7 Combinations of Cytotoxic Drugs, Ionizing Radiation, and Angiogenesis Inhibitors

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7 Combinations of Cytotoxic Drugs,

Ionizing Radiation, and Angiogenesis Inhibitors

Carsten Nieder and Nicolaus H. Andratschke

C. Nieder, MD

N. H. Andratschke, MD

Department of Radiation Oncology, Klinikum rechts der Isar der Technischen Universität München, Ismaninger Strasse 22, 81675 München, Germany

7.1

Introduction

Many years after the fundamental hypotheses about delivery of oxygen and nutrients to malignant tumors via formation of new blood vessels (angiogenesis) were published, drugs developed to inhibit angiogenesis have now entered routine clinical practice, after landmark randomized trials have identified suitable combination regimens in metastatic colorectal cancer and advanced non- squamous non-small cell lung cancer (Table 7.1).

Previously, monotherapy, e.g., with matrix metalloproteinase inhibitors, failed to improve the results.

The fundamental principles of anti-angiogenic approaches and their consequences for the devel-

opment and growth of solid tumors were published by Folkman (1971, 1986, 1995). Tumor angiogenesis results from imbalance between pro-angiogenic factors, e.g., vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), and endogenous anti-angiogenic factors such as angiostatin and endostatin (Folkman 1995; O’Reilly et al. 1996, 1997; Endrich and Vaupel 1998; Ferrara and Alitalo 1999; Kerbel 2000; Carmeliet and Jain 2000; Yancopoulos et al. 2000). A large body of experimental data indicate that application of either inhibitors of pro-angiogenic factors or administration of anti-angiogenic factors reduce the formation of new blood vessels. As a result, tumors grow at a slower rate or even decrease their size. However, in most cases no permanent tumor control can be achieved; therefore, the combination of anti-angiogenic strategies with cytotoxic agents such as chemotherapy, ionizing radiation, or both represents a promising approach to increase the cure rates of solid tumors ( Folkman 1971; Teicher et al. 1992;

Denekamp 1993; Folkman 1995; Siemann et al.

2000; Koukourakis 2001; Rosen 2002; Kal et al.

2004; Jain 2005; Xu et al. 2005). This chapter is focused on the VEGF pathways as an illustrative example of the therapeutic principles and efficacy.

Further data can be found in the disease-specific chapters such as 15.6.4 (pancreatic cancer) and 20.4.3 (gynecological cancers).

Angiogenesis is not restricted to tumors but can also be found in many other physiological and patho- logical conditions, e.g., normal growth, wound heal- ing, proliferative retinopathies, rheumatoid arthri- tis, and inflammation (for review see Folkman 1995; Carmeliet and Jain 2000); hence, inhibition of angiogenesis combined with other treatment may be associated with increased normal tissue reactions. In addition, several angiogenic growth factors were shown to reduce radiation-induced long-term toxicity, e.g., in the spinal cord and parotid glands (Andratschke et al. 2004, 2005; Thula et al. 2005);

therefore, the therapeutic ratio of anti-angiogenic

CONTENTS

7.1 Introduction 103

7.2 VEGFs in Tumor Angiogenesis 104 7.3 VEGF Inhibition Strategies 105 7.4 Combination of VEGF Inhibition with Radiotherapy 106

7.4.1 Tumor Models 106

7.4.2 Mechanisms Underlying the Enhanced Effect of Anti-VEGF Agents when Combined with Irradiation 108

7.4.3 Impact of Anti-VEGF Strategies on Radiobiological Hypoxia 108

7.4.4 Sequencing of Anti-VEGF Strategies Combined with Radiotherapy 109

7.4.5 Side Effects of Anti-VEGF Compounds Combined with Radiotherapy 109

7.5 Trimodal Treatment with Anti-Angiogenic Agents, Radiotherapy, and Chemotherapy 110

7.6 Conclusion 110 References 111

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strategies in combination with radiotherapy needs to be thoroughly determined for safe translation of this approach into clinical practice.

7.2

VEGFs in Tumor Angiogenesis

VEGFs are potent mitogenes for endothelial cells and key mediators of tumor angiogenesis (for review see Dvorak et al. 1995; Kerbel et al. 1998; Ferrara 1999; Carmeliet 2000; Carmeliet and Collen 2000; Carmeliet and Jain 2000; Karkkainen and Petrova 2000; Kerbel 2000; Veikkola et al.

