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Growth factor receptors as targets for lung cancer therapy
Growth Factor Receptors As Targets for Lung Cancer Therapy Dao M. Nguyen and David S. Schrump Dysregulated signal transduction of growth factor receptors contributes to the process of malignant transformation by promoting cell proliferation, motility, and invasion through extracellular matrix as well as angiogenesis. Epidermal growth factor receptors (EGFR), and to a lesser extent HER2/neu, is overexpressed in the majority of nonsmall cell lung cancer (NSCLC) compared with normal tissue, making them ideal targets for the development of novel therapeutics for this disease. Multiple clinical trials have demonstrated that antireceptor strategies employing antagonistic monoclonal antibodies or low molecular weight tyrosine kinase inhibitors against EGFR are well tolerated and occasionally result in objective clinical responses in patients with advanced NSCLC. This report provides an overview of the molecular basis and the preclinical evidence supporting clinical development of anti-EGFR therapy as well as results of phase I–III clinical trials of these compounds in treating patients with solid tumors including NSCLC. Key words: Growth factors, lung cancer, molecular therapy.
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ung cancer is the second-most common malignant disease in both men and women in the United States. It is estimated that lung cancer [(20% small cell lung cancer (SCLC), 80% nonsmall cell lung cancer (NSCLC)] will account for 171,900 new cases and 157,200 deaths in 20031 with case-to-mortality ratio of almost one, which underscores of the lack of an effective therapy for this disease. The overwhelming majority of SCLC presents as stages III or IV for which multidrug chemotherapy regimens and thoracic/cranial radiotherapy is the only available treatment modality with an overall 5-year survival rate of less than 25%.2 Surgical resection offers the best chance for cure in patients with early stages (I and II) NSCLC. About 70% of newly diagnosed cases of NSCLC, unfortunately, present as either locally advanced (stage III) or metastatic disease (stage IV), for which combinations of chemotherapy and radiation are the standard-of-care treatment strategy. Overall, the rate of meaningful tumor response that can be translated to increased survival by standard cytoFrom the Section of Thoracic Oncology, Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD Address reprint requests to Dao M. Nguyen, Room 2B07, Building 10, 10 Center Drive, Bethesda, MD 20892. 1043-0679/04/1601-0003$30.00/0 doi:10.1053/j.semtcvs.2003.12.002
toxic chemotherapy regimens administered to patients with stage IV NSCLC remains low, with an overall 2-year survival rate of only 11%.3 It is clear that a new paradigm/direction for lung cancer therapeutics is needed. Growth factors and their receptors, particularly those of the epidermal growth factor receptor (EGFR) superfamily, have been shown to play crucial roles in carcinogenesis. More importantly, a large percentage of solid tumors express high levels of growth factor receptors (GFRs). This observation provides the rationale for the research efforts designed to develop anti-GFR compounds as part of a novel anticancer therapeutic strategy for a variety of solid malignancies including lung cancer.
Molecular Biology of Growth Signaling Pathways Of all the GFRs expressed on lung cancer cells, those belonging to the EGFR superfamily (EGFR/erbB1 and HER2/neu/erbB2) are the best studied and most extensively exploited as targets for development of novel therapeutics for this disease.4 EGFR-mediated signal transduction, serving as a model for molecular signaling by other tyrosine kinase membrane receptors, will be described in detail in this review. The EGFR superfamily consists of four members: EGFR (erbB1), HER2/neu (erbB2), erbB3,
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Figure 1. Tyrosine kinase GFR-dependent signal transduction pathways, biological response and relevant targeted pharmacologic inhibitors
and erbB4. These surface receptors are composed of a ligand-binding extracellular domain, a transmembrane segment, and an intracellular domain that has tyrosine kinase (TK) activity. Binding of ligands (EGF, TGF-␣, amphiregulin, and others) to the extracellular domain of EGFR results in formation of homodimers (EGFR-EGFR) or heterodimers (EGFR and other members) that result in the stimulation of intrinsic receptor tyrosine kinase activity. This leads to autophosphorylation of multiple tyrosine residues located in the intracellular domain of the receptors. The phosphorylated intracellular domain then serves as a docking site for various adaptor molecules (such as SOS, Grb2) as well as functional proteins (PI3K p110 subunit). These in turn activate multiple parallel intracellular signaling pathways (such as those mediated by ERK1/2, JNK, PI3K/ AKT, STAT, PKC). The biological responses from such diverse intracellular signalings include cell proliferation, survival, motility, invasion, adhesion, and angiogenesis (Fig 1). Overexpression
