3 The Role of Blood Pool Contrast Media in the Study of Tumor Pathophysiology

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3 The Role of Blood Pool Contrast Media in the Study of Tumor Pathophysiology

Laure S. Fournier and Robert C. Brasch

L. S. Fournier, MD

R. C. Brasch, MD, Professor of Radiology and Pediatrics Center for Pharmaceutical and Molecular Imaging, Depart- ment of Radiology, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143-0628, USA

3.1

Introduction

Characterizing the biological properties of individual tumors has become a major goal for non-invasive imaging. With growing enthusiasm, the use of serial MRI to monitor the pharmacokinetics of paramagnetic contrast media is being explored to functionally characterize tumors, particularly tumor vascularity. Analysis and kinetic modeling of the dynamic tumor enhancement response after contrast medium administration is being applied to generate quantitative estimates of microvascular characteristics, particularly the fractional blood volume and the microvascular permeability of tumor vessels. Understandably, it is commonly hoped by many MR imagers that the currently available and widely used gadolinium-based contrast media, all belonging in the class of small molecular contrast media (SMCM

<1000Daltons) will be satisfactory for characterization of microvessels with dynamic contrast-enhanced imaging. But it should be emphasized that endothelial permeability to solutes of substantially different size cannot

be equated. Unfortunately, SMCM are too small to optimally exploit the well-recognized hyperpermeability of neoplastic microvessels; such hyperpermeability, with rare exception, has been consistently demonstrated in relation to larger macromolecular solutes. Smaller solutes in the size range of commercially available gadolinium chelates are known to diffuse across vascular endothelium in both normal and neoplastic tissues; a notable exception being the blood–brain barrier. Thus, the differentiation of normal from neoplastic tissue by the MRI assay of tissue SMCM leakage is problematic. A macromolecular contrast media (MMCM) is favored to probe and quantitatively estimate by MRI the elevated macromolecular permeability of tumor microvessels.

Another obstacle to quantitative MRI tumor characterization pursued with SMCM is the highly variable and unpredictable vascular extraction fraction, both in normal and in neoplastic tumor tissues.

MMCM are also referred to as blood pool contrast media (BPCM). While BPCM/MMCM are now being clinically developed and positioned for governmental approval, prototype MMCM are being applied with considerable success in a host of experimental tumor models. The pre-clinical results show strongly positive and signiﬁ cant correlations between MMCM- assayed tumor vascular permeability and tumor blood volume with pathologic tumor grade, tumor angiogenesis as assayed by the histologic microvascular density, and with tumor response to multiple forms of anti-angiogenesis therapy. In groups of tumors characterized both by SMCM and MMCM- enhanced MRI, the correlations between MRI assays and histopathologic endpoints are consistently superior using the macromolecular-enhanced imaging.

3.2

What Are Blood Pool Contrast Media and What Is Their Appeal?

Blood pool contrast media (BPCM) are not strictly deﬁ ned; which compounds should be included within

CONTENTS

3.1 Introduction 39

3.2 What Are Blood Pool Contrast Media and What Is Their Appeal? 39 3.3 Biological Aspects of Tumor Vessel Hyperpermeability 40

3.4 Shortcomings of Small Molecular Contrast Media for Characterizing Tumor Vessels 41

3.4.1 What Are the Determinants of PS? 41 3.5 Applications of Blood Pool Contrast Media 42 3.5.1 Angiography 42

3.5.2 Characterizing Individual Tumor Biology 44 3.5.3 Monitoring Tumor Response to Treatment 47 3.6 Conclusion 50

References 50

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this category may depend upon whom one asks. Yet, a qualitative defi nition is universally accepted: a blood pool contrast medium for MRI is an enhancing, typically paramagnetic, formulation, that after intravascular administration remains largely in the vascular space for an extended time. For instance, one could choose to defi ne a BPCM as an agent with a plasma half-life of more than 60 min. Typically, such plasma retention would be associated with molecules having molecular weights greater than 50,000 Daltons (Vexler et al. 1994). However, plasma half-life not only varies with molecular weight but also depends on molecular shape and charge, and on the animal species used for the evaluation. Other MRI scientists might wish to include relatively smaller molecules in the category of BPCM and might defi ne “prolonged vas-

cular retention” as a net plasma half-life greater than that observed for typical small molecular gadolinium contrast media (SMCM) represented by gadopentetate (MW=547 Daltons). For reference, the plasma half- life of gadopentetate in rats is 13 min and approximately 20 min in humans (Weinmann et al. 1984).

Using such a broad deﬁ nition of BPCM would permit inclusion of contrast-enhancing formulations ranging from 5000−50,000 Daltons and larger. Obviously, the pharmacokinetic properties and thus the clinical utility of BPCM could vary substantially across this broad range of molecular sizes. Importantly for the reader, all BPCM should not be considered equivalent or interchangeable. The speciﬁ c agent and its properties must be considered in any discussion of kinetics, or diagnostic application (see “Appendix”).