2000; Yancopoulos et al. 2000; Byrne et al. 2005).

The VEGFs represent a family of distinct proteins, e.g., VEGF (VEGF-A), VEGF-B, VEGF-C, VEGF-D, VEGF-E, and isoforms (e.g., VEGF₁₂₁ and VEGF₁₆₅).

Some family members are involved in lymphangiogenesis (VEGF-C and VEGF-D). Down-regulation of VEGF-C, e.g., by endostatin, might therefore inhibit lymph node metastasis (Fukumoto et al. 2005). The VEGF expression can be demonstrated in the vast majority of tumors and is usually elevated above normal tissue levels. Until tumors reach a volume of about 1 mm³, tumor cells are supplied by diffu- sion from the surrounding tissues (Folkman 1971;

Ausprunk and Folkman 1977; Folkman 1986). At larger volumes, impaired supply with oxygen and nutrients and accumulation of metabolites occur in the tumor. This is accompanied by important changes in the tumor microenvironment, e.g., hypoxia, hypoglycemia, and acidosis, which result in up-regulated production and release of HIF-1D, VEGF, and other angiogenic factors by the tumor cells (angiogenic switch, see Chap. 20.3.1 for further details). These factors represent potential targets

Table 7.1. Randomized clinical trials of chemotherapy plus/minus anti-angiogenic agents

Reference Compound Tumor Patient no. Results

Leighl et al.

(2005)

Paclitaxel/carboplatin plus BMS-275291 (MMPI) or placebo

NSCLC stage IIIB/IV 774 Outcome not improved, toxicity increased Bissett et al.

(2005)

Cisplatin/gemcitabine plus prinomastat (MMPI) or placebo

NSCLC stage IIIB/IV or recurrent

362 Outcome not improved, more treatment interruption Sparano et al.

(2004)

Marimastat (MMPI) or placebo after ﬁ rst-line chemotherapy

Metastatic breast cancer

179 Outcome not improved, toxicity increased Bramhall et al.

(2002a)

Gemcitabine plus marimastat or placebo

Unresectable pancreatic cancer

239 Outcome not improved, well tolerated

Bramhall et al.

(2002b)

Maintenance marimastat vs placebo after max. 1 5-FU-based chemotherapy regimen

Non-resectable gastric and gastro-esophageal cancer

369 2-year survival 3 vs 9%

in favor of marimastat (p=0.07), PFS sign. Longer Stadler et al.

(2004)

SU 5416 plus dexamethasone vs dexamethasone

Hormone-refractory prostate cancer

36 Outcome not improved

Szczylik et al.

(2005)

Sorafenib vs best supportive care Advanced renal cell carcinoma

769 PFS sign. improved (interim analysis)

Miller et al.

(2005)

Capecitabine plus/minus bevacizumab

Metastatic breast cancer

462 Sign. higher response rate (20 vs 9%), PFS, and OS not improved

Sandler et al.

(2005)

Paclitaxel/carboplatin plus/minus bevacizumab

Non-squamous NSCLC stage IIIB/IV

878 Sign. improvement in response rate, PFS and OS Kabbinavar et al.

(2005)

5-FU/LV plus bevacizumab or placebo

Metastatic colorectal cancer

209 PFS sign. better, OS better but not signiﬁ cant Hurwitz et al.

(2004)

5-FU/LV/irinotecan plus placebo vs bevacizumab

Metastatic colorectal cancer

813 Sign. improvement in OS, PFS, and response (ﬁ rst line) Giantonio et al.

(2005)

5-FU/LV/oxaliplatin (FOLFOX4) vs FOLFOX4 plus bevacizumab vs bevacizumab alone

Previously treated advanced colorectal cancer

829 Sign. improvement in PFS and OS

MMPI matrix metalloproteinase inhibitor, NSCLC non-small cell lung cancer, 5-FU 5-ﬂ uorouracil, LV leucovorin, PFS progres- sion-free survival, OS overall survival

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for anti-cancer treatment (Abdollahi et al. 2005;

