PERSPECTIVES. Integration of EGFR inhibitors with radiochemotherapy

Transcription

1 OPINION Integration of EGFR inhibitors with radiochemotherapy Mukesh K. Nyati, Meredith A. Morgan, Felix Y. Feng and Theodore S. Lawrence Abstract Laboratory studies that led to the development of epidermal growth factor receptor (EGFR) inhibitors indicated that such inhibitors would be effective when given to patients with tumours that are driven by activated EGFR. However, initial clinical studies have shown modest responses to EGFR inhibitors when used alone, and it has not yet been possible to clearly identify which tumours will respond to this therapy. As a result, EGFR inhibitors are now used in combination with radiation therapy, chemotherapy and, more recently, with concurrent radiochemotherapy. In general, these clinical trials have been designed without much preclinical data. What do we need to know to make these combinations successful in the clinic? Clinicians have combined chemotherapy and radiation therapy since the 1980s. Two non-mutually exclusive potential mechanisms have been proposed to explain why radiochemotherapy might be successful. The first is that there would be a favourable interaction between chemotherapy and radiation that improved the effectiveness of radiation therapy (that is, radiosensitization). In the laboratory, this is usually defined as synergistic by isobologram (BOX 1) or related analysis 1,2, which mathematically shows that the observed effect of using chemotherapy and radiation concurrently is greater than simply adding the two together. Although it is beyond the scope of this Perspective to review the different mathematical methods of analysing drug radiation interactions, it should be noted that synergy cannot be determined simply by normalizing the radiation cell-survival curve for drug-induced cytotoxicity. This is because, for both drugs and radiation, the relationship between dose and cytotoxicity is not linear. In the clinic one is seeking an improved therapeutic index (greater tumour control compared with normal tissue toxicity). A second potential reason to combine radiation and chemotherapy would be to obtain the local effects of radiation with the potential systemic benefit of chemotherapy, which has been called spatial additivity 3. Randomized clinical trials show improved local control and survival through the use of concurrent chemotherapy and radiation therapy for patients with highgrade gliomas and locally advanced cancers of the head and neck, lung, oesophagus, stomach, rectum and anus. It now seems that the chief benefit of radiochemotherapy comes from the first mechanism proposed above: radiosensitization produces improved local control. This conclusion is based on several clinical observations. First, the concurrent administration of chemotherapy and radiation is typically required to increase survival. This has been best shown by a meta-analysis of head and neck cancer sequential (often called induction ) chemotherapy produces little if any improvement in survival, whereas concurrent chemoradiation produces a significant increase in survival 4. Although initial studies indicated that sequential chemotherapy and radiation had some benefit for lung cancer 5, more recent work indicates that concurrent therapy is superior, and it is now the standard treatment 6. The chief exceptions to this rule seem to be lymphoma, in which sequential treatment has been successful owing to the sensitivity of the disease to each individual therapy, and breast cancer, where lumpectomy plus radiation (without chemotherapy) results in local control in excess of 90% at 5 years after treatment. Although concurrent radiochemotherapy has produced important advances both in improved survival and improved organ conservation, it is becoming clear that further advances are being limited by the toxicity of treatment. For example, in the curative treatment of locally advanced head and neck cancer, it is now standard to ask patients to have a tube placed directly into the stomach, as patients routinely develop such severe mucositis (BOX 1) that they are otherwise unable to maintain adequate nutrition. The toxicity of treatment clearly results from the relative lack of specificity of both Box 1 Definitions of terms used Accelerated repopulation The hypothesis that the rate of tumor cell replication increases during the course of radiation, particularly after the fourth week. Isobologram An analysis of drug drug or drug radiation interactions that compares equally effective dose pairs (isoboles) with each agent used alone. This approach is needed when there is a non-linear relationship between concentration or dose and cell survival. Mucositis The inflammation of a mucus membrane. Oral mucositis is a common complication of chemotherapy and radiation therapy. Multivariate analysis A statistical analysis that takes into account potential interactions between variables. Multivariate analysis enables the individual contribution to the outcome of interest of many variables to be determined. 876 NOVEMBER 2006 VOLUME 6