Appendix

Different classes of contrast media (manufacturers and references). ¹Weinmann et al. (1984); ²Schering AG, Berlin, Germany (Henderson et al. 2000); ³Amersham Health, Oslo, Norway (Bonk et al. 2000); ⁴Guerbet, Aulnay-sous-Bois, France (Daldrup- Link et al. 2001); ⁵Epix, Cambridge, MA, USA (Kroft and de Roos 1999); ⁶Bracco, Milan, Italy (Cavagna et al. 2001); ⁷Ogan (1988); ⁸Amersham Health, Oslo, Norway (Hoffmann et al. 2002); ⁹Okuhata et al. (1999); Weissig et al. (2000)

3.3

Biological Aspects of Tumor Vessel Hyperpermeability

Although it is commonly taught in biological sciences to “never say never” and conversely, nothing is “always”

true, it can be stated with some certainty that tumor microvessels when compared to normal non-tumor vessels are more permeable to the transendothelial diffusion of large molecular solutes. Macromolecular

hyperpermeability of tumor vessels has been demonstrated consistently over more than 50 years by numerous, but generally invasive assays; published assays have utilized macromolecular dyes like Evan’s blue, radio-labeled proteins such as ﬁ brinogen and albumin, and ﬂ uorescent-labeled proteins detected with video microscopy (Gerlowski and Jain 1986;

Nagy et al. 1989; Dvorak 1990; Sevick and Jain 1991; Yuan et al. 1993; Jain 1994). Our group at UCSF ﬁ rst detected and quantitatively monitored by MRI

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this macromolecular hyperpermeability of tumors in 1989 when we performed dynamic contrast-enhanced (DCE) MRI in an experimental mouse ﬁ brosarcoma model using a prototype macromolecular contrast agent, albumin-(Gd-DTPA)₃₅(Aicher et al. 1990).

Albumin-(Gd-DTPA)₃₅ is a highly paramagnetic bio-probe with a molecular weight of 92,000 Daltons and a hydrodynamic radius of approximately 6 nm (Ogan 1988). It is interesting to note that even this early attempt to quantitatively assess tumor microvascular permeability previewed the later confi rmed potential of the MMCM-enhanced DCE MRI for monitoring tumor response to therapy. This relatively early investigation showed a signifi cant reduction in tumor vessel leakiness, measured by both MRI and Evan’s blue assays after only 1−2 h of treatment with tumor necrosis factor-alpha (Aicher et al. 1990). Jain (1994), in an excellent review article appearing in Sci- entifi c American, summarized more than a decade of work from his laboratory and from others explain- ing, from an engineering perspective, the nature of macromolecular diffusion from blood within tumor microvessels into the tumor interstitium. Physi- ologically, diffusion dominates the transendothelial exchange of macromolecules in the tumor periphery where interstitial pressure is not as high as that often to be found in the tumor core. Jain (1994) notes that within the tumor core, there is an inhibitory effect from high interstitial pressure on the extravascular leakage/diffusion of solutes. This pathophysiological property of tumor hyperpermeability with respect to macromolecular solutes can be exploited by MMCM- enhanced MRI to characterize this consistent biological feature of malignant tumors.

3.4

Shortcomings of Small Molecular Contrast Media for Characterizing Tumor Vessels To understand the biology underlying the kinetics of MRI contrast media, it is essential to recognize that tumor microvascular hyperpermeability is relatively speciﬁ c to macromolecular solutes. Small molecular solutes such as insulin, glucose, or paramagnetic gadolinium chelates are known to be readily diffus- ible across the endothelial barrier of both normal and neoplastic vessels (Crone and Levitt 1984; Jain 1987). There is no basis from previous invasive studies to anticipate that tumor vessels will leak SMCM while normal vessels in the same organ will not be permeable to the same bio-probes. Two notable

exceptions are found in the microvessels of the brain and the testes; normal vessels in these organs have unusually tight junctions between endothelial cells, limiting diffusion of even SMCM, while tumor vessels in these same organs allow extravascular accumulation of contrast agents.

The degree of transendothelial diffusion for any substance is reﬂ ected in its extraction fraction (E) or by the more complex functional parameter termed the ‘permeability surface area product’ ’ (PS) (Renkin 1959; Crone 1963). As implied by the name, the PS parameter depends on both the local- ized permeability of the vessel and the surface area of the vessel available for transendothelial diffusion.

Intuitively, one can appreciate that diffusion of solutes across the endothelium will also depend on the rate of blood ﬂ ow in the leaky microvessel. One could reasonably predict that with slower ﬂ ow there would be more time for permeable solute molecules to actu- ally escape the blood compartment and diffuse into the extravascular space.

3.4.1

What Are the Determinants of PS?

Physiologists have been fascinated by vascular permeability for generations and more than one kinetic model has been proposed; Renkin (1959) and Crone (1963) described a useful and widely accepted model of this process. The Renkin/Crone equation is as fol- lows:

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where FLR represents fractional leak rate, F ﬂ ow , PV plasma volume , and PS the permeability surface area product.

In the limiting situation in which the value for PS is much smaller than the value for ﬂ ow (F), the Renkin/

Crone equation can be simpliﬁ ed by substituting

for (2)

then (3)

and (4)

In this speciﬁ c situation, realized with macromolecular blood pool contrast agents for which PS and the

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extraction fraction are quite small (less than 0.001 per passage through the vascular bed), the complex exponential Renkin/Crone equation reduces to

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This is most fortunate, because currently there are no easily employed techniques using MRI for measurement of ﬂ ow in tissue microvessels (St. Lawrence and Lee 1998a,b).

Unfortunately for those wishing to estimate PS using SMCM, the extraction fraction and the PS are not small compared to F. The ﬂ ow term cannot be ignored, cannot be easily estimated on a gadolinium- enhanced MRI examination, and cannot reasonably be assumed to be constant in different tissues, tumors, or even in all regions of a given tumor.

