. Sensitivity and specificity of ultrasound imaging for detection and characterization of pathologic processes increased significantly after the application of microbubble-based contrast agents. Different intravenously injectable microbubble-based ultrasound contrast agents with their gas/shell specifications are presented in Table 8.1 [17]. The commonly used ultrasound contrast agent Sonovues (made by Bracco), considered to be safe and well tolerated both in healthy subjects and in patients with chronic obstructive pulmonary disease of varying severity [15], is commercially available as sulfur hexafluoride-filled microbubbles with flexible phospholipid shell. Prior to the administration, a contrast agent suspension is prepared by adding physiological saline to the lyophilisate powder according to the instructions of the manufacturer and complying to clinical aseptic requirements. DCE ultrasound was reported to be a feasible tool for predicting the early therapeutic effects of sunitinib in metastatic renal cell carcinoma patients [39,40]. An update in 2011 on nonhepatic applications of the EFSUMB Guidelines and Recommendations on the

Clinical Applications As well as being a routinely used morphological technique for certain clinical indications (e.g., first-look examination of the abdomen), the use of microbubbles has broadened the applicability of ultrasound. Nontargeted Microbubble-Based Ultrasound Nontargeted ultrasound contrast agents have been routinely used in a clinical setting for several years. Characterization of focal liver lesions and assessment of renal and portal venous vasculature are

FIGURE 8.3 Ultrasound images of hepatocellular carcinoma (HCC; grade II) in a patient. Grayscale ultrasound image reveals an isoechoic HCC nodule (a). Arterial phase (16 s) shows homogenous hyperenhancement of the nodule (b), whereas portal phase (113 s) (c) and late portal phase (240 s) (d) show less and more pronounced hypoenhancement, respectively, compared to the adjacent liver tissue. Source: Reprinted from [38], with kind permission of Springer Science +Business Media.

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TABLE 8.1 Microbubble-Based Ultrasound Contrast Agents for Intravenous Injection Trademark Name

Code Name

Albunex

Manufacturer

Formulation (Shell/ Filling Gas)

Mallinckrodt

Human albumin/air

Bisphere

PB127

Point Biomedical

Polymer bilayer: albumin/air

Definity

MRX-115

Bristol-Myers Squibb

Phospholipid/ perfluoropropane

Schering

Galactose/air

Cavcon

Lipid/air

DMP-115 Echovist

SH U454

Filmix Imavist (imagent)

AFO150

Imcor Surfactant/ Pharmaceuticals perfluorohexane-air

Levovist

SH U508A

Schering

Galactose-palmitic acid/ air

Myomap

AIP 201

Quadrant Healthcare

Recombinant albumin/air

Optison

FS069

Amersham Health Inc.

Perflutren protein-type A/perfluorobutane

Perflubron

PFOB

Alliance Perfluorooctyl bromide Pharmaceuticals

Quantison

Quadrant Healthcare

Recombinant albumin/air

Sonazoid

NC100100 Amersham Health

Lipid/perfluorobutane

Sonogen

QW7437

Sonus Surfactant/ Pharmaceuticals dodecafluoropentane

SonoVue

BR1

Bracco

Phospholipid/sulfur hexafluoride

(taken with minor modification from [17], with kind permission of Springer Science +Business Media.)

Clinical Practice of Contrast Enhanced Ultrasound [41] both explains rationale for proceeding with the investigation and reports on the extremely rare and limited risks for patients that are examined utilizing contrastenhanced ultrasound. These guidelines and recommendations include general considerations (including the dose of contrast agent and data storage) and issues regarding the equipment (e.g., sensitivity, tissue suppression, and resolution), investigator training, terminology (e.g., arterial or venous phase in regard to timing of enhancement, the degree of enhancement, and contrast distribution), and safety aspects. The recently published French Multicenter Support for Innovative and Expensive Techniques Study aimed to assess the feasibility of DCE ultrasound for the evaluation of response to antiangiogenic therapy in solid tumors [42]. The study included patients with the following tumor types: metastatic breast cancer,

melanoma, colon cancer, gastrointestinal stromal tumors, renal cell carcinoma, and primary hepatocellular carcinoma. Firstly, an initial morphologic, B-mode ultrasound examination was performed, followed by the DCE ultrasound using Sonovues. Patients were treated with sunitinib, sorafenib, bevacizumab, and imatinib. The study was conducted in 19 oncology centers in France, with standardized DCE settings for all centers (including the training of radiologists and implementation of strict rules for the evaluation of the results). The authors found DCE ultrasound to be a feasible method for evaluation of antiangiogenic response in selected tumors that could be obtained in different imaging centers (only 3% of the examinations performed by these centers were found to be not interpretable).