of EGFR or HE2/neu and dysregulation of the downstream signal transduction pathways play a critical role in tumorigenesis.5 While normal bronchial mucosa weakly expresses EGFR mainly confined to the basal layer, bronchial squamous metaplasia and dysplasia overexpress EGFR.6-8 EGFR overexpression (higher than levels of receptor expression in surrounding normal tissues) is very frequent in NSCLC (84% of squamous cell carcinoma, 65% of adenocarcinoma and large cell carcinoma), while none is detected in small cell lung cancer.6 In addition to NSCLC, high levels of EGFR expression have been documented in other solid tumor malignancies including colon, prostate, head/ neck, ovary, and breast. In contrast to the HER2/ neu overexpression identified in breast cancer that is most frequently caused by gene amplification, the molecular mechanism of EGFR overexpression in lung cancer cells is unclear. Coexpression of EGFR ligand TGF-␣, frequently seen in NSCLC and prostate cancer, can mediate a
Growth Factor Receptors in Lung Cancer Therapy
constitutive activation of the receptor via an autocrine/paracrine loop.5 The association between EGFR overexpression and poor prognosis has been extensively investigated in multiple studies with inconclusive results. Recent meta-analysis, however, has indicated that EGFR expression might be a poor prognostic factor for survival in NSCLC, but the amplitude of the impact is small and subjected to publication bias.9 About 33% of NSCLC (adenocarcinoma and large cell carcinoma but not squamous cell carcinoma) express high levels of HER2/neu. While gene amplification is the common mechanism responsible for high levels of HER2/neu expression (3⫹/4⫹) in 30% of breast cancers that are susceptible to Herceptin-based therapy,10 HER2/neu overexpression in lung cancer is much less intense (2⫹) and gene amplification is rare. Immunohistochemical staining of tumor NSCLC using the FDA-approved Herceptest indicated that ⱖ2⫹ HER2/neu expression was observed in 20% to 30% of cases.10 The predictive value of HER2/neu overexpression and poor clinical outcome in patients with NSCLC has been investigated but the results remain controversial.6 Despite the lack of a clear understanding of the prognostic relevance of EGFR family members in NCSLC, their overexpression patterns do make them suitable for molecularlybased therapy. In addition to members of the EGFR superfamily, other GFRs have been described in cultured lung cancer cell lines as well as tumor tissues. These include platelet-derived growth factor receptor (PDGFR),11 the hepatocyte growth factor/scatter factor (HGF/SF) receptor (c-Met)12,13 and the insulin-like growth factor (IGF-R)14 as well as c-kit.15 In-depth description of the molecular biology and the therapeutic potentials of these receptors and their cognate growth factors are beyond the scope of this review. Preclinical studies have been published describing the expression of these receptors in both NSCLC and SCLC and their putative roles in promoting cancer development.11,15,16 Of note, is the expression of PDGFRs and c-kit on lung cancer cells for which there exist several potent TKIs for this group of GFRs, particularly the wellstudied drug Imatinib mesylate (Gleevec, formerly STI571, Novartis Pharma AG, Basel, Switzerland).17,18
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Molecular Therapy for Lung Cancer by Targeting the Growth Signaling Pathways The signal transduction activity of GFRs can be abrogated by: 1) inhibition of receptor expression using gene therapy (antisense approach); 2) antagonistic monoclonal antibodies that prevent binding of ligands to receptors or; or 3) pharmacologic (low-molecular weight) receptor-selective tyrosine kinase inhibitors. The later two approaches are the preferred strategies currently under intense clinical development. Some of these anti-EGFR agents (tyrosine kinase inhibitors Iressa, Tarceva and monoclonal antibody IMC-225) are now in phase III clinical trials for patients with advanced-staged NSCLC. The underlying rationale for developing EGFR-targeted therapy is that differential expression of EGFR in tumor cells versus normal tissues can provide an exploitable therapeutic window in cancers known to overexpress EGFR. Both treatment strategies have been shown to be safe in clinical trials involving hundreds of patients. Tyrosine kinase inhibitors are administered orally, making them ideal for chronic maintenance therapy. Functionally, these agents can indirectly block erbB2 signaling via inhibition of erbB1/erbB2 heterodimers as well as other lateral signaling pathways that converge with the erbB1 pathway.7,19 Monoclonal antibodies, on the other hand, have to be administered intravenously. They, however, have a very long half-life. Functionally, these antibodies, in addition to blocking ligand binding to receptors, induce downregulation of surface receptors as well as incite antibody-dependent cell-mediated cytotoxicity.20 It is not possible at this point to predict which of these two well-tolerated approach will be more effective. In addition to membrane receptors, all of the intracellular intermediaries of EGFR-dependent signaling pathways have been selected as potential molecular targets for clinical development.21 Some of the pharmacologic antagonists are in early phase clinical trials (Table 1).