Not withstanding the fact that estimating fl ow in tumor microvessels by MRI methods is problematic, tumor microvascular fl ow can be estimated invasively using labeled microspheres. Daldrup and colleagues (1998) used a combination of invasive microsphere assays and non-invasive dynamic contrast-enhanced MRI acquisitions in a group of human breast tumors (MDA-MB-435) implanted in the mammary fat pads of athymic rats (Daldrup et al. 1998b). By estimating the fractional leak rate (FLR) and the fractional plasma volume (fPV) from the MRI tumor enhancement response to gadopentetate and by measuring the microvascular blood fl ow (F) from the microsphere accumulation, they were able to quantitatively estimate the extraction fractions in this series of tumors with respect to gadopentetate.

The tumor gadopentetate extraction fractions were neither consistent nor small; E values were all in the range of 20%–48%. In fact, those experienced with DCE MRI using this contrast agent and the trend for strong rapid tumor enhancement, might predict such a relatively high rate of transendothelial diffusion for gadopentetate. The E of gadopentetate can be even higher than in these tumors, for example 55% in normal myocardium (Svendsen et al. 1992;

Haunso et al. 1980). Returning to the Renkin/Crone equation, for gadopentetate the extraction fraction is clearly not small compared to the PS. Thus, for gadopentetate (and similar SMCM), the equation cannot be simpliﬁ ed algebraically to eliminate the ﬂ ow (F) term, and the PS value cannot be reliably estimated for this class of small molecular MRI contrast media.

These considerations argue for the development and

use of MMCM/BPCM for assessing the permeability of tumor microvessels. The same agents can also be used to advantage for blood volume measurements and angiographic imaging.

3.5

Applications of Blood Pool Contrast Media The potential applications of blood pool contrast media for tumor evaluations are still being deﬁ ned, but no less than three clinically feasible and attractive goals are emerging. To be discussed separately will be angiography, characterizing individual tumor biology including differentiation of benign from malignant tumors and tumor grading, and monitoring of tumor response to treatment.

3.5.1

Angiography

First, it is useful to consider angiography with conventional small molecular contrast media (SMCM).

For these agents having molecular weights less than 1000 Daltons, there is a fast transendothelial diffusion resulting in a rapidly declining vessel-to-tissue contrast (Taupitz et al. 2000). Bolus tracking is also recommended when using SMCM to ensure that the image acquisitions are obtained with optimal timing, ideally just after the bolus arrives in the vessels (Bonk et al. 2000). However, the requirement for relatively fast acquisitions, within the short dura- tion of high vessel-to-background ratio, excludes the possibility of using higher spatial resolution pulse sequences, which entail longer acquisition periods.

MMCM/BPCM, on the other hand, provide a prolonged enhancement of blood within vessels related to their low transendothelial extraction fraction and slower blood clearance rates (Kroft and de Roos 1999; Kauczor and Kreitner 2000).

Several macromolecular BPCM formulations have been tested for their potential to angiographically deﬁ ne both large vessels and to characterize tumors generally. A 20-kDa hexamethylene diamine co-polymer (Bonk et al. 2000), and a carboxymethyl-dextran (P717, Guerbet) with a molecular weight of 52 kDa (Daldrup-Link et al. 2001), when evaluated in rabbits showed stable blood enhancement for 40–60 min in the abdominal aorta and pelvic vessels. In comparison, liposomes ﬁ lled with gadolinium chelates, much larger than the polymeric molecules, produced

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a substantial shortening of blood T1 for over 4 h (Weissig et al. 2000). Although the vessel deﬁ nition was considered good with all three of these BPCM, the MRI seemed to slightly underestimate the vascular diameter compared to measurements by conventional radiographic angiography, for example in pelvic vessels (Bonk et al. 2000).

Of course, there are additional important considerations, beyond blood half-life, in the choice among BPCM. For example, when using the 20-kDa carboxymethyl-dextran formulation (p717), the enhancement in the liver did not clear until 10 days, considered to be an undesirable long retention. Similarly, the whole body retention of albumin-(Gd-DTPA)₃₅ is considered undesirably high and long, 18% at 3 weeks (White et al. 1989) for this much-used prototype BPCM; such retention discouraged the development of this compound as a clinical pharmaceutical. However, this 92-kDa prototype compound has several near ideal pharmacokinetic properties including a volume of distribution equal to the plasma volume and a blood half-life of 90 min (Schmiedl et al. 1987). Albumin- (Gd-DTPA)₃₅ has been used frequently to demonstrate the range of potential beneﬁ ts for MMCM-enhanced MRI (Fig. 3.1); Schwickert and coworkers (1995) showed detailed rodent angiograms using albumin- (Gd-DTPA)₃₅ which persisted essentially unchanged visually for 88 min after administration.

Additional intermediate size gadolinium-based blood pool contrast agents have been tested for magnetic resonance angiography. Gadomer-17 (Schering AG, Berlin), for example, has a molecular weight of 17.5 kDa, and has been used successfully in pigs to angiographically study of coronary and pulmonary arteries, with a noticeable increase of signal-to-noise (SNR) and contrast-to-noise (CNR) ratios between pre- and post-contrast images (Li et al. 2001; Abol- maali et al. 2002). However, such intermediately sized molecules serve as reliable markers of the blood pool only during the ﬁ rst passes. Being smaller than serum proteins, they diffuse progressively into the interstitial space, and their kinetics tend to resemble those of extracellular agents with a bi-exponential plasma disappearance. Though this plasma clearance characteristic might not diminish MR angiography, it tends to limit the usefulness of these intermediate-sized agents for tissue characterization, since they would tend to overestimate tissue blood volume (Kroft and de Roos 1999).