2 radiation and chemotherapy for cancer cells as opposed to rapidly dividing normal cells. Therefore, the introduction of molecularly targeted therapy has been greeted with substantial excitement by both radiation and medical oncologists. By targeting aberrant growth-factor or pro-angiogenic pathways that are specific to cancer cells rather than all rapidly proliferating cells, molecularly targeted therapies offer the potential to improve outcome without increasing toxicity. The best known example is probably imatinib, which targets the BCR ABL kinase that is expressed in chronic myeloid leukaemia (CML) and, to a lesser extent, gastrointestinal stromal tumours (GIST). Imatinib as monotherapy produces long-lasting responses with minimal toxicity 7 9. However, imatinib for CML has proved to be the exception rather than the rule, and the most common solid tumours (breast, lung and colon) show only a modest 10 15% transient response rate to monotherapy with molecularly targeted agents. Therefore, the greatest use of molecularly targeted therapies has been in combination with chemotherapy or radiation therapy. Although adding molecularly targeted therapy to chemotherapy or radiation has produced some successes, it has also produced some spectacular failures. An important point of this Perspective is the hypothesis that these inconsistent results might be due, at least in part, to the fact that these new clinical trials might have actually used molecularly targeted therapies in a manner that is antagonistic with chemotherapy. This Perspective will approach the question of how to combine molecularly-targeted therapy with standard radiochemotherapy by focusing on how to combine epidermal growth factor receptor (EGFR) inhibitors with radiation and chemotherapy. We will also make some preliminary remarks about combining EGFR inhibition with radiochemotherapy regimens, as there is, in fact, already a multi-institutional randomized clinical trial under way (RTOG 0522) that compares radiochemotherapy (cisplatin and radiation) with radiochemotherapy plus cetuximab for the treatment of head and neck cancer 10. As this is undoubtedly the first of many such trials, it would be our hope that subsequent studies could benefit as much as possible from informative preclinical studies. EGFR and inhibitors of EGFR EGFR is a 170 kda transmembrane glycoprotein comprised of an extracellular ligand-binding domain (621 amino acids) and an intracellular protein tyrosine kinase domain (542 amino acids) connected by a Box 2 The main downstream signalling pathways regulated by EGFR Epidermal growth factor receptor (EGFR) phosphorylation can directly or indirectly activate signal transducer and activator of transcription 1 (STAT1), STAT3 and STAT5. The activated STAT proteins translocate into the nucleus and directly regulate gene expression crucial for cell survival, proliferation, transformation and oncogenesis 102. EGFR activates phosphatidylinositol 3-kinase (PI3K) that phosphorylates phosphatidylinositol 4,5-biphosphate to form phosphatidylinositol 3,4,5-triphosphate, which then activates Akt by binding at its pleckstrin homology (PH) domain. Phosphorylated Akt has several effects, both in the cytoplasm and in the nucleus, which include the inhibition of proapoptotic factors such as BAD (BCL2 antagonist of cell death), procaspase-9 and the Forkhead (FKHR) family of transcription factors (FOXO). Akt-mediated activation of mammalian target of rapamycin (mtor) is also important in stimulating cell proliferation, and vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α (HIF1α) are important in angiogenesis 103,104. The Ras MAPK (mitogen-activated protein kinase) pathway is activated after the activation of EGFR through two mechanisms, either through the direct recruitment of GRB2 (growth factor receptor-bound protein 2) at the SH2-domain or through the indirect recruitment of GRB2 through the SHC adaptor. Ras is activated by phosphorylation that is mediated by son of sevenless (SOS). Activated Ras binds to Raf, which in turn triggers the phosphorylation of MEK1/2 (mitogen-activated protein kinase kinase 1/2) and ERK1/2 (extracellular signal-regulated kinases 1/2). Phosphorylated ERK1/2 translocates into the nucleus and activates various transcription factors such as ELK1. Various cytoskeletal proteins are also activated by ERK1/2 105,106. PLCγ (phospholipase c-γ) binds through its SH2 domain to phosphorylated EGFR tyrosine kinase to become active. Once activated, PLCγ hydrolyses phosphatidylinositol 4,5-biphosphate to diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 mediates calcium release from intracellular stores, affecting a host of Ca2 + -dependent enzymes, whereas DAG is a cofactor for the activation of the serine/threonine kinase protein kinase-c (PKC). The activation of PKC results in cell-cycle progression, transformation, differentiation and apoptosis 107. The figure shows signalling pathways that are activated by EGFR. Important signalling pathways regulated by EGFR are represented with important functions highlighted in coloured boxes. The signalling cascade does not include all the known components of a given pathway, and cross-talk is not shown for clarity. Examples of inhibitory signals are also shown, such as PTEN (phosphatase and tensin homologue), which can dephosphorylate PI3K and MKP1 (MAPK phosphatase 1), which can dephosphorylate ERK1/2 signalling pathways. The signalling cassettes presented in the figure regulate the activity of many cytoplasmic targets. In addition, these signalling pathways also regulate the activity of transcriptional factors directly or indirectly. CCND1, gene encoding cyclin D1; CDKN1A, gene encoding p21; JAK, Janus kinase; TGFα, tranforming growth factor-α. NATURE REVIEWS CANCER VOLUME 6 NOVEMBER