Targeted Microbubble-Based Molecular Ultrasound Although preclinical investigations of the applicability of molecular ultrasound in tumor detection and evaluation of response to antitumor therapy in recent years yielded encouraging results, it was not followed by the transfer to the clinic (Figure 8.4). This is partly due to the lack of ultrasound contrast agents that are both approved regarding their safety profile and considered suitable to fulfill the task of targeting tumorspecific structures. Currently, the majority of targeted ultrasound applications are tested in research settings. However, several recently published studies focusing on the feasibility of clinically translatable ultrasound contrast agents suggest that clinical use of targeted microbubbles may soon become a reality. Anderson et al. [44] investigated the use of a microbubble ultrasound contrast agent that relied on the biocompatible, covalent conjugation chemistry. Authors reported that a microbubble contrast agent covalently bound to RGD peptide might be used for ultrasound molecular imaging of αvβ3 integrin, quantitatively and with low MI. The BR55 compound, contrary to the vast majority of targeted microbubbles for molecular ultrasound, does not rely on biotin/streptavidin coupling strategy or an antibody for binding. A study published by Pysz et al. aimed to test the suitability of human kinase insert domain receptor (KDR)-targeted microbubbles for assessment of VEGFR2 expression in mouse tumor vasculature. Study results suggest this approach is feasible and, importantly, a clinically translatable option to assess antiangiogenic treatment [45]. Of course, contrast agents devoid of biotin/streptavidin chemistry are not the only prerequisite for clinical translation. Efforts are needed to standardize quantification of a signal coming specifically from targeted microbubbles.

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FIGURE 8.4 In vivo ultrasound molecular imaging of orthotopic pancreatic ductal adenocarcinoma (PDAC) xenografts in mice and corresponding ex vivo immunofluorescence analysis. Transverse ultrasound images obtained in contrast mode after intravenous injection of thymocyte differentiation antigen 1 (Thy1)-targeted contrast microbubbles show strong imaging signal in human Thy1-positive tumor and background signal in both types of control tumors (scale bar 5 mm; color-coded scale is shown for ultrasound molecular imaging signal in arbitrary units). Note low imaging signal after control microbubbles in all tumor types (green circles, region of interest). Corresponding immunofluorescence micrographs of merged double-stained sections (murine CD31, red; human Thy1, green) confirm human Thy1 expression on neovasculature in Thy1positive tumors (yellow), while both negative control tumors did not show human Thy1 staining on the neovasculature (scale bar 50 μm). AsPC1: human pancreatic ductal adenocarcinoma cell line. Source: Reprinted from [46].

CONCLUSION Ultrasound imaging is a widely used method in research and clinical settings. It is a valuable part of the larger repertoire of imaging methods that are nowadays used, including MRI, CT, PET, and SPECT. Unenhanced ultrasound is well established particularly in abdominal pathology. Contrast-enhanced ultrasound is characterized by the use of microbubbles that, due to their nonlinear response after insonation, enable the filtration of specific signal from the background signals. Microbubble particles remain in the intravascular space after intravenous administration and, due to their size, may pass through the pulmonary capillary network. Different microbubble-based contrast agents are nowadays used based on the properties of their gaseous core and the shell that encapsulates it. The advantage of microbubbles as contrast agents is that they are relatively easily targeted to molecules of interest expressed on tumor endothelial cells. Since various tumor types develop their own vasculature at a certain stage of their growth, targeting of microbubbles to the structures expressed on tumor blood vessels has emerged as a method feasible to specifically detect and quantify these target structures. The vast majority of experimental microbubbles are based on biotin/

streptavidin chemistry, which is not biocompatible. Novel, biocompatible targeted microbubbles are therefore needed for clinical translation. Recent success in introducing biocompatible microbubble-based ultrasound contrast agents in preclinical settings gives hope that application of contrast agents that enable reliable quantification of factors expressed by tumors in patients, especially for therapy response assessment, is a matter of the near future.