MS-325 (Epix, Cambridge, MA) is a small gadolinium chelate (957 Da), which binds reversibly to circu- lating albumin, forming a macromolecular complex (Kroft and de Roos 1999). The percentage of binding varies with species but is approximately 95% at equilibrium in humans and rabbits. Despite the presence of some small unbound molecules diffusing into the interstitial space and cleared by glomerular ﬁ l- tration, MS-325 produces a strong vascular enhancement in patients for a length of time sufﬁ cient to accurately depict stenoses and ulcerations in carotid arteries (Bluemke et al. 2001; Grist et al. 1998; Li et al. 1998; Stuber et al. 1999), with an SNR and CNR in the carotid vessels which decreased by only 10%

between 5 and 50 min post-contrast.

Another serum protein-binding gadolinium chelate (94% binding at equilibrium), B-22956/1 (Bracco, Milan, Italy) with a molecular weight of 1060 Da also has shown promise in MR angiography (Cavagna et al. 2001, 2002), due to its strong binding to albumin and the slow elimination from the plasma in humans.

Although it is not an issue for MR angiography, the presence of unbound molecules may limit the usefulness of this type of agent for tumor microvascular characterization. The unbound molecule, though in small proportion, presents a much higher extraction fraction and transendothelial transport rate than the albumin-bound complex, and its very fast kinetics can mask the slower exchanges of the macromolecular form.

Other even larger macromolecules have shown potential utility as angiographic contrast agents.

Fig. 3.1. Anterior view of a rat MR angiography (maximum intensity projection), performed 10 min after injection of albumin-(Gd-DTPA)₃₅ at a dose of 0.03 mmol Gd/kg, on a 2T MRI system. Large and relatively small veins and arteries can be deﬁ ned

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Clariscan (Feruglose, Amersham, UK), representa- tive of ultrasmall superparamagnetic iron oxide (USPIO) particles, has been used extensively in pilot studies as a vascular imaging agent in animals and patients. Other preparations of iron oxide particles have also been evaluated for their potential in angiographic studies; for example, Taupitz and colleagues (2000) tested a VSOP (very small superparamagnetic iron oxide particles), with hydrodynamic diameter of 8 nm, which yielded a highly contrasted depiction of even small thoracic and abdominal vessels in rats and rabbits for up to 50 min. Improved MR angiography is a goal attainable with virtually all of the examined blood pool contrast media, and may contribute to the deﬁ nition of tumor vascular characteristics, with a better delineation of the feeding vessels of the tumor and their relationship with host vasculature. For example, BPCM-enhanced angiography during biopsies would highlight the large vessels so that they could be avoided during biopsy, and would ease the time pressure inherent to rapidly deteriorating vascular contrast typically observed with SMCM (Kauczor and Kreitner 2000).

3.5.2

Characterizing Individual Tumor Biology

Malignant tumors differ from benign lesions in several regards, notably in having a more active recruitment of neovascularity. This acceleration of angiogenesis is essential for the exponential growth and metastasis of the tumor cells (Folkman 1992). Tumor vessels differ from normal tissue vessels by their structural irregularity (abnormal endothelial cell contours and peculiar branching patterns), heterogeneity (ﬂ ow, diameter, and spacing), and leakiness to macromolecular solutes (Jain 1988; Less et al. 1991; Baish and Jain 2000; Eberhard et al. 2000). Recent data from scanning electron microscopy (EM) show that tumor endothelial cells overlap one another and are loosely interconnected, leaving gaps ranging from 0.3 to 4.7 µm (Fig. 3.2) (McDonald and Foss 2000;

Hashizume et al. 2000). These gaps likely account for the macromolecular hyperpermeability that leads to extravasation of plasma proteins, considered nec- essary for angiogenesis, as well as transendothelial passage of tumor cells, required for hematogenous metastases (Heuser and Miller 1986; Dvorak et al. 1988; Hashizume et al. 2000).

This endothelial characteristic of macromolecular hyperpermeability in malignant tumors can be

exploited by the use of dynamic contrast-enhanced imaging to identify and grade the abnormal tumor microvessels. Stated in the simplest of terms, malignant tumors should leak macromolecular contrast media and benign tumors should not leak.

In clinical oncology, a neoplastic lesion is usu- ally evaluated for its aggressiveness by performing a biopsy followed by histopathologic microscopic examination. However, this method is invasive and can only sample a small percentage of the entire lesion, possibly missing the most aggressive part of the tumor and leading to an erroneous evaluation.

Non-invasive imaging can complement the histopathologic information and may in some respects surpass it as a means to grade tumor properties.

Imaging can be performed on living tissues, on multiple occasions, allows for evaluation of the entire (and often heterogeneous) tumor, and provides both morphologic and physiologic data.

Biopsy tumor specimens are graded by their speciﬁ c histopathologic characteristics. In breast tumors, for example, the Scarff-Bloom-Richardson (SBR) grading method is used to deﬁ ne the presence and degree of malignant characteristics for a tumor (Bloom and Richardson 1957; Scarff and Torloni 1968). The SBR score sums the microscopic evaluations of three separate morphologic elements:

frequency of mitotic ﬁ gures, nuclear polymorphism, and glandular/tubular formation, each scored from ‘1’

to ‘3’. Benign tumors, were they scored like the malignant lesions, would have an SBR score of ‘3’ (summing the minimum score of ‘1’ in each category), whereas malignant tumors can have scores ranging from ‘3’

to ‘9’.