3 Table 1 EGFR-targeting monoclonal antibodies in clinical use Agent and manufacturer Molecule Specificity Half life Comments Cetuximab (IMC-C225, Erbitux); ImClone Systems/ Bristol-Myers Squibb ABX-EGF (Panitumumab, rhumab-egfr); Amgen EMD72000 (Matuzumab); EMD Pharma ICR62; The Institute of Cancer Research, UK MDX-447; Merk and Medarex Mouse human chimeric mab EGFR 97h 68 FDA approved for the treatment of CRC in combination with irinotecan, and in combination with radiation therapy for SCCHN or as a single agent for the treatment of patients with recurrent or metastatic SCCHN for whom previous platinum-based therapy has failed 121,122 Fully human mab EGFR 16 days 123 Phase I/II for NSCLC, CRC, renal and oesophageal cancer Mouse human chimeric mab Rat mab Mouse human chimeric mab EGFR 4 days 126 Phase II trials for NSCLC, gynecological, pancreatic and oesophageal cancers 126,127 EGFR and EGFRvIII EGFR and CD64 Not known Phase I for NSCLC 128 Not known Phase I for refractory glioblastoma multiforme* *See the clinical trials web site for more information. CRC, colorectal carcinoma; EGFR, epidermal growth factor receptor; FDA, US Food and Drug Administration; NSCLC, non-small-cell lung cancer; SCCHN, squamous cell carcinoma of the head and neck. small transmembrane-anchoring region (23 amino acids) 11. Within the broad family of receptor tyrosine kinases, EGFR belongs to the type I subfamily (or ERBB tyrosine kinase receptors) that also includes ERBB2 (also known as HER2 and NEU), ERBB3 (also known as HER3) and ERBB4 (also known as HER4). The binding of a ligand, such as EGF, transforming growth factor-α (TGFα) or amphiregulin causes the EGFR to dimerize with itself or with another member of the ErbB family of receptors, leading to receptor-linked tyrosine kinase activation and the activation of downstream signalling cascades that are crucial for normal cell growth and proliferation (BOX 2). Because aberrant EGFR activation has been associated with uncontrolled cell proliferation, EGFR is an attractive and logical target for cancer therapy 12. In the past two decades, strategies that target either the extracellular ligand-binding domain or the intracellular kinase domain of the receptor have been developed Although the disruption of EGFR function can be accomplished by several methods, two approaches have been most extensively investigated in the laboratory and clinical settings. The first approach uses monoclonal antibodies (mabs) directed against the receptor to prevent ligand from binding to the extracellular domain of the receptor. This approach prevents receptor dimerization and activation, and ultimately induces receptor degradation. Several EGFR antibodies have been developed and are in various phases of clinical testing (TABLE 1). The second approach to disrupting EGFR function involves the use of small-molecule tyrosine kinase inhibitors (TKIs), which bind to the ATP-binding pocket of the cytoplasmic domain of the receptor and inhibit receptor phosphorylation, which ultimately blocks downstream signalling cascades. An increasing number of such agents have entered into clinical development in the past decade (TABLE 2). Both of these anti-egfr approaches have a cytostatic effect on tumour cells in vitro by blocking cell-cycle progression and proliferation. However, their in vivo activity seems to be greater than might be anticipated from the in vitro results, which could be due to additional direct or indirect effects on angiogenesis, invasion and metastasis and increased tumour cell death Although overexpression of the EGFR protein is certainly a poor overall prognostic marker 12,27, the relative roles of EGFR protein expression, gene amplification and receptor mutation in predicting specific response to EGFR TKIs are less clear. The relationship between EGFR expression and gefitinib (TABLE 2) sensitivity has been retrospectively analysed in a few studies. In the IDEAL (Iressa Dose Evaluation in Advanced Lung Cancer) trials for non-small-cell lung cancer (NSCLC), no correlation was shown between EGFR expression and response. This finding was also confirmed in other studies In analogy to ERBB2 amplification and sensitivity to trastuzumab, many have looked for a relationship between EGFR gene amplification and sensitivity to EGFR TKIs. In one study, fluorescence in situ hybridization (FISH) was used to assess EGFR gene copy number in tumours from 102 patients with advanced NSCLC. High EGFR gene copy numbers were significantly associated with better response, disease control rate, time to progression and survival; the association with survival remained significant after multivariate analysis 31 (BOX 1). Another more recent study by Hirsch et al. concluded that increased EGFR gene copy numbers were associated with improved outcome after gefitinib therapy in patients with bronchoalveolar carcinoma 32. In addition, high EGFR copy numbers (as assessed by real-time PCR) were associated with increased response rate and time to progression, but not with increased survival in a Japanese patient cohort 33. Because EGFR mutations were shown to be a more important predictor of survival in this Japanese population, it has been proposed that the relative significance of EGFR gene amplification and mutation as predictive factors of response to EGFR TKIs might depend on the patient population 34. The role of EGFR mutations in the response to small-molecule TKIs has been intensively investigated. Two main categories of EGFR mutations have been identified: deletion mutations of the extracellular domain and somatic mutations in the tyrosine kinase domain. A deletion mutant EGFRvIII is the most prevalent naturally occurring form of EGFR mutation, and is found in most glioblastomas and medulloblastomas (also called EGFRvIII or de2 7EGFR or EGFR) 35,36. The deletion of exons 2 7 (amino acids 2 273) results in a truncated protein that lacks ligand-binding domain L1 and most of the cysteine-rich S1 region of the extracellular domain. As a result, EGFRvIII is not activated by its ligand; however, it is constitutively activated and is not internalized, which results in constitutive long-term signalling. A recent study showed that the presence of EGFRvIII might sensitize glioblastomas to EGFR TKIs 37. The other category of EGFR mutations involves changes in the tyrosine kinase domain. Two clinical studies found that gefitinib caused significant tumour regression in a small subset of non-smoker, female patients from Japan with adenocarcinoma 878 NOVEMBER 2006 VOLUME 6