Diaz-Gonzalez et al. 2005). In addition to envi- ronmental factors, other triggers, such as mechani- cal stress, immune/inflammatory responses, and genetic alterations (e.g., activation of oncogenes or deletion of tumor-suppressor genes that control production of angiogenesis regulators), may influence the expression of VEGF (Kerbel et al. 1998; Okada et al. 1998; Carmeliet and Jain 2000). Secreted VEGFs bind to specific receptors (VEGFR-1/Flt-1, VEGFR-2/KDR, VEGFR-3/Flt-4), which are almost exclusively expressed on the surface of endothelial cells (Jakeman et al. 1992; Veikkola et al. 2000;

Yancopoulos et al. 2000). Within the families of VEGFs and VEGFRs ligand affinity and receptor properties are variable. The VEGFRs consist of distinct structural elements, most importantly an extracellular immunoglobulin-like binding domain and an intracellular tyrosine kinase domain. Bind- ing of VEGF is followed by receptor dimerization, tyrosine kinase activation, autophosphorylation, and eventually activation of a complex cascade of intracellular pathways (Guo et al. 1995; Abedi and Zachary 1997; Karkkainen and Petrova 2000;

Veikkola et al. 2000; Stoletov et al. 2001). The activation of endothelial cells results in vasorelax- ation, increased vascular permeability, endothelial cell migration, proliferation, and survival. New blood vessels originating from surrounding, pre- existing normal vessels generate a tumor neovas- culature that allows further tumor growth, tumor progression, and development of metastasis; however, important differences in vessel structure and organization exist, such as irregular leaky basement membranes (Di Tomaso et al. 2005).

7.3

VEGF Inhibition Strategies

Based on the molecular mechanisms underlying the biological effects of the VEGFs and their receptors, various rationally designed strategies to inhibit VEGF-dependent angiogenesis have been developed (Table 7.2). The best investigated approaches include antibodies against VEGF or VEGFR and VEGF receptor tyrosine kinase inhibitors (RTKI).

Table 7.2. Strategies to enhance the efﬁ cacy of radiotherapy by inhibitors of angiogenesis

Target Mechanism Reference

VEGF ligand Neutralizing monoclonal antibodies Kim et al. (1993), Asano et al. (1995), Borgström et al. (1996), Presta et al. (1997)

Neutralizing chimeric proteins Aiello et al. (1995)

Soluble VEGFR-1 Lin et al. (1998), Goldman et al. (1998), Davidoff et al. (2001) Antisense oligonucleotides Bell et al. (1999), Im et al. (1999)

VEGF receptor Neutralizing monoclonal antibodies Prewett et al. (1999), Fenton et al. (2004), Li et al. (2005) Mutant VEGF receptor 2 (VEGFR-2) Machein et al. (1999)

Ribozyme targeted to mRNA Parry et al. (1999) Antisense oligonucleotides Rockwell et al. (1997)

Tyrosine kinase inhibitors Fong et al. (1999), Laird et al. (2000), Wood et al. (2000), Wedge et al. (2000), Zips et al. (2005)

Others Multitargeted kinase inhibitors, e.g., ZD6474 and SU11657

Williams et al. (2004), Damiano et al. (2005), Huber et al. (2005), Frederick et al. (2006)

Multitargeted growth factor-binding molecules

Sun et al. (2005)

Inhibitors of mTOR Shinohara et al. (2005; for further references see Chap. 9.4) Agents disrupting the tubulin cyto-

skeleton, e.g., ZD6126

Siemann and Rojiani (2005), Wachsberger et al. (2005)

Thalidomide Ansiaux et al. (2005)

HIV protease inhibitor ritonavir Maggiorella et al. (2005)

MN-029 Shi and Siemann (2005)

AMCA (lysine analog interfering with lysine binding sites of plasminogen)

Kal et al. (2004)

Metronomic chemotherapy Kerbel and Kamen (2004)

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Neutralizing antibodies against VEGF or VEGFR2 can reduce the growth rate of tumors and metastases in experimental animals (Kim et al. 1993; Asano et al. 1995; Borgström et al. 1996; Presta et al. 1997;

Prewett et al. 1999; Gorski et al. 1999; Bruns et al.

2000; Lee et al. 2000; Kozin et al. 2001; Gupta et al.