In a rodent model of mammary tumors induced by the single intraperitoneal administration of a chemi- cal carcinogen, N-ethyl-N-nitrosourea (ENU), a spec- trum of tumors develop over months, paralleling the spectrum of breast tumors encountered clinically in women. Several research groups have used this ENU model to evaluate and compare different contrast media for DCE MRI in the characterization of mammary neoplasms (Daldrup et al. 1998a; Su et al. 1998;

Helbich et al. 2000; Turetschek et al. 2001a).

Daldrup and coworkers (1998a) used this model to determine if DCE MRI enhanced with either an MMCM [albumin-(Gd-DTPA)₃₅] or an SMCM (gadopentetate dimeglumine) could differentiate benign from malignant tumors, and furthermore, if MRI results could predict the histopathologic SBR score.

MRI-assayed microvascular parameters including the coeffi cient of endothelial permeability, defi ned as K^trans (refl ecting leakiness), and the fractional plasma

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volume, fPV, (reﬂ ecting richness of vascularity), were estimated in each tumor for each contrast agent using a simple two compartment tissue model comprising the blood and interstitial water of the tumor tissue.

Correlations were sought between MRI-assayed characteristics and pathologic status including assignment to benign or malignant status and SBR scores. MRI- assayed permeability (K^trans) estimated using the macromolecular albumin-(Gd-DTPA)₃₅, showed a signifi - cant difference between benign fi broadenomas and malignant carcinomas (p<0.05). All ten benign tumors had K^trans values of zero, whereas all tumors showing measurable permeability to this macromolecular contrast agent, (K^trans>0), were diagnosed pathologically as carcinomas. There was a slight overlap in pathol- ogy for tumors having no measurable MRI leakiness to macromolecules; other than the ten benign tumors, fi ve of 23 carcinomas had no MRI measurable macromolecular permeability, but these fi ve tumors also had the lowest possible SBR scores (‘3’–‘4’). In this series of 33 tumors, a positive MRI-assayed endothelial permeability value, as estimated with macromolecular albumin-(Gd-DTPA)₃₅, was a consistent sign of malignancy

with an observed speciﬁ city of 100%. Regarding MRI tumor grading, microvascular permeability to albumin-(Gd-DTPA)₃₅ (K^trans) showed a strong positive correlation with histological tumor grade (r²=0.76;

p<0.001) (Fig. 3.3).

By comparison, in the same tumors, when using gadopentetate dimeglumine as the contrast agent, there was a broad overlap and no signifi cant difference in K^trans values observed between benign fi broadenomas and carcinomas (K^trans of 13.2 versus 13.3, respectively; p>0.99). No correlation between gadopentetate-assayed K^trans or fractional plasma volume and histologic SBR grade was found (r²=0.01 and p>0.95 for K^trans; r²=0.03 and p>0.15 for fPV) (Fig. 3.4). This initial report of positive results using macromolecular contrast media and DCE MRI to characterize and grade ENU-induced mammary tumors was reconfi rmed in multiple studies despite sometimes differing methods of kinetic analysis, but all using albumin-(Gd-DTPA)_x (Su et al. 1998;

Turetschek et al. 2001a,b).

The same chemically induced mammary tumor model has been used to evaluate other BPCM for-

Fig. 3.2a−c. a Scanning electron micrograph of vascular endothelial cells from a mouse mammary tumor, showing irregular cell structure such as cellular overlap, bridges (arrowheads), tunnels, and wide openings in the vessel wall (scale bar represents 15 µm). b Transmission electron micrograph of a mouse mammary tumor blood vessels showing transcellular fenestrae (arrow- heads) of 50−80 nm in diameter visible in the endothelial cell (scale bar represents 0.5 µm). c Transmission electron microscopy of mammary tumor endothelium lining showing intercellular gaps, which may account for the characteristic leakiness of tumor blood vessels (scale bar represents 3 µm). (All images courtesy of Donald M. McDonald).

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mulations and provides a useful means to compare performance of different agents. Using Gadomer-17 (Schering AG, Berlin) with an apparent molecular weight of 17.5 kDa, Daldrup-Link and coworkers (2000) showed a signifi cant difference in MRI-estimated permeability between benign and malignant tumors. However, apparently due to a high variability within both fi broadenoma (benign) and carcinoma (malignant) groups, there was no signifi cant correlation between K^trans or fPV, and histopathologic tumor grade. Similarly, Su and colleagues (1998) reported limited specifi city when using the intermediately-sized Gadomer-17 being able to differenti-

ate between high grade and low-grade carcinomas, but not between low-grade carcinomas and benign tumors.

Turetschek and colleagues (2001a) evaluated MS-325, a small molecule that spontaneously asso- ciates with albumin, for characterization of tumor microvessels assaying plasma volume and permeability in the ENU rodent tumor model. No signiﬁ - cant correlations were found between MRI-estimated characteristics and pathologic tumor grade or microvascular count, a marker of angiogenesis;

the lack of correlation was attributed in part to the inability to resolve the kinetics of the small-unbound MS-325 (25% in rats) and the larger protein-bound complex.

Yet another class of potential BPCM, the ultrasmall superparamagnetic iron oxide (USPIO) particles, has been evaluated in the ENU-induced mammary tumor model (Turetschek et al. 2001d). NC100150 injection (Clariscan, Amersham, UK) yielded a strongly positive correlation between MRI-derived K^trans estimates and histological SBR tumor grade (r=0.82; p<0.001). K^trans also correlated signifi cantly with histologically assessed microvascular density (MVD). In this study of 19 total tumors, fi ve were benign fi broadenomas, all with non-measurable (zero) leakiness to the USPIO. Nine of 14 carcinomas did show measurable permeability to the MRI probe; the other fi ve carcinomas without leakiness showed low aggressive potential as refl ected in low SBR scores. Overall, the tumors that were leaky to USPIO were all carcinomas. The signifi cance of these results in animal tumor models is accentuated because USPIO particles have already been tested extensively in human clinical trials as angiographic and lymph node enhancers with favorable results;

governmental approval for USPIO is anticipated soon (Kernstine et al. 1999; Hudgins et al. 2002;

Varallyay et al. 2002).

