The EGFR (ErbB) Family of Receptors in Cancer

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8 Combinations of Cytotoxic Drugs, Ionizing Radiation and EGFR Inhibitors

Guido Lammering

G. Lammering, MD, PhD

Department of Radiation Oncology and Laboratory of Experi- mental Radiation Oncology, University of Maastricht, 6401 CX Heerlem, The Netherlands

8.1 The EGFR (ErbB) Family of Receptors in Cancer

Growth factors and their receptors play a key role in the development and progression of human cancers.

They are overexpressed or aberrantly expressed in many cancers, which results in unregulated cell sig- nalling, dysregulation of growth, tumour initiation or promotion, and invasion and metastasis, thus contributing to at least four of the six hallmarks of cancer (Hanahan and Weinberg 2000).

Within the growth factor receptors, the ErbB family of receptor tyrosine kinases and related plasma membrane receptors has been identi- fied as critical components facilitating autocrine growth regulation that are typically the result of coordinated co-expression of growth factors and

their receptors (Weinberg 1989; Baselga and Mendelsohn 1994). Although the plasma mem- brane components share important similarities as 170- to 200-kD transmembrane glycoproteins, each ErbB species carries a specific function within the ErbB-receptor Tyr kinase response network (Riese and Stern 1998). ErbB 1 (EGFR) and ErbB 4 are complete receptors with growth factor binding sites in the extracellular NH2-portion and a Tyr kinase domain in the cytoplasmic COOH-terminal portion of the molecule. ErbB 2 represents a constitutively active receptor without a ligand binding domain, and ErbB3 shares ligand specificities with ErbB4 but lacks Tyr kinase activity; therefore, ErbB 2 and ErbB 3 represent important modulators of cellular response to growth factors through heterodimerisa- tion with ErbB 1 and ErbB 4 (Riese and Stern 1998;

Earp et al. 1995).

These different properties of ErbB receptor tyro- sine kinases determine the nature of their interac- tions with defined homo- and heterodimerisation hierarchies and result in receptor activation. The ErbB receptors mediate their proliferative signals through a major cytoprotective signalling pathway involving the adapter proteins (i.e. GrB2 and SHC), GTP exchange factors, such as SOS, phospholipase CJ (PLCJ), Ras, protein kinase C (PKC), Raf, MAPK and PI-3-kinase-dependent pathways. These sig- nalling pathways directly or indirectly affect cell- cycle control and transcription regulation initiating the biosynthetic machinery and cell proliferation (Schmidt-Ullrich et al. 1999).

8.1.1 EGFR

All cells of epithelial origin as well as many cells from mesenchymal derivation express the EGFR. A primary function of the EGFR revolves around its capacity to influence cellular growth, proliferation and differentiation. In recent years, many reports have confirmed an overexpression of EGFR in epi-

CONTENTS

8.1 The EGFR (ErbB) Family of Receptors in Cancer 115

8.1.1 EGFR 115

8.1.2 EGFR Variations 116

8.2 EGFR and Treatment Resistance 116 8.3 EGFR and Irradiation 117

8.4 Approaches to Inhibit EGFR During Irradiation 118

8.4.1 The Antibody and Dominant-Negative Approach 118

8.4.2 The Small Molecule Approach 118 8.5 Combination of EGFR Inhibitors and Irradiation 119

8.6 Combinations of Cytotoxic Drugs, Irradiation and EGFR Inhibitors 121

8.6.1 C225 or EGFR Tyrosine Kinase Inhibitors and Radiochemotherapy 121

8.7 Conclusion 122 References 123

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thelial tumours. As is the case with other growth factor receptors, increased EGFR activation can result from higher levels of ligand (such as EGF), EGFR gene amplification, increased transcription or mutations that cause unregulated receptor sig- nalling. A correlation between EGFR overexpres- sion and disease stage, disease progression, patient survival and response to therapy has been put forth for a variety of the most common human malig- nancies (Wells 2000). Although this correlation between EGFR overexpression and poor clinical outcome appears convincing, a direct cause and effect relationship has yet to be firmly established.

It is certainly possible that the specific reliance of a particular cell type or tissue on the EGFR pathway for growth is more important than the arithmetic quantification of EGFR.