2002; Winkler et al. 2004); however, considerable het- erogeneity in response to anti-VEGF or anti-VEGFR antibodies has been observed. In some tumors treated with anti-VEGF antibodies a nearly complete inhibition of angiogenesis, a decline in vessel permeability, and a decrease in vascular density compared with control tumors has been demonstrated by intravital microscopy and histology (Borgström et al. 1996;

Yuan et al. 1996). Recent data suggest that VEGFR2 blockade increases tumor oxygenation, pericyte cov- erage of brain tumor blood vessels, and degradation of their pathologically thick basement membrane ( Winkler et al. 2004). To generate VEGF antibodies suitable for clinical trials, mouse monoclonal antibodies were humanized in order to combine high affinity to human VEGF with little or no immunogenicity (Presta et al. 1997; Ferrara and Alitalo 1999).

In a phase-I clinical trial including 25 patients with advanced solid tumors no dose-limiting toxicity up to the highest dose of recombinant human anti-VEGF monoclonal antibody (bevacizumab, 10 mg/kg body weight) was observed (Gordon et al. 2001); however, in this trial three episodes of tumor bleeding were noted. In two additional patients with known pulmo- nary metastasis minor hemoptysis occurred. It was not possible to discriminate whether these bleedings were caused by the treatment or by the underlying disease. In combination with chemotherapy rhumab- VEGF (3 mg/kg body weight) was safely administered without hemorrhagic complications ( Margolin et al.

2001). In a multicenter trial 99 patients with advanced non-small cell lung cancer were randomized to standard chemotherapy or standard chemotherapy plus rhumabVEGF (DeVore et al. 2000). Response rate and time to progression were increased after combined treatment. In six patients, four of them with a cen- tral tumor location, severe tumor bleeding occurred, which was fatal in four patients.

VEGF-dependent angiogenesis can also be sup- pressed by RTKIs. Agents such as SU5416, ZD4190, and PTK787/ZK222584, competitively inhibit ATP- dependent phosphorylation of tyrosine residues of the VEGF receptor resulting in a decrease in proliferation and survival of endothelial cells in vitro, angiogenesis in vivo, growth rate of tumor, and metastasis (Fong et al. 1999; Shaheen et al. 1999; Drevs et al.

2000; Laird et al. 2000; Mendel et al. 2000; Wedge

et al. 2000; Wood et al. 2000; Schuuring et al. 2005).

Although these compounds were designed to inhibit VEGFR2, some of them show also activity against other receptors involved in angiogenesis (Laird et al. 2000). In a clinical phase-I study on single-agent SU5416 treatment in patients with advanced malignant disease, the dose-limiting toxicity was vomit- ing, nausea, and severe headache (Cropp et al. 1999;

Rosen et al. 1999; Stopeck 2000). In a subsequent phase-I clinical trial SU5416 combined with cisplatin and gemcitabine in a total of 19 patients with solid tumors, an unexpectedly high rate of thrombem- bolic events was observed (Kuenen et al. 2002).

These discouraging results may be a consequence of the specific regimen of chemotherapy applied. This illustrates the importance of further, detailed investigations of interactions and optimal scheduling of administration (Marx et al. 2002). Of special importance in this context are pre-clinical investigations into normal tissue effects of combined approaches, exploiting suitable animal models.

7.4

Combination of VEGF Inhibition with Radiotherapy

7.4.1

Tumor Models

Whether anti-VEGF strategies in combination with radiotherapy can be used to improve tumor response was investigated in a number of experimental models (Table 7.3). In these studies different human and murine tumors, grown in experimental animals, were treated with various anti-VEGF compounds combined with single-dose or fractionated irradiation. Although VEGF inhibition alone had only a modest or no significant impact on tumor growth, the combination with irradiation consis- tently resulted in improved outcome. For example, in the study published by Gorski et al. (1999) anti-VEGF₁₆₅ antibody alone did not reduce tumor growth rate in U87 human glioblastoma xenografts, whereas the combination of 40 Gy given in eight fractions with anti-VEGF₁₆₅antibody 3 h before each fraction increased tumor growth delay compared with irradiation alone. In another study performed by Kozin and colleagues (2001) two different human xenograft tumors were treated with an anti-VEGFR2 antibody combined with fractionated irradiation. In both tumor models the combined treatment resulted

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in a statistically significant decrease of the dose necessary for permanent local tumor control compared with tumors that were only irradiated. This result demonstrates that anti-VEGFR antibody treatment increases the inactivation of clonogenic tumor cells and supports the hypothesis that anti-VEGF strategies may improve the outcome of curative radiotherapy; however, in the majority of the experiments listed in Table 7.3, tumor growth delay was

the experimental end point. Importantly, total dose, dose per fraction, overall treatment time, and the sequence of the modalities in combined treatment may impact on the outcome of combination treatment; hence, further pre-clinical studies in relevant animals models addressing these parameters need to be conducted to investigate whether the benefit of the combined therapy holds true for clinically relevant irradiation schedules.