A preliminary study was performed in women to evaluate the capacity of USPIO particles for breast tumor characterization. Although the qualitative tumor enhancement evident to the eye was relatively low with the USPIO at a dose of 2 mg Fe/kg, making tumor detection more diffi cult than with gadopentetate, Daldrup-Link and colleagues (2002) found a signifi cant difference in enhancement patterns and kinetic analyses between carcinomas (n=9), and benign lesions including fi broadenomas and mastopathic lesions (n=10) (Daldrup-Link et al.

2002). However, there was no signiﬁ cant difference in enhancement proﬁ les between these same two groups using the small-molecular gadopentetate. The lack of

Fig. 3.4. Plot showing the lack of correlation between the endothelial transfer coefﬁ cient (K^PS=K^trans) after intravenous injection of gadopentetate, and histologic tumor grade quan- tiﬁ ed according to the Scarff-Bloom-Richardson method in benign (circle) and malignant tumors (triangle). (Adapted from Daldrup et al. 1998a)

Small molecular agent (Gadopentetate)

K^PS (Endothelial

transfer coefﬁ cient)

µl/h/cm³)

Histologic SBR score

Fig. 3.3. Plot showing strong positive and signiﬁ cant correla- tion between the endothelial transfer coefﬁ cient (K^PS=K^trans) after intravenous injection of albumin-(Gd-DTPA)₃₅ and his- tologic tumor grade in benign (circle) and malignant tumors (triangle). (Adapted from Daldrup et al. 1998a)

K^PS (Endothelial

transfer coefﬁ cient)

µl/h/cm³

Macromolecular contrast agent (Albumin-Gd-DTPA)₃₀

Histologic SBR score r² = 0.76

p < 0.001

r² = 0.76 p < 0.001

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signiﬁ cance was attributed to the broad overlap in gadopentetate enhancement patterns between the two groups, results similar to those observed in the animal mammary models. The results of this clinical study in women with breast tumors supports a unique role of blood pool contrast media for tumor characterization, allowing the differentiation of benign from malignant lesions. Initial tumor detection may be best accom- plished by other means such as radiographic mam- mography or SMCM-enhanced MRI.

MMCM-enhanced MRI can also non-invasively assay tumor angiogenesis, the process by which cancers recruit new vessels growing in from the non- tumor host tissue. Although there is no single “gold standard” assay for angiogenesis, the counting of immunohistochemically stained endothelial cell clusters within a given area of the tumor to yield the microvascular density (MVD) has been used widely as a surrogate marker of the angiogenesis process (Weidner 1995). Clinical series have shown that MVD correlates with the presence of metastases at time of diagnosis and with decreased patient survival in numerous types of malignancies including breast, lung, prostate, bladder, ovary, and head and neck carcinomas. Of note, the status of tumor cell differentiation, for example, the SBR score, and that of concomi- tant tumor angiogenesis do not necessarily correlate.

In fact, tumor grade and angiogenesis are considered independent biological characteristics.

Using albumin-(Gd-DTPA)₃₅ in two groups of xenograft human breast carcinomas grown in athymic rats, van Dijke and coworkers (1996) evaluated the potential of MMCM-enhanced MRI to assay angiogenesis. The ﬁ rst group of tumors was angiogenically less active and showed a slower growth rate with distinctly and signiﬁ cantly lower MVD values (MVD<50; p<0.001). In contrast, the more aggres- sive and rapidly growing group of tumors had MVD values ranging from 80 to 305. MRI-derived tumor plasma volumes and permeability estimates increased exponentially with increasing microvascular density, and there was a strong positive correlation between MRI-assayed microvascular characteristics and MVD (r²=0.8; p<0.001) (Fig. 3.5).

The authors discussed that MRI estimated angiogenesis might be superior to the pathologic MVD assay, which reﬂ ects only the number of vessels, because the MRI technique reﬂ ects both vessel number and size through the plasma volume assay, and the functional characteristics of the vessels through the permeability essay. Indeed, MRI samples the entire tumor, is non- invasive, is not operator-dependent, and can be used repeatedly in the same subject.

3.5.3

Monitoring Tumor Response to Treatment

Beyond assessing individual tumor biology at time of diagnosis, MMCM-enhanced MRI with quantitative estimates of microvascular blood volume and permeability have been shown effective to deﬁ ne the responses of individual tumors to various forms of treatment. In this sense, MMCM-derived MRI measurements are treatment response biomarkers.

In current clinical practice, monitoring of tumor treatment response is typically assessed on imaging examinations by the measurement of tumor size.

Generally such size evaluations are performed at 6- or 8-week intervals. Adding to the problem of a long delay, tumor size is a rather indirect and imprecise morphologic sign of treatment effectiveness. Defi n- ing additional and more direct biological signs of response to treatment would be highly desirable, particularly if that response were detectable non-invasively and before other measurable changes such as tumor shrinkage or necrosis. However, not only do clinicians need a means to defi ne biological effectiveness soon after the initiation of a given treatment, there is also an urgent requirement in the fi eld of oncologic pharmaceutical development for a sensi- tive treatment response marker for a broad spectrum of therapeutic agents undergoing development.