Preclinical Studies Growth Factor Receptor Antagonists Antireceptor mouse monoclonal antibodies (mAb) have been developed to bind to the easily accessible extracellular domain of the receptor
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Table 1. Selected Agents Targeting GFR-Dependent Signal Transduction Pathways Currently in Clinical Trials for Solid Tumors Including NSCLC Class
Agent
Target
Antibodies
Cetuximab ABX-EGF EMD-72000 RH3 MDX-447 Trastuzumab Gefitinib Erlotinib GW0126 CI-1033 EKB-569 PI-166 ISIS-2503⫹ R 115777⫹⫹ SCH 66336 BMS-214662 ISIS-5132 L-779-450 BAY-43-9006 PD 184352 17-AAG FK228
EGFR EGFR EGFR EGFR EGFR EGFR EGFR EGFR EGFR, HER2/neu EGFR, HER2/neu, HER4 EGFR EGFR, HER2/neu Ras Ras Ras Ras Raf Raf Raf MEK Multiple (HSP90)* Multiple (HDACI)**
GFR-TKI
Others
Phase of Development Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase
2, 2 1 2 1 2 3 3 1, 1, 1 1, 2 2, 2 2, 2 1 2 1 1, 1,
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2 2 2 3 3
2 2
⫹, Antisense oligonucleotide. ⫹⫹, Farnesyl transferase inhibitor. *HSP90: heat shock protein 90. **HDACI: histone deacetylase inhibitor.
and compete with ligand binding to the receptor. To eliminate immunologic response to xeno-immunoglobulins, humanized (chimeric human: murine) antibodies have been produced. The two prime examples of monoclonal antibodies are antiHER2/neu Herceptin (Trastuzumab) and anti-EGFR IMC-225 (Cetuximab). In addition to preventing ligand binding and activation of receptor phosphorylation, these antibodies also induce antibody-mediated cell-dependent cytotoxicity as well as antibody-mediated receptor dimerization and downregulation. Receptor tyrosine kinase inhibitors (RTKI) are low molecular weight compounds, identified by highthroughput screenings of natural and synthetic chemicals that compete, reversibly or irreversibly, with ATP for binding to the tyrosine kinase portion of the receptor thereby inhibiting the receptor’s catalytic activity. Extensive preclinical in vitro and in vivo studies have been performed to evaluate the antitumor properties of these agents. Treating cancer cells of diverse histology with either EGFR TKIs or MAbs results in dose-de-
pendent reduction of phosphorylated (activated) EGFR, AKT, ERK1/2 without effecting overall protein levels. This indicates that these compounds functionally inhibit the kinase activity as well as the downstream signalings of EGFR. This was paralleled with elevated levels of the cdk 2 inhibitor p27, hypophosphorylation of the Rb protein, reduction of cdk4 and cell arrest at G1 phase of the cell cycle.19,22,23 Induction of apoptosis has been reported with anti-EGFR mAb treatment in vitro24 but this is the exception rather than the rule with this treatment modality. GFR antagonists also result in a significant decrease in tumor cell-derived production of angiogenic growth factors such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and interleukin-8.25-28 This correlates with a reduction of microvessel density and increase of apoptosis of endothelial cells in human tumor xenografts.27-29 GFR blockade also results in significant reduction of the expression and activity of several matrix metalloproteinases (MMPs), most notably MMP-9, and a decrease of cancer cell motility and invasive-
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ness through an in vitro Matrigel matrix.30,31 This corresponds with inhibition of tumor growth and metastasis formation in vivo. The inhibitory effects on invasion, metastasis, angiogenesis may explain why anti-GFR treatments are often more effective in vivo and in vitro. GFR blockade has been shown to enhance tumor cell response to cytotoxic agents of different classes (cisplatin, doxorubicin, paclitaxel, topotecan) in an additive or supraadditive (synergistic) fashion both in tissue culture as well as in animal models of human cancer xenografts.32-37 Even though considered an EGFR-targeted therapy, it is peculiar to observe that the growth inhibitory response of cancer cells to Iressa, both in vitro and in vivo preclinical studies, and as monotherapy or in combination with cytotoxic agents, can not be reliably predicted by the levels of EGFR expression on the cell membrane.38-41 EGFR levels may not be the most accurate biomarker predictive of cellular responsiveness to EGFR blockade. Phosphorylation of EGFR and/or downstream effectors such as AKT or ERK1/2 (indicative of activation) in target cells may be of more value in determining their susceptibility to GFR inhibition regardless of the total levels of EGFR.