8.1.2 EGFR Variations

Overexpression of EGFR is sometimes associ- ated with the expression of mutated species. One mutant EGFR, named EGFRvIII, lacks a portion of the extracellular ligand-binding domain, leading to a constantly active tyrosine kinase (Huang et al.

1997). EGFRvIII is not found in normal tissues but is expressed on the cell membrane in certain tumours including gliomas, prostate, breast, non-small cell lung, colorectal and ovarian cancers (Moscatello et al. 1995). Mutations of the EGFR kinase domain have also been reported, and recent studies indicate that its frequency is rare in most types of human cancer apart from that of lung adenocarcinoma (Sihto et al. 2005). Studies of a large number of lung tumour patients identified a frequency of 24%

for EGFR mutations in the tyrosine kinase domain (exons 18–21) with a higher incidence for female, young, non-smoking patients. Importantly, these mutations seem to identify distinct subsets of lung cancer patients with an increased response to an EGFR-inhibiting drug called gefitinib (Pao et al.

2004).

8.2 EGFR and Treatment Resistance

Alterations in chemosensitivity have been noted in preclinical studies of EGFR-overexpressing tumour cell lines. Indeed, higher levels of expres-

sion of drug-resistance-related proteins, such as topoisomerase II and p-glycoprotein, are found in untreated EGFR-positive renal tumours. Ogawa et al. (1993) measured EGFR expression and cisplatin sensitivity in tumour tissues from 84 patients with lung cancer. The EGFR expression was significantly higher in tumours that were resistant to cisplatin compared with cisplatin-sensitive tumours (Ogawa et al. 1993). Similarly, patients with ovarian cancer who have EGFR-positive tumours or increased transforming growth factor (TGF)-D expression have a lower rate of response to chemotherapy with cisplatin compounds compared with patients with lower EGFR levels (Fischer-Colbrie et al. 1997).

Santini et al. (1991) reported that patients with head and neck tumours in which EGFR expression levels were >100 fmol/mg protein had a lower probability of response to chemotherapy than did patients with EGFR levels <100 fmol/mg protein.

Furthermore, an association between EGFR expression and clinical radioresistance has been reported in patients with cancer. Ang et al. (2004) and Giralt et al. (2002) reported a correlation between EGFR overexpression and response to radi- otherapy in human head and neck cancers or rectal cancer, respectively. The EGFR expression was a sig- nificant and independent prognostic indicator for overall survival and recurrence-free survival after radiation therapy in patients with astrocytic glio- mas (Zhu et al. 1996). Pillai et al. (1998) noted that patients who had residual or recurrent disease after radiotherapy for cervical cancer had more EGFR expression than those patients who were disease- free. Other authors found an inverse correlation between EGFR expression and radiocurability in murine carcinomas (Akimoto et al. 1999). Treat- ment of EGF protected cells against radiation in cul- ture, whereas treatment with an antibody against EGFR induced radiosensitisation (Balaban et al.

1996). While preclinical studies indicate that EGFR inhibition can sensitise many tumour cells to ion- izing radiation, in vitro sensitisation with cell lines may not reflect the prognostic implications of EGFR overexpression in vivo.

Together, the currently available data suggest that higher levels of EGFR may be associated with chemo- and/or radioresistance in some tumours.

These findings therefore stimulated the research

of targeted modulation of EGFR function as a new

therapeutic strategy; however, the current data are

insufficient to suggest using EGFR expression as a

predictor of response to chemo- and/ or radiother-

apy in general. It is probably more valuable to con-

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sider modifying EGFR activity to enhance chemo- and/or radiotherapy.

8.3 EGFR and Irradiation

Over the past decade, molecular biological approaches applied to radiobiological questions have uncovered several mechanisms by which cells respond to ionizing radiation (Schmidt-Ullrich et al. 2003). The DNA damage responses are argu- ably the most critical, although it is unclear if they can account for the variability in response observed between different tumours and patients. Radiation effects on protein expression and activation of cel- lular signalling are also important. Three princi- pal consequences of EGFR activation have been shown to be important for tumours and radiation response:

1. Proliferation and cell cycle effects. The inﬂ uence of EGFR on tumour cell proliferation has been mainly established by studies investigating the capacity of anti-EGFR agents to slow tumour pro- liferation and modulate cell cycle phase distribu- tion (Huang et al. 1999, 2002).