4 Table 2 Small-molecule inhibitors of the EGFR tyrosine kinase in clinical use Agent and manufacturer Gefitinib (ZD1839, Iressa); AstraZeneca Erlotinib (OSI 774, Tarceva); Genentech Inc. PKI166 (CGP 75166); Novartis Pharma CI-1033 (canertinib); Pfizer EKB-569; Wyeth Research Lapatinib (GW572016, Tykerb); GlaxoSmithKline Molecule and receptor binding Anilinoquinazoline; reversible Anilinoquinazoline; reversible Specificity Half life Comments EGFR 48h 129 In June 2005 the FDA approved new labelling for gefitinib for patients currently receiving and benefiting from gefitinib, or patients who have previously received and benefited from gefitinib EGFR 36h In November 2004 the FDA approved erlotinib for the treatment of patients with locally advanced or metastatic NSCLC after failure of at least one previous chemotherapy regimen 130 ; in November 2005 the FDA approved erlotinib for locally advanced, unresectable or metastatic pancreatic carcinoma treatment in combination with gemcitabine Pyrrolo-pyrimidine EGFR 12h 131 Phase I trials for advanced solid malignancies are underway 131 Anilinoquinazoline; irreversible 3-cyanoquinoline; irreversible Thiazolylquinazoline; reversible Pan ERBB 5h 132 Phase II completed EGFR and ERBB h 135 Phase II for patients with gefitinib-resistant NSCLC 136 EGFR and ERBB2 24h 137 Phase III for various neoplasms 137 EGFR, epidermal growth factor receptor; FDA, US Food and Drug Administration; NSCLC, non-small-cell lung cancer. of the lung 38,39. They established a strong correlation between responsiveness to gefitinib and somatic mutations in the EGFR gene, particularly L858R (exon 21) and exon 19 short in-frame deletions in the tumour cells. Further cell culture and clinical studies showed that cells that contained these mutations respond at a higher level to EGF stimulation and also respond significantly better to gefitinib and erlotinib compared with wild-type receptor-expressing cells (FIG. 1). A more detailed discussion of the role of EGFR mutations in sensitivity to smallmolecule TKIs can be found elsewhere 40,41. In summary, a substantial amount has been learned about the methods by which EGFR can become constitutively activated. However, as is described in more detail below, the overall response rate to monotherapy with these inhibitors is only in the range of 10 15%, and there are still no clear markers, beyond EGFR mutations in NSCLC, to predict response to EGFR inhibitors. In an attempt to improve the effectiveness of EGFR inhibitors, investigators have attempted to combine these inhibitors with radiation and/or chemotherapy. EGFR inhibitors and radiotherapy Rationale for combining EGFR inhibitors with radiation. Initial studies showed that prolonged exposure of head and neck cancer cells to EGF could increase the effects of radiation 42,43. In retrospect, it is probable that this radiosensitivity was achieved through EGF-induced EGFR degradation (see below). Additional early studies showed that monoclonal antibodies that target EGFR could increase radiationinduced apoptosis 44. Milas and others showed an inverse correlation between EGFR expression levels and radiation response, further stimulating interest in combining EGFR inhibitors and radiation. This relationship between EGFR expression and poor prognosis was subsequently confirmed in human head and neck carcinoma samples 48. There are several compelling reasons to explore the combination of EGFR inhibitors and radiation therapy. In addition to the correlation between EGFR expression and radioresistance, there are several compelling reasons to explore the combination of EGFR inhibitors and radiation therapy. FIG. 2 shows the effect of cytostasis, cytotoxicity, radiosensitization and repopulation on the surviving fraction of irradiated cells. EGFR inhibitors commonly produce cytostatic effects (which are characterized by G1 arrest 49,50 ) and, less frequently, cytotoxicity and radiosensitization (see below). However, a purely cytostatic agent could significantly potentiate the effectiveness of a fractionated course of radiation by preventing tumour cell repopulation (FIG. 2). If EGFR inhibitors could also produce cytotoxicity and radiosensitization, their effect on decreasing cell survival would be further increased. Additional interest in combining EGFR inhibitors with radiation was generated by the demonstration of radiation-induced EGFR activation in vitro. Confluent cells in culture treated with ionizing radiation rapidly show increased levels of phosphorylated EGFR This was proposed to promote cellular proliferation and DNA-damage repair capability, therefore counteracting the toxic effects of radiation therapy 52, and might explain a phenomenon known as accelerated repopulation (BOX 1). Specifically, ionizing radiation induces EGFR import into the nucleus, where it interacts with DNA-protein kinase (DNAPK) and subsequently results in increased DNAPK activity. Cetuximab inhibits this radiation-induced activation of DNAPK, as well as EGFR nuclear import, DNA repair and radiation survival 55. Clinically, it has been observed that protraction of radiation seems to decrease tumour control, which would be consistent with the idea that cells are stimulated to undergo proliferation potentially owing to the radiationinduced stimulation of EGFR. Therefore, the inhibition of radiation-induced receptor activation might sensitize cancer cells to the lethal effects of ionizing radiation (FIG. 3). Although several studies have shown that radiation can cause a rapid and transient increase in EGFR phosphorylation, it remains to be seen if this is the case for human tumours in a clinical setting. This issue is of great importance, as most of the results published so far on this topic are from confluent or serum-starved cell-culture models. As tumours are heterogeneous, NATURE REVIEWS CANCER VOLUME 6 NOVEMBER