Acknowledgments This work was funded by the Deutsche Forschungsgemeinschaft (DFG), SFB-TR 23 (D.K., T.B.).

Abbreviations Au AsPC1 CE CT DCE EFSUMB FAK HCC ICAM-1 KDR MadCAM1

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Arbitrary units Human pancreatic ductal adenocarcinoma cell line Contrast-enhanced Computed tomography Dynamic contrast-enhanced European Federation Criteria in Solid Tumors Focal adhesion kinase Hepatocellular carcinoma Intercellular adhesion molecule-1 Kinase insert domain receptor Mucosal addressin cellular adhesion molecule

136 MI MRI PDAC PET PFC Pyk2 RECIST RGD SPAQ SPECT Thy1 UICC VCAM-1 VEGF VEGFR WHO

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Mechanical index Magnetic resonance imaging Pancreatic ductal adenocarcinoma Positron emission tomography Perfluorocarbon Proline-rich kinase-2 Response Evaluation Criteria in Solid Tumors Arginine-glycine-aspartic acid Sensitive particle acoustic quantification Single-photon emission computed tomography Thymocyte differentiation antigen 1 International Union Against Cancer Vascular cell adhesion molecule-1 Vascular endothelial growth factor Vascular endothelial growth factor receptor World Health Organization

Glossary Angiogenic Switch The induction of a tumor vasculature, a step required to allow tumor progression. Occurs when the balance of pro-angiogenic and antiangiogenic factors moves in the direction of pro-angiogenic outcome, resulting in the transition from dormant avascularized to outgrowing vascularized tumor and possible subsequent malignant progression. Ultrasound Elastography Ultrasound technique that provides information regarding mechanical properties of the tissue (e.g., their hardness and stiffness). RGD Sequence Arg-Gly-Asp (arginine-glycine-aspartic acid) sequence; cell attachment site of a number of adhesive extracellular matrix and cell surface proteins. Various integrins recognize this sequence in their adhesion protein ligands.

References [1] Pysz MA, Willmann JK. Targeted contrast-enhanced ultrasound: an emerging technology in abdominal and pelvic imaging. Gastroenterology 2011;140(3):785 90. [2] World Health Organization (WHO). WHO handbook for reporting results of cancer treatment. Geneva (Switzerland): World Health Organization Offset Publication; 1979. [3] Hayward JL, Carbone PP, Heuson JC, Kumaoka S, Segaloff A, Rubens RD. Assessment of response to therapy in advanced breast cancer: a project of the Programme on clinical oncology of the international union against cancer, Geneva, Switzerland. Cancer 1977;39(3):1289 94. [4] Therasse P, Arbuck S, Eisenhauer E, Wanders J, Kaplan R, Rubinstein L, et al. New guidelines to evaluate the response to treatment in solid tumors. J Natl Cancer Inst 2000;92:3. [5] Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45(2):228 47. [6] Scha¨berle W. Ultrasonography in vascular diagnosis. Berlin Heidelberg: Springer-Verlag; 2011. [7] Widder B, Go¨rtler M. Doppler- und duplexsonographie der hirnversorgenden arterien. Heidelberg: Springer; 2004. [8] Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144(5):646 74. [9] Weinberg RA. The biology of cancer. Taylor and Francis; Garland Science; 2007.