2. Anti-apoptosis and survival. The EGF has been demonstrated in some cell lines to prevent apop- tosis or promote survival in cells that overexpress EGFR (Rodeck et al. 1997). Many studies on inhi- bition of EGFR stimulation suggest that activation of growth factor receptors, such as EGFR, may have a role in promoting cell survival in some tumours (Modjtahedi et al. 1998).

3. Angiogenesis. Several oncogenic growth factors and their receptors, including EGF and EGFR, are thought to play a role in tumour angiogenesis as evidenced by numerous studies (Fox et al. 1996).

The EGFR activation has been shown to upregu- late VEGF production, whereas EGFR inhibition signiﬁ cantly effects tumour angiogenesis and reduces VEGF expression (Huang and Harari 2000).

The exact mechanisms by which the EGFR family pathway mediates resistance to radiation have been investigated over the past years. Recent studies have been able to demonstrate that irradiation of tumour cells at clinically relevant dose levels can result in an immediate activation of the EGFR family of recep- tors, and that repeated radiation exposures of 2 Gy lead to an increased expression of EGFR (Schmidt-

Ullrich et al. 1994, 1996). The underlying molecu- lar mechanisms of the radiation-induced activation of EGFR remain to be understood. First studies indicate, however, that reactive oxygen species after ionizing radiation inhibit protein-tyrosine phos- phatases, such as SHP-2, which then specifically phosphorylate the Tyr-992 residue of EGFR (Sturla et al. 2005).

The radiation-induced activation of EGFR is defined by a several-fold increase in the tyrosine phosphorylation with a secondary activation of existing signalling transduction cascades. These pathways, which mainly include protein kinases, induce cytoprotective and cytotoxic responses from cell survival to cell death. Cytoprotective responses include signalling cascades of the mitogen-activated protein kinase (MAPK) and the phosphatidyl-inosi- tol-3 phosphate kinase (PI3K), which activate the biosynthetic cascade and therefore possibly stimu- late cell proliferation. Considering the increased biosynthetic activity of rapidly proliferating tumour cells, it can be assumed that this will improve the capacity for DNA damage repair. Interestingly, emerging data suggest a novel mechanism by which EGFR after irradiation might improve the capac- ity for DNA repair. Ionizing radiation, but not EGF, induces the import of EGFR into the nucleus. In the nucleus, EGFR then activates the DNA-dependent kinase (DNA-PK) to improve the DNA repair capac- ity (Dittmann et al. 2005).

The radiation-induced activation of the EGFR family of receptors leads to a dose-dependent prolif- erative response, which can be observed after single, as well as repeated, radiation exposures (Contessa et al. 1999; Reardon et al. 1999). This cellular pro- liferative response after repeated radiation expo- sures leads to increased renewal of tumour clono- gens (Withers et al. 1988; Fowler 1991). It has therefore been concluded that the radiation-induced activation of EGFR is involved in the mechanism of accelerated proliferation or repopulation. Because the radiation-induced proliferative, as well as the improved DNA repair responses of tumour cells, counteract the toxic effects of radiotherapy, they are defined as the EGFR-family-induced cytoprotective responses of tumour cells.

Taken together, the radiation-induced cytopro-

tective responses of tumour cells might potentially

be induced at the level of the cell membrane through

activation of the EGFR family and other involved

molecules (Fig. 8.1); therefore, the blockade of

EGFR function could result in radiosensitisation in

tumour cells.

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8.4 Approaches to Inhibit EGFR During Irradiation

Since EGFR and the other ErbB receptors have emerged as promising targets in radiotherapy, exten- sive research activity explores potential procedures to target EGFR, the other ErbB receptors and/or its downstream pathways for radiotherapy to enhance radiation action in human carcinomas and malig- nant gliomas. Over the past several years, it has been recognised that antibodies, small-molecule inhibitors and genetic modulation can be used therapeutically to disturb EGFR and/or ErbB signalling at the cellular level during irradiation. These approaches include monoclonal antibodies and dominant-negative EGFR directed against the receptors and synthetic tyrosine kinase inhibitors that act directly on the cytoplasmic domain of EGFR and/or other ErbB molecules.