5 Figure 1 A schematic view of EGFR showing the crucial features required for kinase activity and key mutations identified in patients with non-small-cell lung carcinoma. Epidermal growth factor receptor (EGFR) contains a highly conserved glycine rich ATP-binding motif GXGXXG in the C terminus 108. Once activated by ligand binding, the receptor undergoes rapid internalization and degradation. Mutation at K721 (ATP-binding domain) causes the receptor to become inactive so that it is neither internalized nor degraded 109,110. Ligand binding can induce homoor hetero-dimerization, which can activate many sites within the C terminus such as Y992, Y1045, Y1068, Y1086, Y1148 and Y1173 (shown in red), or SRC non-receptor kinase can phosphorylate Y845 and Y1101 (shown in purple). Y845 phosphorylation stabilizes the activation loop, maintains the enzyme in an active state, and regulates signal transducer and activator of transcription 5b (STAT5b) activity 111. Phospholipase Cγ (PLCγ)-mediated signalling is stimulated by PLCγ binding to a phosphorylated Y992 site. The phosphorylation of Y1045 creates a docking site for CBL, which enables receptor ubiquitylation and degradation 112. The phosphorylation of Y1068 and Y1086 facilitates the binding of the SH2 domain of growth factor receptor-bound protein 2 (GRB2). This binding results in mitogen activated protein kinase (MAPK) activation through the Ras-signalling pathway 113. SHP1 phosphatase can bind to the phosphorylated Y1173 domain, which leads to EGFR dephosphorylation 114. Several mutations in the extracellular domain of EGFR have been identified, among the most common is an 801bp in-frame deletion that produces a truncated form known as variant III, which is present in about 20 50% of glioblastomas with EGFR amplification. This deletion leads to the expression of constitutively activated proteins. EGFRvIII is thought to be a promising target for therapy, as it is tumour-specific and occurs in the membrane of glioblastoma cells with EGFR amplification 37. Several mutations in the intracellular domain of EGFR have been identified that are associated with gefitinib or erlotinib sensitivity in patients with non-small-cell lung cancer. These mutations are restricted to four exons (exons 18 21) of the tyrosine kinase domain. The two most common mutations, deletions of four amino acids in exon 19 and a single point mutation in exon 21, L858R, account for about 85% of all mutations in EGFR (shown in green boxes). Other mutations are reported (shown in yellow). In some patients more then one point mutation in EGFR has been identified 37 39,95,115. At least one report suggests that the different mutations might produce differential sensitivity to an apparently similar class of drug, gefitinib and erlotinib 116. Most of the tumours that respond initially to tyrosine kinase inhibitors tend to acquire resistance through secondary mutations. One such example is a point mutation in exon 20, T790M, which confers resistance to both gefitinib and erlotinib 117. S, sensitive; R, resistant. portions of the tumour (especially in the case of slow-growing tumours) might reflect this phenomenon. However, tumours with aberrant EGFR expression are usually rapidly dividing and radio-resistant, which might present different conditions from the confluent-culture model. Although some evidence indicates that EGFR is rapidly phosphorylated in response to radiation in tumour xenografts 56,57, this phenomenon still needs to be explored in rapidly dividing portions of the tumour. Preclinical and clinical studies that combine EGFR inhibitors and radiation. Various preclinical studies have shown that EGFR inhibitors can increase radiation sensitivity in both in vitro and in vivo model systems 20,25, Although most studies have reported additive effects resulting from the combination of EGFR antagonists and radiotherapy in vitro, the same combination has been reported to produce even greater (synergistic) effects in xenograft models 20,23,25,63,64. This might be because EGFR inhibitors and radiation potentially affect several downstream signalling pathways 65. These include pathways that regulate cellular proliferation and apoptosis, which would be evident both in vitro and in vivo, and pathways that regulate angiogenesis 20,64 and tumour invasion 66,67, which would only be detectable in tumour xenograft models. Although preclinical studies have highlighted the potential therapeutic gains that could be achieved by adding EGFR inhibitors to radiation, the best validation of this combination has been from the results of clinical trials in patients with unresectable head and neck cancer. In a phase I trial at the University of Alabama, 16 patients were treated with this combination and 13 patients achieved a complete response, 2 a partial response and there was 1 non-evaluable case 68. More recently, a phase III clinical trial showed that, in a cohort of 424 patients with locoregionally advanced squamous cell carcinoma of the head and neck, the addition of cetuximab nearly doubled the median survival of patients from 28 to 54 months 880 NOVEMBER 2006 VOLUME 6