Table 7.3. Pre-clinical in vivo studies testing anti-VEGF strategies in combination with radiotherapy. (Modiﬁ ed from Zips and Baumann 2003)

Compound Mechanism of VEGF inhibition

Tumor Treatment schedule End point Reference

SU5416 RTKI^a Murine SCC VII^b 5u2 Gy plus concomitant SU5416

Growth delay +^c Ning et al.

(2002) SU6668 RTKI Murine SCC VII 5u2 Gy plus concomitant

SU6668

Growth delay + Ning et al.

(2002)

SU6668 RTKI Human mammary

carcinoma SCK

Two applications of SU6668 prior to irradiation with a single dose of 15 Gy

Growth delay + Grifﬁ n et al.

(2002)

SU5416 RTKI Murine maligant

glioma GL261

Six fractions of 3 Gy with concomitant SU5416

Growth delay + Geng et al.

(2001) ZK 222584/

PTK 787

RTKI Human SW480 colon carcinoma

12 Gy in four fractions with concomitant ZK222584/

PTK787

Growth delay + Hess et al.

(2001)

A.4.6.1 Anti-VEGF antibody

Human glioblastoma U87, human colon adenocarcinoma LS174T

Single dose under normoxic or hypoxic conditions after antibody treatment

Growth delay + Lee et al.

(2000)

DC101 Anti-

VEGFR2 antibody

Human small cell lung cancer 54A, human glioblastoma U87

Five fractions in 5 days with six applications of DC101 every third day

Growth delay, tumor control rate

+ Kozin et al.

(2001)

DC101 Anti-

VEGFR2 antibody

Human SCC-1 or SCC-6 carcinoma

3 Gy twice weekly for 3.5 weeks with twice weekly DC101 for 3 weeks

Growth delay + Li et al.

(2005)

DC101 Anti-

VEGFR2 antibody

Mca-4 and Mca-35 mammary adenocarcinoma

Five fractions of 6 Gy with DC101

Growth delay + Fenton et al.

(2004)

Anti-VEGF₁₆₅ Anti-human VEGF₁₆₅ antibody

Human SCC SQ20B, Seg-1 human adenocarcinoma, human glioblastoma U87

Anti-VEGF₁₆₅ prior to irradiation with four or eight fractions

Growth delay + Gorski et al.

(1999)

Anti-VEGF₁₆₅ Anti-murine VEGF₁₆₅ antibody

Murine Lewis lung carcinoma

40 Gy in two fractions with anti-VEGF₁₆₅ prior to irradiation

Growth delay + Gorski et al.

(1999)

Anti-VEGF₁₆₅ Anti-human VEGF₁₆₅ antibody

Seg-1 human adenocarcinoma, human glioblastoma U87

Anti-VEGF₁₆₅ plus 20 Gy in four fractions (Seg-1) or 40 Gy in eight fractions (U87)

Growth delay + Gupta et al.

(2002)

aRTKI receptor tyrosine kinase inhibitor.

bSquamous cell carcinoma.

cBetter outcome with combined treatment compared to control tumor.

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7.4.2

Mechanisms Underlying the Enhanced Effect of Anti-VEGF Agents when Combined with

Irradiation

The observation that anti-VEGF compounds alone only modestly impact tumor growth rate while in combination with irradiation anti-VEGF agents con- sistently improve the response of tumors, suggests a radiosensitizing effect. This might be restricted to endothelial cells, because VEGF receptors are almost exclusively expressed on this cell type (Jakeman et al. 1992; Ning et al. 2002). This notion is supported by the fact that neither unirradiated nor irradiated tumor cells were affected by anti-angiogenic compounds (Gorski et al. 1999; Hess et al. 2001;