8.4.1 The Antibody and Dominant-Negative Approach

A number of antibodies have been generated against EGFR. Among the most promising is IMC-

225 (Cetuximab), a humanised M225, which has a higher affinity for EGFR with a longer half-life (Mendelsohn 1997; Goldstein et al. 1995). C225 was found to compete with EGF binding, inhibit EGF-induced tyrosine kinase-dependent phos- phorylation, and downregulate EGFR expression by inducing receptor internalisation. Another mono- clonal antibody 806 is a novel EGFR antibody with significant antitumour activity that recognises the mutant EGFRvIII and a subset of EGFR found in cells that overexpress EGFR (Perera et al. 2005).

Another approach currently in preclinical and clinical development is the functional inhibition of the ErbB-receptor tyrosine kinase network through gene therapeutic expression of a dominant-negative EGFR mutant called EGFR-CD533. EGFR-CD533, a mutant of EGFR, lacks the entire cytoplasmic domain of 533 amino acids and confers no trans- formation or proliferation-promoting activity (Kashles et al. 1991; Redemann et al. 1992). This dominant-negative EGFR exerts its effect at the pro- tein level through the formation of non-functional receptor complexes with the ErbB-receptor tyrosine kinase family (Kashles et al. 1991; Lammering et al. 2001).

8.4.2 The Small Molecule Approach

There exist a growing number of EGFR/ErbB inhibi- tory small molecule compounds in various stages of preclinical and already clinical development. For tyrosine kinase inhibitors to be used therapeuti- cally, they must be highly specific. The inhibitors studied to date have varying levels of specificity.

Several quinazoline derivates have been developed that are more selective for EGFR than for other tyrosine kinase receptors. They act by competitively inhibiting ATP binding (Fry et al. 1994). ZD1839 (Gefitinib, Iressa) is an anilinoquinazoline with an IC50 of 20 nM for the EGFR tyrosine kinase. It binds reversibly to EGFR tyrosine kinase, whereas EKB-569 is an irreversible inhibitor of EGFR. OSI- 774 (Erlotinib, Tarceva) is a quinazoline analogue with a nanomolar IC50 for reversible inhibition of EGFR activity and high specificity for the receptor (Woodburn 1999).

Besides these selective approaches to target EGFR only, several dual inhibitors have also been developed. CI-1033 (Canertinib) is a quinazoline that irreversibly inhibits EGFR and ErbB-2 tyro- sine kinases. PKI-166 and GW-572016 (Lapatinib)

Fig. 8.1. Model of the radiation-induced EGFR-family-mediated pro-proliferative and cytoprotective responses to irradiation

Radiation activation

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represent potent pyrrolopyrimidines competitively reversibly binding to the ATP binding site of EGFR and ErbB-2 tyrosine kinases. The compound ZD 6474, on the other hand, is a potent VEGF receptor- 2 tyrosine kinase inhibitor with additional activity against the EGFR (Table 8.1).

8.5 Combination of EGFR Inhibitors and Irradiation

Studies using A431 human carcinoma xenografts in nude mice, combining C225 with local tumour radiation, demonstrated enhanced tumour radiore- sponse by a factor of 1.6 when a single dose of C225 was used and 3.6 when three doses of C225 were used (Milas et al. 2000). Other groups confirmed the significantly enhanced clinical response in a wide spectrum of epithelial human tumour xeno- grafts that received the combination of radiation plus C225 (Huang et al. 1999; Saleh et al. 1999).

Other authors recently demonstrated encouraging effectiveness of C225 when administered systemi- cally in an intracranial model of an EGFR-ampli- fied glioblastoma. The median survival of these animals increased by at least 900% (Eller et al.

2005). Interestingly, the interactions of C225 and radiation in general were more profound in vivo than initially predicted by in vitro studies, sug- gesting that mechanisms beyond simple prolifera-

tive growth inhibition act in the in vivo setting (Harari and Huang 2001).

Multiple mechanisms seem to underlie the enhancement of tumour response to radiation by C225 involving both direct and indirect interaction with tumour cells. They may include C225-induced inhibition of DNA damage repair, enhanced radio- sensitivity originating from specific disturbances in cell-cycle phase distribution, enhancement of radiation-induced apoptosis, inhibition of tumour angiogenesis and effects on tumour cell migration and invasion capacity (Harari and Huang 2002).