6 (compared with radiotherapy alone). This study represented the first significant success achieved by the addition of an EGFR antagonist to radiotherapy. This positive clinical trial has stimulated substantial interest in optimizing the combination of EGFR inhibitors and radiation. Several important questions need to be resolved. First, does the method through which EGFR is inhibited matter? There are no direct comparisons in clinical trials. Our preclinical studies in head and neck cancer suggest that the combination of radiation with cetuximab might be superior to gefitinib, probably because of the ability of cetuximab to produce long-term suppression of EGFR owing to receptor internalization and degradation, but this clearly requires further study in other model systems and clinical trials. Second, do combinations of EGFR inhibitors make sense? Two recent preclinical studies have shown that the combination of either erlotinib or gefitinib with cetuximab can provide more effective suppression than either one used alone 69,70, but whether this will be true in the clinic remains to be seen. A third crucial question is how to predict which patients are likely to respond to treatment. Only a fraction of patients with any given cancer will have tumours that are primarily driven by activated EGFR; so far no test clearly identifies this population. EGFR inhibitors and chemotherapy Substantial preclinical efforts have also been directed towards combining EGFR inhibitors with chemotherapy There are at least three potential mechanisms by which EGFR inhibitors might interact with chemotherapy: through the cell cycle, through EGFR signalling or through DNA repair. We will organize our discussion around these three potential mechanisms. Cell cycle. As chemotherapeutic agents are usually less effective against cells that do not divide soon after drug exposure, the cell-cycle distribution within a tumour can be an important determinant of the outcome of drug therapy. This raises the possibility that EGFR inhibitors, which can cause G1 arrest through upregulation of the cyclin-dependent kinase inhibitors p27 and p21 (REFS 49,50,74,75), could actually be antagonistic to cell-cycle-dependent chemotherapy. More specifically, this would lead to the hypothesis that giving chemotherapy before an EGFR inhibitor would be more effective than the reverse schedule. To test this rationale, the order of drug delivery for the combination of chemotherapy and EGFR inhibitors has been investigated 76,77. In exploring the optimum sequencing for the combination of gemcitabine and gefitinib 77, we recently found that gemcitabine followed by gefitinib was superior to the opposite drug order, in part because the first sequence optimized gemcitabine cytotoxicity by allowing for an S-phase arrest, whereas the reverse sequence inhibited gemcitabine-mediated cell death by arresting cells in G1. The superiority of gemcitabine followed by gefitinib over the reverse order was also confirmed in xenografts. Sequence-dependent synergistic cytotoxicity has also been found when gefitinib, cetuximab or ZD6474 has been combined with either a platinum derivative, a taxane or a topoisomerase I inhibitor 76,78,79. It is not clear why the sequence of chemotherapeutic agent before EGFR inhibitor is crucial in the case of cytotoxic agents that are not necessarily S-phase specific. At least in the case of the platins it has been suggested that the mechanism(s) of synergy involves the inhibition of DNA repair (discussed below). Furthermore, not only does the synergistic antiproliferative activity of EGFR antagonists depend on sequence (chemotherapeutic agent first), but so does the induction of apoptosis and accumulation of the surviving cells in G2/M 76,79. The arrest of cells in G2 after chemotherapy treatment might permit cells an opportunity to repair damaged DNA and avoid apoptosis. Therefore, it would seem that the accumulation of cells in G2/M is probably not the mechanism of synergy between EGFR inhibition and chemotherapy. The EGFR signalling pathway. In addition to cell-cycle effects, another mechanism by which cytotoxic chemotherapeutic agents and EGFR inhibitors might interact is through the modulation of the EGFRsignalling cascade itself. EGFR phosphorylation occurs in response to various cytotoxic drugs, including gemcitabine 77, cisplatin 80, oxaliplatin 81, 5-fluorouracil 81, paclitaxel 82, doxorubicin 83 and irinotecan 78. The phosphorylation of EGFR by oxaliplatin or 5-fluorouracil treatment alone correlates with the synergistic inhibition of cell viability and cell growth by gefitinib 81. The blockade of EGFR activation in response to paclitaxel by gefitinib promotes apoptosis and suppresses tumour growth 82. At the same time, one must consider the context in which EGFR phosphorylation occurs. It has long been known that EGFR phosphorylation occurs after exposure to Figure 2 Potential mechanisms of synergy between EGFR inhibitors and radiation. Although each daily radiation treatment kills a fraction of the cells, some cells grow back by the next day, which attenuates the effectiveness of radiation treatment. If an epidermal growth factor receptor (EGFR) inhibitor has only a selective cytostatic effect and blocks regrowth between fractions, the result would be a dramatic increase in radiation efficacy. The benefit of the inhibitor would be even greater if it caused tumour cell cytotoxicity and/or radiosensitization. normal ligands