The need for MRI treatment biomarkers is in no place more evident than for angiogenesis inhibitors.

This class of anti-cancer drugs is known to generally retard tumor growth, but not to cure or totally eradicate lesions; therefore, the drug may be effective but the tumor persists, albeit inhibited from attract- ing additional blood vessels (Fenton et al. 2001).

An imaging biomarker may be superior to tumor

Fig. 3.5. Correlation between MRI-assayed permeability (K^PS=K^trans) and pathologically determined microvascular density in two populations of xenograft breast tumors, a slow- growing group (circles) and a more aggressive rapidly-grow- ing group (triangles). (Adapted from van Dijke et al. 1996)

Microvascular Density (MVD) Permeabillity

(K^PS) ml/hr/ml of

tissue

r² = 0.80 Least aggressive subtype More aggressive subtype

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sizing for documentation of angiogenesis inhibitory effect. These biomarker changes may be detectable hours after treatment initiation, rather than in weeks or months needed typically to defi ne substantial changes in tumor size. An early evaluation of therapeutic effi cacy would allow physicians to adapt treatment regimens and doses based on individual response, optimally associate anti-angiogenic with other anti-tumor therapies, and interrupt an eventu- ally ineffi cient treatment, reducing morbidity.

Several reports indicate that MMCM-enhanced MRI-derived microvascular characteristics can be used effectively to monitor the biological effectiveness of angiogenesis inhibitory treatment (Cohen et al. 1995; Schwickert et al. 1996; Pham et al. 1998;

Su et al. 1999; Gossmann et al. 2000; Clement et al.

2001; Roberts et al. 2001, 2002; Turetschek et al.

2001c; Allegrini et al. 2002; Gossmann et al. 2002;

Petrovsky et al. 2002). Vascular endothelial growth factor (VEGF) , also known as vascular permeability factor (VPF) is considered a central signaling molecule in the complex process of tumor angiogenesis (Dvorak 2000). VEGF/VPF has multiple stimulatory effects, all tied to angiogenesis, including endothelial cell mitogenesis, endothelial cell migration, cell survival, and increased endothelial permeability.

The VEGF-induced hyperpermeability of cancer microvessels, 50,000 times stronger than that induced by histamine, leads to an extravasation of macromolecular proteins that form a favorable substrate in the tumor interstitium into which the new vessels grow. It was therefore relevant to design an experiment which would probe the potential of MRI-assayed microvascular responses for the detection of anti-angiogenic effect in tumors.

The fi rst anti-angiogenic drug tested in our center was a human antibody directed against VEGF (Avas- tin, Genentech, South San Francisco, CA) (Pham et al. 1998). A signifi cant (p<0.01) suppression (>75%) of MRI-assayed microvascular hyperpermeability, expressed as the coeffi cient of permeability surface area product (K^trans), was observed in a human breast cancer (MB-MDA-435) grown in athymic rats following a 1-week course of three 1-mg doses of human anti-VEGF antibody. With appropriate controls, the anti-VEGF antibody was shown to reduce tumor weight, growth rate (Fig. 3.6), and MRI-assayed permeability to the blood pool contrast agent (Fig. 3.7).

In a follow-up experiment (Brasch et al. 2000), MMCM-enhanced MRI demonstrated a reduction in permeability, induced by anti-VEGF antibody as early as 24 h after only a single dose. A signiﬁ cant (p<0.01) reduction in tumor microvascular macromolecular

permeability to levels less than 40% of baseline was recorded.

In a parallel manner, subsequent studies showed anti-VEGF antibody induced reductions in MRI- assayed permeabilities in models of human ovarian carcinoma and cerebral glioblastoma multiforme (Gossmann et al. 2000, 2002). In the case of intraperitoneal human ovarian cancers (SKOV-3) grown in athymic rats, after ﬁ ve 1-mg doses of anti-VEGF antibody administered every 3 days, permeability assayed by MMCM-enhanced MRI was seen to decrease signiﬁ cantly in treated tumors, while saline- treated control ovarian tumors exhibited an increase in permeability to MMCM (Fig. 3.8).

Consistent with the hypothesis that elaboration and deposition of VEGF/VPF by ovarian cancer cells into the peritoneal cavity leads to microvascular hyperpermeability, macromolecular extravasation,

Fig. 3.6. Tumor growth for controls and animals treated with 1-mg doses of anti-VEGF antibody every third day over a period of 1 week. Notice the substantial slowing of tumor growth in the anti-VEGF antibody-treated group. (Adapted from Pham et al. 1998)

Number of days after tumor inoculation Tumor Volume

(mm³)

Controls

Anti-VEGF treated

Fig. 3.7. MRI-assayed permeability for controls (inactive immunoglobin) and animals treated with anti-VEGF antibody following a 1-week course of treatment. Notice a signiﬁ cant reduction in MRI-estimated PS whether assayed for the whole tumor or exclusively in the tumor rim or center. (Adapted from Pham et al. 1998)

Tumor Region of Interest Center Rim

Whole Permeability

Surface Factor (PS) µl/cm³ · h

control anti-VEGF

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and ascites (Nagy et al. 1989), there was a signiﬁ cant reduction in the measured ascites accompanying the reduced permeability in the angiogenically-inhibited tumors (Fig. 3.9).