[10] Ba¨uerle T, Merz M, Komljenovic D, Zwick S, Semmler W. Drug-induced vessel remodeling in bone metastases as assessed by dynamic contrast enhanced magnetic resonance imaging and vessel size imaging: a longitudinal in vivo study. Clin Cancer Res 2010;16(12):3215 25. [11] Merz M, Komljenovic D, Zwick S, Semmler W, Bauerle T. Sorafenib tosylate and paclitaxel induce anti-angiogenic, antitumour and anti-resorptive effects in experimental breast cancer bone metastases. Eur J Cancer 2011;47(2):277 86. [12] Kircher M, Willmann J. Molecular Body Imaging: MR Imaging, CT, and US. Part I. Principles. Radiology, 2012;263(3):633 43. [13] Kiessling F, Huppert J, Palmowski M. Functional and molecular ultrasound imaging: concepts and contrast agents. Curr Med Chem 2009;16(5):627 42. [14] Reinhardt M, Hauff P, Briel A, Uhlendorf V, Linker R, Maeurer M, et al. Sensitive particle acoustic quantification (SPAQ) a new ultrasound-based approach for the quantification of ultrasound contrast media in high concentrations. Invest Radiol 2005;40(1):. [15] Deshpande N, Needles A, Willmann JK. Molecular ultrasound imaging: current status and future directions. Clin Radiol 2010;65(7):567 81. [16] Piscaglia F, Bolondi L. Italian Society for Ultrasound in Medicine and Biology (SIUMB) Study Group on Ultrasound Contrast Agents. The safety of Sonovue in abdominal applications: retrospective analysis of 23188 investigations. Ultrasound Med Biol 2006;32(9):1369 75. [17] Quaia E (Ed). Contrast Media in Ultrasonography. Springer; 2005. [18] Deng CX, Lizzi FL. A review of physical phenomena associated with ultrasonic contrast agents and illustrative clinical applications. Ultrasound Med Biol 2002;28(3):277 86. [19] Liu J, Levine AL, Mattoon JS, Yamaguchi M, Lee RJ, Pan X, et al. Nanoparticles as image enhancing agents for ultrasonography. Phys Med Biol 2006;51(9):2179 89. [20] Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 2003;3(6):401 10. [21] Kiessling F, Fokong S, Koczera P, Lederle W, Lammers T. Ultrasound microbubbles for molecular diagnosis, therapy, and theranostics. J Nucl Med 2012;53(3):345 8. [22] Kaufmann BA, Lewis C, Xie A, Mirza-Mohd A, Lindner JR. Detection of recent myocardial ischaemia by molecular imaging of P-selectin with targeted contrast echocardiography. Eur Heart J 2007;28(16):2011 7. [23] Kaufmann BA, Sanders JM, Davis C, Xie A, Aldred P, Sarembock IJ, et al. Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1. Circulation 2007;116(3):276 84. [24] Schumann PA, Christiansen JP, Quigley RM, McCreery TP, Sweitzer RH, Unger EC, et al. Targeted-microbubble binding selectively to GPIIb IIIa receptors of platelet thrombi. Invest Radiol 2002;37(11):587 93. [25] Thakur M, Lentle BC. Report of a summit on molecular imaging. Radiology 2005;236(3):753 5. [26] Merz M, Komljenovic D, Semmler W, Bauerle T. Quantitative contrast-enhanced ultrasound for imaging antiangiogenic treatment response in experimental osteolytic breast cancer bone metastases. Invest Radiol 2012;47(7):422 9. [27] Bagi CM, Christensen J, Cohen DP, Roberts WG, Wilkie D, Swanson T, et al. Sunitinib and PF-562,271 (FAK/Pyk2 inhibitor) effectively block growth and recovery of human hepatocellular carcinoma in a rat xenograft model. Cancer Biol Ther 2009;8(9):856 65. [28] Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 2005;23(5):1011 27.