Gupta et al. 2002; Dings et al. 2005). The VEGFs are potent mitogens and survival factors for endothelial cells (see section 7.2). Irradiation of endothelial cells in vitro results in decreased proliferation and decreased clonogenic survival (De Gowin et al. 1974;

Hei et al. 1987; Haimovitz-Friedman et al. 1991;

Fuks et al. 1994; Gorski et al. 1999; Hess et al. 2001;

Kermani et al. 2001; Gupta et al. 2002; Brieger et al. 2005). When VEGF is added to culture media, the effects of irradiation on proliferation and survival of endothelial cells were attenuated. On the other hand, VEGF inhibitors reduce these effects of VEGF on irradiated endothelial cells (Gorski et al. 1999;

Hess et al. 2001; Gupta et al. 2002). In studies using another angiogenic growth factor, bFGF, increased clonogenic survival of irradiated bovine aortic endothelial cells was observed (Haimovitz-Friedman et al. 1991; Fuks et al. 1994). In these studies the slope of the dose–survival curves was not significantly different between endothelial cells incubated with or without bFGF; however, the shoulder region of the dose effect curve was almost completely eliminated when no bFGF was added to culture media. An inter- esting hypothesis of the mechanism of anti-VEGF combined with irradiation was proposed by Gorski and colleagues (1999) based on the fact that many cell types, including tumor cells, respond to irradiation with an increased release of growth factors such as VEGF or bFGF. These growth factors may in an autocrine and/or paracrine manner increase endothelial cell survival after irradiation (Witte et al. 1989;

Haimovitz-Friedman et al. 1991; Gorski et al.

1999; Gupta et al. 2002). The authors hypothesized that the inhibition of VEGF can abrogate the effects of radiation-induced VEGF on irradiated endothelial cells in tumors. This may result in increased radiation-induced endothelial cell kill which even-

tually leads to impaired tumor vascularization. As a result, the tumor cell production rate decreases and/or the tumor cell loss increases which may result in improved tumor response after irradiation. Fur- ther experimental studies are required to elucidate the complex interactions of the endothelial cell and the tumor cell compartment after a combination of anti-angiogenic compounds with irradiation.

7.4.3

Impact of Anti-VEGF Strategies on Radiobiological Hypoxia

Theoretically, anti-angiogenic agents might increase tumor hypoxia. Anoxic cells are less sensitive to irradiation compared with normoxic cells by a factor of about three (Gray et al. 1953; Wright and Howard- Flanders 1957); thus, inhibitors of angiogenesis, by decreasing vessel density or vessel function, might increase the proportion of poorly oxygenated clonogenic tumor cells and thereby decrease the efficacy of irradiation. A negative effect of anti-angiogenic agents on the results of irradiation was observed in an experiment by Murata and colleagues (1997).

The authors investigated the effect of TNP-470 on local tumor control after single-dose and fractionated irradiation of murine mammary carcinoma in C3H/He mice. The radiobiological hypoxic fraction of clonogenic tumor cells, as determined from the tumor control data after single-dose irradiation, was not affected significantly by TNP-470; however, for fractionated irradiation under normal blood flow conditions, tumors treated with TNP-470 were more resistant compared with control tumors. In contrast, no differences were observed for fractionated irradiation under clamp hypoxia. The authors concluded that inhibition of angiogenesis by TNP-470 impairs reoxygenation during fractionated irradiation and thus increases radioresistance of tumors. In a study on human colon carcinoma growing in nude mice, another anti-angiogenic compound, suramine, was also found to increase radiobiological hypoxia (Leith et al. 1992). Yet, both TNP-470 (Zhang et al. 2000) and suramine do not specifically inhibit VEGF or VEGFR but decrease angiogenesis at least in part by other mechanisms. Whether anti-VEGF drugs similarly increase radiobiological hypoxia was addressed in two experimental studies using anti- VEGF and anti-VEGFR antibodies (Lee et al. 2000;

Kozin et al. 2001). Tumor growth delay after graded single radiation doses given under normal blood flow conditions was compared with tumor growth delay

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after irradiations applied under clamp hypoxia. In both studies anti-VEGF and anti-VEGFR antibodies increased tumor growth delay compared with control tumors irrespective of the tumor oyxgenation status at the time of irradiation. These results suggest that VEGF inhibition does not increase the radiobiological hypoxic cell fraction in tumors. The discrep- ancy of reduced vessel density on the one hand but lack of increase in hypoxia, on the other hand, may be attributed to an impact on the quality of vascular organization by selective ablation of immature tumor blood vessels, a decrease in the number of oxygen consuming cells, and a decrease in vessel permeability (Benjamin et al. 1999; Lee et al. 2000).