Huang and Harari (2000) reported that treatment with C225 could cause redistribution of DNA-PK from the nucleus to the cytosol resulting in reduced radiation-induced DNA damage repair. C225 signif- icantly inhibits formation of new vessels, suggesting a direct inhibitory effect of C225 on tumour angio- genesis possibly through inhibition of mRNA and protein production of angiogenic factors (Milas et al. 2000; Perrotte et al. 1999). In contrast, the mechanisms by which C225 inhibits EGFR at the protein level are not fully understood. For example, it has not been investigated whether the radiosen- sitising effects of C225 might also be explained by the blockade of EGFR heterodimers formation and receptor crosstalk, which would support a criti- cal involvement of other EGFR family members and other growth factors and their receptors in the enhanced radiosensitivity.

Functional inhibition of the ErbB-receptor tyro- sine kinases through dominant-negative EGFR (DN-

Table 8.1. EGFR/ErbB inhibitory small molecule compounds. TKI tyrosine kinase inhibitors

TKI compounds Target Action Tested with

radiotherapy

Reported mechanisms of enhanced radiation response

Reference

ZD1839 (geﬁ nitib) EGFR Competitive ATP In vitro/in vivo Antiproliferation, DNA damage, apoptosis, antivascular

Solomon et al. (2003);

Williams et al (2002);

Tanaka (2005) OSI774 (erlotinib) EGFR Competitive ATP In vitro/in vivo Cell cycle, apoptosis,

repopulation, DNA damage

Chinnaiyan et al. (2005)

EKB569 EGFR Irreversible (Cys 773) − − −

PKI-166 EGFR,

ErbB 2

Competitive ATP − − −

GW-572016 (lapatinib)

EGFR, ErbB 2

Competitive ATP In vitro Radiosensitisation Zhou et al. (2004)

CI-1033 (canertinib) EGFR, ErbB 2

Irreversible (Cys 773) In vitro/in vivo Radiosensitisation Rao et al. (2000);

Nyati et al. (2004) ZD6474 (zactima) EGFR,

VEGFR2

Competitive ATP In vitro/in vivo Antiproliferation, tumour perfusion, antiangiogenesis

Damiano et al. (2001);

Williams et al. (2004)

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EGFR) has also been shown to significantly sensitise a broad spectrum of tumour cells independent of the varying ErbB-receptor expression levels. The fur- ther developed approach of DN-EGFR application in human xenograft tumours also effectively induced tumour radiosensitisation, providing further proof of the promise of EGFR inhibition to enhance radio- sensitivity in human tumours (Lammering et al.

2001).

Just as with C225, there is solid preclinical data regarding the capacity of ZD1839 and CI-1033 to enhance radiation efficacy in both in vitro and in vivo model systems (Huang et al. 2002; Raben et al. 2001; Rao et al. 2000; Williams et al. 2002).

ZD1839 has been proven to enhance the cytotoxic- ity of radiation across a spectrum of human cancer cell lines including lung, pancreas, malignant glioma, colon and head and neck (Stea et al. 2003;

Solomon et al. 2003; Baumann et al. 2003). In a more recent publication, however, ZD1839 failed to act as a radiosensitiser in vitro in concomitant association with radiation. The drug brought about additive to subadditive interaction with radiation with regard to growth inhibition, clonogenic death and induction of apoptosis, but it did not hinder the rejoining of radiation-induced DNA double- strand breaks in any cell line tested (Giocanti et al. 2004). This finding is in contrast to another more recent presentation at the Annual Meeting of the American Association for Cancer Research 2005, strongly supporting the inhibitory effect of ZD1839 on the repair of double-strand breaks after ionizing radiation in two non-small cell lung cancer (NSCLC) cell lines (Tanaka 2005). These conflicting data highlight the growing body of evi- dence of the different efficacy of anti-EGFR strate- gies on different cellular characteristics. It could well be that successful modulation of radiosensi- tivity through inhibition of DNA repair with the help of anti-EGFR approaches depends on specific molecular characteristics such as the ras genotype (Toulany et al. 2005).

For human colon carcinoma xenografts, the combination of ZD1839 with radiation enhanced the therapeutic effect compared with radiation alone by a factor of 1.6 (Williams et al. 2002).