II. MOLECULAR IMAGING

137

REFERENCES

[29] Murukesh N, Dive C, Jayson GC. Biomarkers of angiogenesis and their role in the development of VEGF inhibitors. Br J Cancer 2010;102(1):8 18. [30] Avraamides CJ, Garmy-Susini B, Varner JA. Integrins in angiogenesis and lymphangiogenesis. Nat Rev Cancer 2008;8(8): 604 17. [31] Bachmann C, Klibanov AL, Olson TS, Sonnenschein JR, RiveraNieves J, Cominelli F, et al. Targeting mucosal addressin cellular adhesion molecule (MAdCAM)-1 to noninvasively image experimental Crohn’s disease. Gastroenterology 2006;130(1):8 16. [32] Villanueva FS, Jankowski RJ, Klibanov S, Pina ML, Alber SM, Watkins SC, et al. Microbubbles targeted to intercellular adhesion molecule-1 bind to activated coronary artery endothelial cells. Circulation 1998;98(1):1 5. [33] Ferrante EA, Pickard JE, Rychak J, Klibanov A, Ley K. Dual targeting improves microbubble contrast agent adhesion to VCAM-1 and P-selectin under flow. J Control Release 2009;140 (2):100 7. [34] Lee DJ, Lyshchik A, Huamani J, Hallahan DE, Fleischer AC. Relationship between retention of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeted ultrasonographic contrast agent and the level of VEGFR2 expression in an in vivo breast cancer model. J Ultrasound Med 2008;27(6):855 66. [35] Willmann JK, Lutz AM, Paulmurugan R, Patel MR, Chu P, Rosenberg J, et al. Dual-targeted contrast agent for US assessment of tumor angiogenesis in vivo. Radiology 2008;248(3):936 44. [36] Palmowski M, Huppert J, Ladewig G, Hauff P, Reinhardt M, Mueller MM, et al. Molecular profiling of angiogenesis with targeted ultrasound imaging: early assessment of antiangiogenic therapy effects. Mol Cancer Ther 2008;7(1):101 9. [37] Korpanty G, Carbon JG, Grayburn PA, Fleming JB, Brekken RA. Monitoring response to anticancer therapy by targeting microbubbles to tumor vasculature. Clin Cancer Res 2007;13 (1):323 30. [38] Loria F, Loria G, Basile S, Crea G, Randazzo D, Frosina L. Contrastenhanced ultrasound of hepatocellular carcinoma: correlation

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

between enhancement pattern and cellular differentiation on histopathlogy. Updates Surg 2012;64(4):247 55. Lassau N, Koscielny S, Albiges L, Chami L, Benatsou B, Chebil M, et al. Metastatic renal cell carcinoma treated with sunitinib: early evaluation of treatment response using dynamic contrastenhanced ultrasonography. Clin Cancer Res 2010;16(4):1216 25. Williams R, Hudson JM, Lloyd BA, Sureshkumar AR, Lueck G, Milot L, et al. Dynamic microbubble contrast-enhanced US to measure tumor response to targeted therapy: a proposed clinical protocol with results from renal cell carcinoma patients receiving antiangiogenic therapy. Radiology 2011;260(2):581 90. Piscaglia F, Nolsoe C, Dietrich CF, Cosgrove DO, Gilja OH, Bachmann Nielsen M, et al. The EFSUMB Guidelines and Recommendations on the Clinical Practice of Contrast Enhanced Ultrasound (CEUS): update 2011 on non-hepatic applications. Ultraschall Med 2012;33(1):33 59. Lassau N, Chapotot L, Benatsou B, Vilgrain V, Kind M, Lacroix J, et al. Standardization of dynamic contrast-enhanced ultrasound for the evaluation of antiangiogenic therapies: the French multicenter Support for Innovative and Expensive Techniques Study. Invest Radiol 2012;47(12):711 6. Kaneko OF, Willmann JK. Ultrasound for molecular imaging and therapy in cancer. Quant Imaging Med Surg 2012;2(2): 87 97. Anderson CR, Hu X, Zhang H, Tlaxca J, Decleves AE, Houghtaling R, et al. Ultrasound molecular imaging of tumor angiogenesis with an integrin targeted microbubble contrast agent. Invest Radiol 2011;46(4):215 24. Pysz MA, Foygel K, Rosenberg J, Gambhir SS, Schneider M, Willmann JK. Antiangiogenic cancer therapy: monitoring with molecular US and a clinically translatable contrast agent (BR55). Radiology 2010;256(2):519 27. Foygel K, Wang H, Machtaler S, Lutz AM, Chen R, Pysz M, et al. Detection of pancreatic ductal adenocarcinoma in mice by ultrasound imaging of thymocyte differentiation antigen 1. Gastroenterology 2013;145(4):885 94.

II. MOLECULAR IMAGING