Examination of FSAII tumors grown in mice demonstrated that anti-angiogenic treatment with thalidomide caused reoxygenation within 2 days, accompanied by a reduction of interstitial fluid pressure and increase in perfusion (Ansiaux et al. 2005).

7.4.4

Sequencing of Anti-VEGF Strategies Combined with Radiotherapy

The VEGF inhibitors can be administrated before (neoadjuvant), during (simultaneous) and/or after (adjuvant) a course of fractionated radiotherapy. No conclusion on the optimal sequence can be drawn from the studies listed in Table 7.3. Current data indicate that VEGF inhibitors have only minor effect on tumor volume when given alone. Neoadjuvant application should have only little effect on the number of clonogenic tumor cells that need to be killed by irradiation; thus, a substantial improvement of the results of radiotherapy by neoadjuvant VEGF inhibition can only be expected from different mechanisms such as more functional vascularization and abrogation of resistance-inducing microenviron- mental parameters. Table 7.3 shows that simultaneous application of VEGF inhibitors with fractionated irradiation improved the radiation response of several tumor models. These results might be caused by abrogation of the radioprotective effects of radiation-induced VEGF on endothelial cells. So far, the experimental results do not support that neoadjuvant or simultaneous application of VEGF inhibitors increases tumor hypoxia. To circumvent the possi- bility of increased hypoxia, anti-angiogenic agents may be applied after the end of fractionated irradiation. In a study by Murata and colleagues (1997) TNP-470 applied after a course of ten fractions in 2 weeks significantly increased tumor growth delay

compared with irradiation alone. In experiments by Zips et al. (2005), different combination schedules of a VEGFR tyrosine kinase inhibitor with fractionated irradiation of human squamous cell carcinomas in nude mice were compared. Short-term neoadjuvant and simultaneous administration showed no effect on tumor growth delay, whereas long-term adjuvant treatment resulted in prolonged tumor growth delay (but not increased cure rates). Arguments for adjuvant administration of VEGF inhibitors include the following: tumors may shrink after irradiation and smaller tumors are more likely to respond to anti- angiogenic compounds than larger tumors (Griffin et al. 2002). Irradiated vessels appear to be more sensitive to VEGF inhibition, which is supported by the observation that in vitro irradiated endothelial cells show an increased VEGFR2 expression (Kermani et al. 2001). An obvious disadvantage of the adjuvant sequence is that the radioprotective effect of VEGF on endothelial cells will not be counteracted.

Certainly, further studies to determine the optimal sequence of VEGF inhibition in combination with fractionated irradiation are needed.

7.4.5

Side Effects of Anti-VEGF Compounds Combined with Radiotherapy

In most of the studies listed in Table 7.3 the combination with irradiation was well tolerated. Skin reactions after irradiation of the subcutaneously growing experimental tumors on the hind leg or flank of the animals were not increased; however, after whole- body irradiation in combination with anti-VEGFR2 antibody DC101 40% of 54A tumor-bearing mice developed ascites and half of them died (Kozin et al.

2001). Histology revealed focal segmental glomeru- lonecrosis associated with increased expression of VEGFR2 on the surface of glomerular endothelial cells in the kidneys (Kozin et al. 2002). This indi- cates that, although angiogenesis in adult organisms is almost exclusively confined to tumor tissue, inhibition of VEGF-dependent processes may result in increased normal tissue reactions after irradiation.

This might be explained by the fact that VEGF does not only stimulate angiogenesis but is also important for survival and maintenance of normal function of endothelial cells. Such results underline the importance of further studies of normal tissue reactions, particularly those dominated by a long-term vascular component, after combined anti-VEGF strategies with radiotherapy.