Growth delay assays with ZD1839 in combination with radiation for human squamous cell carci- nomas revealed a significant synergistic tumour growth inhibition effect (Huang et al. 2002). In line with these data on ZD1839 and radiotherapy, Baumann et al. (2003) also found improvement of growth delay with BIBX1382BS, which is an

inhibitor like ZD1839 in combination with radia- tion for FaDu human squamous cell carcinoma;

however, they could also clearly demonstrate that despite the antiproliferative activity in these rap- idly repopulating FaDu cells and the significantly increased tumour growth delay, local tumour con- trol was not improved. These findings highlight that significant effects on tumour growth delay do not necessarily reflect the efficacy of anti-EGFR small molecule inhibitors in combination with radiation on curative potential (Krause et al.

2005). Furthermore, the efficacy of this combina- tion on tumour cell radiosensitisation might not only purely be dependent on the presence of EGFR, but also on other cellular molecular characteris- tics, which are not easily understood at present ( Toulany et al. 2005).

Preclinical studies in ErbB-overexpressing human breast cancer cells identified a supra- additive effect of CI-1033 with fractionated radia- tion (Rao et al. 2000). Besides the mechanism of inhibition of EGFR-induced cancer cell prolifera- tion, small molecule tyrosine kinase inhibitors also seem to potentiate the antitumour activity of radiation in part by effects on cellular apoptosis and angiogenesis (Huang et al. 2002). This dem- onstrated inhibition of neoangiogenesis in human tumour xenografts may result in improved blood supply to the tumour, leading to reoxygenation and increased radiosensitivity, thereby improving radiocurability. In contrast to C225, small-mole- cule tyrosine kinase inhibitors of EGFR might also be active against the naturally occurring mutant EGFRvIII, since EGFRvIII has an intact, intracel- lular kinase domain. This is particularly relevant because of the emerging importance of EGFRvIII in several human carcinomas and malignant glio- mas (Moscatello et al. 1995; Wikstrand et al.

1997).

Taken together, the currently available data regarding the combination of EGFR inhibi- tors and irradiation have convincingly shown that both the antibody and the small molecule approach to target EGFR are able to afford poten- tiation of radiotherapy through inhibition of tumour repopulation, DNA damage repair, cell cycle kinetics, antiapoptosis and angiogenesis.

The individual response of anti-EGFR strategies

in combination with radiotherapy, however, may

be explained by complex specific molecular and

cellular characteristics, which include mutations

at the level of EGFR itself or at the level of down-

stream effectors.

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8.6 Combinations of Cytotoxic Drugs, Irradiation and EGFR Inhibitors

The significant interest and investigation of EGFR as a molecular target for radiotherapy or chemother- apy and the translation of discoveries in molecular biology into clinically relevant therapies has led to further preclinical investigations of the efficacy of combining anti-EGFR approaches, not only with radiotherapy, but also with radiochemotherapy, since radiochemotherapy is already considered the standard therapy for many epithelial tumours in the clinic.

The mode of combining chemotherapeutic drugs with radiation takes advantage mainly of the sensitis- ing effects of drugs on cell kill by radiation. The addi- tional use of agents that counteract molecular deter- minants or processes responsible for resistance of cancer cells to radiation or chemotherapeutic drugs, however, might further improve efficacy without the expense of an increased normal tissue toxicity;

therefore, first investigations have been undertaken to combine anti-EGFR agents with radiochemother- apy regimen. These investigations not only help us to better characterise the synergistic possibilities of anti-EGFR approaches with the combination of chemotherapy and radiotherapy, they also identify insufficient and antagonistic combination treat-

ments, which should be avoided. Most preclinical data on the combined use of anti-EGFR, chemother- apy and radiotherapy are currently only available for C225 and ZD1839, which are also the most clinically developed of the compounds described.

8.6.1 C225 or EGFR Tyrosine Kinase Inhibitors and Radiochemotherapy

In both in vitro and in vivo preclinical studies, C225 was shown to enhance the antitumour effects of the chemotherapeutic drugs doxorubicin, cisplatin, pacl- itaxel, topotecan and gemcitabine (Mendelsohn and Fan 1997; Ciardiello et al. 1999; Inoue et al. 2000).