such as EGF, which leads to cell growth and, ultimately, to physiological receptor degradation after proliferation is completed 84. However, under conditions of prolonged cellular stress (such as persistent deoxyribonucleotide pool depletion, which is produced by gemcitabine) EGFR phosphorylation can lead to EGFR degradation and cell death. We recently showed that, in response to cytotoxic doses of gemcitabine, head and neck cancer cells undergo phosphorylation-dependent receptor degradation through ubiquitylation and activation of the proteosome 85 (FIG. 3). The blockade of EGFR degradation through the proteasome inhibitor MG132 protected against gemcitabine-mediated cytotoxicity. These findings raise the possibility that the use of proteasome inhibitors such as bortezomib with gemcitabine might produce antagonism, and this combination should be approached with caution. The mechanism by which this chemotherapy-induced EGFR phosphorylation occurs is unknown. It has been proposed that EGFR signalling might serve as a cell-survival response in cells exposed to cytotoxic stress 19. The activation of EGFR in response to cisplatin occurs in an SRC-dependent manner 80. The inhibition of EGFR activation (directly or by SRC inhibition) increases cisplatininduced cell death, indicating that EGFR activation by cisplatin is a survival response. NATURE REVIEWS CANCER VOLUME 6 NOVEMBER

7 Figure 3 The effect of radiation and chemotherapy on EGFR signalling. After stimulation by irradiation or certain chemotherapeutic agents, epidermal growth factor receptor (EGFR) can activate downstream signalling pathways that can promote cell survival or cell death. Several examples are highlighted in this figure. Radiation: in addition to stimulating the pathways activated by epidermal growth factor (EGF) (as described in the text), radiation can trigger the translocation of phosphorylated EGFR (pegfr) into the nucleus 55,118. This process coincides with the transport of Ku70/80 and protein phosphatase 1 into the nucleus (not shown), which results in increases in DNA-dependent protein kinase (DNAPK) levels, the repair of DNA-strand breaks and cell survival. Cetuximab, but not gefitinib, blocks nuclear transport of pegfr. Chemotherapy: two kinds of chemotherapy-induced effects are shown. One, represented by doxorubicin treatment, begins with the phosphorylation of EGFR at the tyrosine 845 site, which results in the translocation of p 845 EGFR to the mitochondria where the receptor binds the cytochrome c oxidase subunit II (MTCO2) 119,120. By stabilizing MTCO2, EGFR binding prevents the activation of apoptosis. The second, represented by gemcitabine, also begins with the phosphorylation of EGFR. In this case, EGFR phosphorylation initially activates Akt 77 but subsequently promotes the ubiquitylation (Ub) of the receptor, which leads to its degradation along a proteosome or lysosome pathway 85. pegfr degradation results in the downregulation of the survival signal pakt, leading to apoptosis. Blocking EGFR degradation at various steps of this pathway reduces gemcitabine-mediated cytotoxicity. Whether an EGFR-activating insult leads to cell survival or cell death might ultimately be determined by the severity and duration of the stress. EGFR phosphorylation results in the activation of signalling cascades that activate Akt, which is a known anti-apoptotic kinase, probably indicating an EGFR-mediated survival mechanism. By blocking this survival signal, EGFR inhibitors could increase the effectiveness of chemotherapy and result in synergistic cytotoxicity. DNA repair. Another mechanism of synergy between EGFR inhibition and chemotherapy is through the inhibition of DNA repair. Chemotherapeutic agents induce various types of DNA damage, including strand breaks, DNA adducts, and inter- and intra-strand DNA crosslinks, and in response cells activate signalling pathways that regulate DNA repair, cell cycle and survival. The repair of cisplatininduced DNA inter-strand crosslinks is inhibited by gefitinib 86,87. Furthermore, gefitinib treatment is accom-panied by increased interaction between EGFR and DNAPK, reduced DNAPK enzymatic activity and protein expression 87,88. Similarly, the removal of oxaliplatin DNA adducts is inhibited by gefitinib, with maximal effects observed in a schedule-dependent manner (oxaliplatin first) 79. Together, these studies indicate that the inhibition of DNA repair by EGFR inhibition might contribute to the synergy between EGFR inhibition and chemotherapy. This potential mechanism of interaction deserves further exploration. Rational trial design Unfortunately, the strong rationale and supportive preclinical biology of combining EGFR inhibitors with chemotherapy has not translated into clinical success. In the INTACT1 (Iressa NSCLC Trials Assessing Combination Therapy 1), INTACT2 and TRIBUTE (Tarceva Responses In Conjunction with Paclitaxel and Carboplatin) phase III clinical trials, the addition of an EGFR TKI to conventional chemotherapy resulted in no survival benefit in the treatment of NSCLC, despite the inclusion of thousands of patients and the use of different anti-egfr agents in these studies The results of a phase III trial conducted in patients with head and neck cancer treated with cisplatin plus cetuximab versus cisplatin alone showed a significant improvement in objective response rate (26% and 10%, respectively) 92 but, again, no improvement in survival. A recent phase III clinical trial of erlotinib plus gemcitabine for patients with advanced pancreatic cancer did produce a significant increase in overall survival compared with patients that received gemcitabine