Other contrast agents have been shown to be poten- tially useful for the defi nition of changes in tumor microvessels induced by anti-VEGF antibody treatment. For example, in a human breast cancer rodent model the intermediately sized molecule, gadomer- 17, showed signifi cant changes both in permeability (K^trans) and in fractional blood volume (fPV) after treatment (Roberts et al. 2001). In a similar study, the serum protein-binding molecule B22956/1 also demonstrated a decrease in vascular permeability (K^trans) refl ecting the biological effectiveness of the antibody on tumor vessels (Roberts et al. 2002).

Dynamic MMCM-enhanced MRI has examined other anti-angiogenic drugs known to diminish tumor growth and metastatic spread for measurable effects on microvessels. Turetschek and coworkers (2001c) studied the effect of an inhibitor of the tyrosine kinase VEGF receptor (PTK787/ZK222584, Novartis, Basel, Switzerland) in athymic rats bearing human MB-MDA-435 breast adenocarcinomas.

MRI-assayed microvascular characteristics were evaluated to determine whether they could reﬂ ect treatment efﬁ cacy, and were compared to tumor size and microvessel density, respectively the clinical and pathological methods used to evaluate biological response. Two macromolecular contrast agents were tested, albumin-(Gd-DTPA)₃₅ and USPIO (SHU555C, Schering AG, Berlin, Germany), to generate quantitative estimates of tumor blood volume and microvascular permeability.

With both albumin-(Gd-DTPA)₃₅ and USPIO, a decrease in estimated permeabilities was observed in the treated group after VEGF receptor inhibition, whereas there was an increase in MRI-estimated permeabilities for the control group (Fig. 3.10). These microvascular responses correlated with the observed slowing in the treatment group of tumor growth and with the signiﬁ cantly reduced microvascular density.

Petrovsky and colleagues (2002) tested macromolecular contrast-enhanced MRI for detection of vascular changes after treatment with another but similar tyrosine kinase inhibitor. Treated animals received this agent, VEGF-RTKI AG013925 (Pﬁ zer, San Diego, CA) at the dose of 25 mg/kg b.i.d.

for 12 days. Using a large polylysine contrast agent, shielded by methoxy polyethylene glycol (MPEG) chains, and labeled with gadolinium chelates, they showed a signiﬁ cant decrease (>50%) in the vascular volume fraction of MV-522 human colon carcinoma

Fig. 3.8. Mean coefﬁ cients of endothelial transport in human ovarian carcinomas implanted in nude rats, estimated by dynamic albumin-(Gd-DTPA)₃₅enhanced MRI, before and after treatment in control animals receiving saline solution and treated animals receiving ﬁ ve 1-mg doses of anti-VEGF antibody every third day. (Adapted from Gossmann et al. 2000)

Fig. 3.9. Volume of ascites at necropsy in rodents bearing intra- peritoneal human ovarian SKOV-3 carcinomas measured for saline-treated control animals and those receiving ﬁ ve 1-mg doses of anti-VEGF antibody every third day. These differ- ences in volume of ascites corresponded to anti-VEGF antibody-induced reductions in MRI-estimated microvascular permeability. (Adapted from Gossmann et al. 2000)

Fig. 3.10. Coefﬁ cient of endothelial transport (K^PS=K^trans) before and after treatment in control animals receiving saline solution and treated animals receiving two daily doses of 50 mg/kg of tyrosine kinase inhibitor, both by oral gavage for 7 days. (Adapted from Turetschek et al. 2001c)

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xenografts in mice after only three inhibitor doses (1.5 days) of the treatment, predictive of a 2.5-times decrease in tumor volume visible after 2 weeks.

In a similar experiment, yet another tyrosine kinase inhibitor, ZD4190 (AstraZeneca Pharmaceuti- cals, Macclesﬁ eld, UK), was tested on the human PC-3 prostate carcinoma implanted in nude mice. A signif- icant decline (>40%) in MRI-estimated permeability was detected after only two inhibitor doses administered in 24 h, when comparing the treated group and the control group (Clement et al. 2001).

MMCM-assayed microvascular status has been applied with success for monitoring responses to other forms of therapy, in addition to anti-angiogenic drugs, and for other diseases in addition to malignancies. For example, acute and highly signiﬁ cant increases in microvascular blood volume and permeability to macromolecular albumin-(Gd-DTPA)₃₅ were reported following a single administration of gamma radiation (30 Gy) to a mammary tumor model (Cohen et al. 1995). The diseased tissue response to treatment for conditions as diverse as arthritis (Jiang et al. 2002), spinal cord injury (Philippens et al. 2002), and oxygen-induced pulmonary ﬁ brosis can be monitored by MMCM-enhanced MRI (Brasch et al. 1993).

3.6

Conclusion

In summary, there is a growing body of evidence sup- porting the development and use of quantitative MRI tumor microvascular bioassays, achievable through the application of macromolecular blood pool contrast media, to characterize individual tumors and non-neoplastic disease processes that affect microvessels. It is reasonable to anticipate that the clinical introduction of a dynamic MMCM-enhanced MRI bioassay could lead to better defi nition of tumor biological properties including the more precise identifi cation of malignancy, the grading of malignancy, the angiographic demarca- tion of tumor feeding vessels, the presence and degree of accelerated angiogenesis, and perhaps clinically most important, a means to rapidly and non-invasively evaluate tumor response to treatment. Critical to the realiza- tion of the full potential of MMCM-enhanced MRI will be the identifi cation, development, and ultimately the governmental approval of macromolecular blood pool contrast agents that combine favorable characteristics for strong image enhancement, prolonged intravascular retention, complete and timely bodily elimination, and safety.

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