Interactions between C225 and chemotherapy may be based on comparable mechanisms, as described for radiotherapy, such as inhibition of DNA repair mechanisms, alteration in growth factor production, promotion of apoptotic cell death and anti-angiogen- esis. Based on these previous studies and the known radiosensitising effect of C225 as well, Buchsbaum et al. (2002) hypothesised that the combination of C225 with gemcitabine, which also represents a known radiosensitiser, and radiotherapy against human pancreatic cancer cells and tumour xenografts, would provide greater efficacy than either treatment alone or any combination of two treatments. The results

Table 8.2. Preclinical results of EGFR-dependent enhanced radiation responses. DER dose enhance- ment ratio

Approach Fractionation In vitro assay DER In vivo assay DER Reference

DN EGFR Single 1.4−1.6 1.6−1.9 Lammering et al. (2001)

Multiple 1.4−1.8 1.8−3.8

C225 Single 1.2−1.3^a Huang et al. (1999)

Multiple 1.3^a >4.0 Huang and Harari (2000)

Single/multiple 1.2−1.4^a 1.6−3.6 Milas et al. (2000)

ZD1839 Single <1.0 1.5; 1.6 Giocanti et al. (2004);

Solomon et al. (2003);

Multiple 1.2 3.3^a; 4.0

OSI-774 Single 1.3−1.5^a 2.0−3.8^a Chinnaiyan et al. (2005)

Multiple

CI-1033 Single 1.7^a 1.4 Rao et al. (2000)

Multiple 1.0−1.4 >4.0^a Nyati et al. (2004)

ZD6474 Single 1.8−3.0^a 2.4^a Damiano et al. (2005)

Multiple 1.3−1.5 concurrent,

1.6 sequential

GW572016 Single 1.2−1.3^a − Zhou et al. (2004)

aEstimated from data. Method: in vitro, D₃₇ or D₁₀; in vivo, ex vivo clonogen or growth delay

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indeed identified a significantly greater efficacy for the triple combination treatment. The in vitro results indicated a significant inhibition of cell pro- liferation and induction of apoptosis. The xenograft tumours showed a remarkable growth inhibition with a 100% regression for MiaPaCa-2 tumours for more than 250 days and the greatest growth inhibi- tion for BxPC-3 tumours compared with any single or dual treatment. The differences in the magnitude of response between the two cell lines again could relate to differences in molecular characteristics, such as growth factor receptors, and angiogenic factors, such as VEGF. In another study combining C225 with cis- platin and radiotherapy in NSCLC, however, the triple therapy yielded only a nonsignificant advantage in tumour growth control over doublet combinations.

In fact, cisplatin in combination with radiotherapy resulted in more comparable growth delay than cetuximab in combination with radiotherapy; thus, this study provided no rationale for the combined treatment of C225 and cisplatin during radiotherapy in NSCLC (Raben et al. 2005).

In another study on the combined use of C225 with radiochemotherapy, Nakata et al. (2004) pro- vided strong evidence for the potent enhancement of tumour response of C225 to docetaxel when com- bined with radiation. These in vivo growth delay assays were performed with a breast cancer cell line MDA468 and a squamous carcinoma cell line A431.

The addition of C225 to the treatment of docetaxel and radiation (1u10 Gy) in A431 increased the growth delay enhancement factor from 1.94 with docetaxel alone during radiation to an enhancement factor of 3.98. For MDA468 tumours, the enhancement in growth delay through docetaxel during single dose or fractionated radiation reached enhancement factors of 1.3 and 1.9, respectively. The additional application of C225 improved the enhancement in growth delay to factors of 5.2 and 3.2 for single dose or fractionated radiation; thus, docetaxel seems to represent an ideal cytotoxic drug to be used in combination with C225 to achieve further enhancement of tumour radiore- sponse. The mechanisms are multiple and include direct effects on the tumour cells or indirect effects through affecting tumour angiogenesis or tumour microenvironment. Docetaxel has been shown to enhance tumour oxygenation and mitotic arrest, whereas C225 interferes with cellular repair pro- cesses, inhibits the production of important angio- genic factors and improves endothelial cell damage;

thus, C225 and docetaxel might synergistically con- tribute to the radiation-induced damage at the level of the tumour as well as the endothelial cell.