alone, but the median increase in survival was only 14.7 days, and was accompanied by a significant increase in the incidence of diarrhoea 93,94. Why have the overall results of combining chemotherapy with EGFR inhibition been so unimpressive? The most obvious potential answer is patient selection. For example, in one of the best-characterized studies focused on the treatment of refractory lung cancer with erlotinib, EGFR expression was correlated with response but not survival 95. Similarly, there are reports of response to cetuximab in the absence of EGFR staining by immunohistochemistry 96. Furthermore, the strength of EGFR staining using immunohistochemistry does not predict response to EGFR inhibitors combined with chemotherapy 97. Although the focus has naturally been on assessing pretreatment paraffin-embedded specimens, it seems possible that a predictive assay might require pre- and post-treatment specimens to be compared to determine whether the inhibitor has actually inhibited both EGFR phosphorylation and downstream signalling. This concept is supported by the intriguing clinical observation that the development of acne during treatment correlates with response 98. This area deserves intensive investigation, given the great expense of these inhibitors. An added complexity derives from the possibility that the factors that underlie 882 NOVEMBER 2006 VOLUME 6

8 Figure 4 Proposed model for the integration of EGFR inhibitors with radiochemotherapy. The preclinical and/or clinical results presented in this Perspective have suggested that there might be an optimal sequence for combining radiosensitizing chemotherapy with radiation, epidermal growth factor receptor (EGFR) inhibitors with radiation, and radiosensitizing chemotherapy with EGFR inhibitors. Based on these bimodality sequences, we now propose that radiosensitizing chemotherapy should be given before treatment with EGFR inhibitors and radiation concurrently. the response to an EGFR inhibitor alone or combined with chemotherapy might differ from those that produce a response to an inhibitor plus radiation. As noted above, in the case of radiation, all that might be required for synergy is that the inhibitor causes a selective cytostatic effect. Another possible explanation for the overall lack of success of chemotherapy combined with EGFR inhibition might be suboptimal sequencing of chemotherapy with EGFR inhibitors 99. In all of the trials described above, EGFR inhibitors were given concurrently and continuously with chemotherapy. Based on preclinical results, one can speculate that the sequential scheduling of these agents, with cytotoxic drugs administered first, might have given better results. However, it might be important to take into account that, in the case of cetuximab, the long half life of a dose given the previous week might influence the effect of chemotherapy given the following week. If scheduling does turn out to be an important issue in treatment, it is possible that the small-molecule TKIs could offer more flexibility given their much shorter half life. In addition to issues of patient selection and drug schedule, it is important to consider whether the dose of inhibitor has always been optimized. Although studies have been performed that assessed the effect of EGFR inhibitors on EGFR in the skin 100,101, results from skin biopsies and tumour biopsies do not seem to correlate 100. Furthermore, we are not aware of any data that address the issue of whether tumours undergoing radiochemotherapy express EGFR, and whether EGFR activation is inhibited by treatment agents such as cetuximab and erlotinib. Such studies are difficult to perform, but would probably be informative. In this article we have tried to summarize laboratory data that concern EGFR inhibition, radiation and chemotherapy in an attempt to provide a rational basis for the successful integration of all three of these therapies into clinical practice. In our own laboratory we have only recently started such studies, which are associated with significant complexities in conduct and interpretation. However, clinical trials are already proceeding on the basis of the standard paradigm derived from previous chemotherapy trials, in which the new drug is added to the older drug. The logic that underlies this approach is that, although the new combination might well be more toxic than the standard treatment, it is unlikely to be less effective. Therefore, the standard clinical paradigm is tending to produce trials that add the new therapy (EGFR inhibition) to the established therapy (concurrent radiation and chemotherapy). Unfortunately, both the clinical and laboratory data we have summarized above would suggest that the standard clinical paradigm for trial design might not carry us successfully into the era of molecularly targeted therapy, in that it is possible to produce antagonism to chemotherapy by inhibiting EGFR at the wrong time. With these scheduling issues in mind, we have made a tentative proposal, based on the laboratory studies summarized in this Perspective, of how EGFR inhibition might be most rationally combined with radiochemotherapy (FIG. 4). However, this proposal is in no way a substitute for the need for clinical verification. We feel that the effective integration of EGFR into radiotherapy and chemotherapy regimens will require both more preclinical study and, even more importantly, small clinical trials that incorporate biopsies to assess the effects of patient selection, scheduling and dose on the interaction of EGFR inhibition, radiation and chemotherapy. 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