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7.5

Trimodal Treatment with Anti-Angiogenic Agents, Radiotherapy, and Chemotherapy

Kumar et al. (2005) reported that combination treatment with low doses of the phosphatidyl-ino- sitol 3-kinase (PI3K) inhibitor LY294002, cisplatin, and irradiation resulted in significantly higher endothelial cell death as compared with each agent used alone. This combination treatment was equally effective in inducing tumor cell death. Combination treatment also significantly inhibited endothelial cell tube formation in Matrigel as compared with each of the agents used alone. In an in vivo severe combined immunodeficient mouse model, combination treatment with low doses of LY294002, cisplatin, and irradiation significantly inhibited the growth of human oral squamous carcinoma (OSCC-3) as well as prostate cancer (LnCap). The combination therapy was also effective in inhibiting tumor angiogenesis where it showed a greater than 90%

decrease in neovascularization. In contrast, combination treatment showed only a 29% inhibition of physiological angiogenesis.

Another group treated mice bearing Lewis lung carcinoma with combinations of intraperitoneal cisplatin, 5-fluorouracil, and the antiangiogenic agent genistein, together with 10 or 20 Gy of external beam radiotherapy (McDonnell et al. 2004). Ani- mals were sacrificed at day 6 when tumor volume, microvessel density, and serum VEGF were determined. Mean tumor volume in the chemoradiotherapy group was 35% larger than in the chemo- radiotherapy plus genistein group (p=0.04). The addition of genistein produced a significant reduction in tumor microvessel density as well as serum VEGF levels compared with those animals receiving chemoradiation alone.

Huber et al. (2005) found that in vitro and in vivo, the anti-endothelial and anti-tumor effects of the triple therapy combination consisting of SU11657 (a multitargeted small molecule inhibitor of VEGF and PDGF receptor tyrosine kinases), pemetrexed (a multitargeted folate antimetabolite), and ionizing radiation were superior to all single and dual combinations. The superior effects in human umbilical vein endothelial cells and tumor cells (A431) were evident in cell proliferation, migration, tube formation, clonogenic survival, and apoptosis assays.

Triple therapy induced greater tumor growth delay than all other therapy regimens without increas- ing apparent toxicity. When testing different treatment schedules for the A431 tumor, these authors

found that the regimen with radiotherapy (7.5 Gy single dose), given after the institution of SU11657 treatment, was more effective than radiotherapy preceding SU11657 treatment. The SU11657 mark- edly reduced intratumoral interstitial fluid pressure after 1 day.

Among the multiple pathways altered by epidermal growth factor receptor (EGFR) inhibitors, angiogenesis appears to play an important role. Data on combined treatment with radiotherapy, chemotherapy, and EGFR inhibitors are reviewed in Chap- ter 8.6.1.

7.6

Conclusion

Pre-clinical experiments in a variety of tumor models indicate that inhibition of angiogenic growth factors or growth factor receptors in combination with irradiation is a promising concept for the improvement of the radiation response of tumors, although most data relate to tumor growth delay rather than cure.

The mechanisms underlying this approach are not fully understood to date. Both, radiosensitization of endothelial cells by VEGF inhibition as well as sensi- tization of endothelial cells against anti-angiogenic agents by irradiation appear to play an important role. In addition to approaches targeting VEGF and its pathways, a variety of other strategies are under investigation. Recently, evaluation of trimodal combinations has started, because simultaneous radio- chemotherapy is now standard treatment for many types of cancer. Whether such strategies improve the therapeutic window remains to be determined.

So far, systematic data on long-term toxicity in many critical organs are lacking. Future research must include investigations of the efficacy of angiogenesis inhibition (and normalization of the microenvironment) in combination with irradiation and cytotoxic drugs with regard to dose-fractionation parameters, optimization of the dosing and sequence, identifica- tion of predictive factors for response, and studies on normal tissue toxicity in relevant experimental animal tumor and normal tissue models as well as early clinical trials. Examples of such trials in gas- trointestinal tumors are discussed in more detail in Chapter 15.6.4. In brief, preliminary experience suggests that changes in tumor perfusion and interstitial intratumor pressure occur, and that acute toxicity of bevacizumab plus radiotherapy is acceptable. Further data have been published for

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the combination of radiotherapy and temozolomide with thalidomide evaluated in a phase-II trial Chang et al. (2004). This regimen was administered to 67 patients with newly diagnosed glioblastoma. It was relatively well tolerated and showed a median survival of more than 16 months. Whether this represents an improvement over trials without thalidomide (median survival 1316 months) requires prospective confirmation.

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