There rarely exist any data on the preclinical evaluation of the synergistic enhancement of radio- sensitivity through the combined use of EGFR- tyrosine kinase inhibitors and chemotherapeutic agents. Although the rationale for combining EGFR tyrosine kinase inhibitors with radiochemotherapy seems convincing, no studies have yet preclini- cally defined the best sequence and the best com- bination cytotoxic drugs for the triple treatment. In vitro data suggest the application of EGFR tyrosine kinase inhibitors to being given before and during cytotoxic drug exposure, which was also the case for the combination with radiation (Magne et al. 2002);

therefore, the overall recommendation exists that EGFR tyrosine kinase inhibitors be applied before and during radiochemotherapy. Phase-II and phase- III trials have already been started mainly in head and neck and lung cancer patients with ZD1839 or OSI-774 and chemotherapy plus fractionated radio- therapy, without awaiting further preclinical inves- tigations regarding optimised combinations and optimised sequencing.

One remaining concern of all triple-treatment combinations is the lack of knowledge of a further increase in normal tissue toxicity, a major limitation of concurrent radiochemotherapy; thus, studies on the effect of EGFR inhibitors on radiochemotherapy- induced normal tissue injury are urgently needed.

8.7 Conclusion

The rationale for targeting the EGFR family in com-

bination with radiotherapy and/or chemotherapy

has been established and clinical trials are already

in progress. The mechanisms by which the EGFR

family mediates resistance to radiotherapy are mul-

tiple and complex and include proliferative, anti-

apoptotic and pro-angiogenic responses as well as

an enhancement in the cellular capacity for DNA

repair. Importantly, ionizing radiation activates the

EGFR family, which leads to an increased activation

of the existing downstream signalling pathways, like

the MAPK and the PI3K pathway, thereby contribut-

ing to the onset of radioresistance. Although both

approaches to target EGFR, the anti-EGFR antibody

(C225) and the EGFR tyrosine kinase inhibitors

confer a significant improvement in the radiosen-

sitivity of human tumour cells in vitro as well as in

vivo, the antibody approach seems to provide a more

profound enhancement of the tumour response to

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radiation. This suggests that other mechanisms of C225, which modulate the microenvironment in vivo, e.g. inhibition of tumour angiogenesis and induced reoxygenation, also significantly contrib- ute to the effects of the enhanced radiosensitivity induced by C225 in vivo (Krause et al. 2005). Prom- ising in vivo data provide first evidence that C225 and chemotherapeutic drugs, such as docetaxel and gemcitabine, might synergistically attribute to the radiation-induced damage in tumours. In general, however, conflicting data continue to exist regard- ing the varying efficacy of the different anti-EGFR strategies in combination with radiation and/or che- motherapy. One important explanation is that some approaches depend on specific molecular and cel- lular characteristics in order to successfully modu- late radiosensitivity. Specific mutations, either at the level of EGFR or at the level of downstream effectors, might correlate to the individual response and the large variety of response depending on the cancer cell line. In this regard, the PI3K/Akt pathway may represent the most important mediator of the cyto- protective responses induced by EGFR. More inves- tigations at all levels of cellular responses to radia- tion are necessary in order to better understand the variation in the effectiveness of responses to anti- EGFR strategies in combination with radiation. This will also provide more insight into the molecular characteristics, which could predict effectiveness of the anti-EGFR approach to radiosensitisation.

It is also still unclear if it is necessary to achieve complete EGFR blockade or significant inhibition of downstream effector kinases, such as Akt or MAPK, and if other ErbB receptors, or even other receptor families, might counteract the radiosensitisation induced by EGFR blockade due to possible compen- satory mechanisms in fractionated radiation regi- mens. There is also a need for a better understand- ing of the optimal combinations, the best sequence and the normal tissue toxicity of EGFR modulators together with radiation and/or chemotherapy. In addition, emerging opportunities for pharmaco- logical manipulation of other attractive molecular targets, such as the PI3K pathway in the radiation treatment of cancer, are pending and may also ulti- mately prove efficacious in combination with EGFR inhibition during radiotherapy.

The challenge in future research development will be to identify the appropriate inhibitor for the specific individual tumour. This also needs further studies at all levels of translational research and genome-wide screening to identify predictive mark- ers of response for the appropriate inhibitor.

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