Other Agents In addition to specific growth factor receptor antagonists described above, there are other pharmacologic agents that exert potent anticancer property by inhibiting the expression of critical components of the signaling pathways. One example of such a compound is the HSP-antagonist 17-AAG. 17-AAG, at nanomolar concentrations, inhibits the chaperone function of heat shock protein 90 (HSP90), thus mediating a rapid and profound degradation of EGFR, ErbB2, c-Met, Raf, total and phosphorylated AKT as well as other oncoproteins in malignant cells.42-44 Treating cultured NSCLC cells expressing high levels of EGFR and/or HER2/neu with 17-AAG resulted in profound growth inhibition, significant sensitization of cancer cells to paclitaxel, drastic reduction of VEGF and MMP-9 production as well as invasion to Matrigel extracellular matrix in vitro.45 The antiangiogenic property as well as the chemosensitization effect of 17-AAG was confirmed in an animal models of NSCLC xenografts.42,46,47 More recently, our laboratory also described the link between down-regulation of
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EGFR, erbB2, Raf-1 expression and inhibition of signal transduction via Raf-1 and ERK pathway to the growth inhibitory property of the histone deacetylase inhibitor FK228 in NSCLC cells.48
Clinical Studies Iressa/ZD1839 Iressa (Gefitinib, AstraZeneca, London, UK) is an EGFR-TKI that has been studied in four phase I dose-escalation clinical trials in patients with solid tumors. These include 100 patients with advanced NSCLC who failed multiple regimens of cytotoxic chemotherapies.49-52 Patients received Iressa 50-1000 mg/d (14-days on/14-days off or 28-days continuous regimens). The maximum tolerated dose was 700 mg/d, with acneiform rash and diarrhea being the most commonly cited dose-limiting toxicities. Other side effects included fatigue, nausea, anorexia, and elevated liver enzymes. These were, however, mild (grade 1,2) and reversible on cessation of drug administration. Partial response or stable disease lasting for 1 to ⬎ 6 months were observed in 22% to 25% of patients with NSCLC. Pharmacodynamic study of the effect of Iressa on EGFR signaling in skin biopsies of patients undergoing treatment with this EGFR-TKI indicated significant reduction of phosphorylated EGFR, MAPK ERK1/2, proliferation antigen Ki67, and elevation of p27/KIP1 similar to those observed in preclinical studies. Similar pharmacodynamic studies evaluating pre- and on-therapy tumor biopsies are currently underway. A possible correlation between EGFR-TKI biologic effects in skin versus tumor may exist which allows accurate prediction of potential benefits early in the course of therapy from skin biopsy.22 The encouraging results from these phase I studies led to two phase II studies (IDEAL-1 and IDEAL-2) of Iressa 250 mg/d and 500 mg/d involving 426 patients with advanced, pretreated NSCLC at two different sites (United States and Japan).53,54 EGFR expression on tumor cells was not an inclusion criterion for enrollment to these trials. It is important to note that the IDEAL trials included prospective evaluation of quality of life/improvement of disease-related symptoms as endpoints of the study. These were measured using the Functional Assessment of Cancer Therapy–Lung (FACT-L) questionnaire and the asso-
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ciated Lung Cancer Subscale (LCS). There was no significant difference in efficacy between 250 mg/d and 500 mg/d in both trials. In IDEAL-1, Iressa 250 mg/d produced an objective response in 18%, stable disease in 36%, an overall symptom improvement rate of 40% (69% in patients with a tumor response), and improvement of quality of life in 24%. Multivariate analysis identified the following as positive prognostic indicators for response to Iressa: female sex, Japanese ethnic background, and adenocarcinoma. It is not yet known if EGFR status of tumor influences the efficacy of EGFR-TKI. In the more heavily pretreated IDEAL-2 population, similar treatment yielded 12% objective response rate, 31% stabilization of disease, as well as 43% and 34% improvement of symptoms and quality of life respectively. These phase II studies indicated a correlation between improvements of NSCLCrelated symptoms and quality of life with tumor response with symptom response uniquely predictive of progression-free and overall survival.55 The lower-dose cohorts reported better tolerance with mild diarrhea and skin rash as the most common side effects. The positive data obtained from these phase II trials has led to the approval of Iressa in Japan, the USA and Australia for use in patients with advanced NSCLC previously treated with chemotherapy.56 Based on these favorable phase II results and preclinical data demonstrating supraadditive antitumor effects of the combination of EGFR TKI and cytotoxic chemotherapeutics35,41 in NSCLC, two phase I trials of Iressa ⫹ chemotherapy were performed. In the first trial, Iressa (250 mg/mL or 500 mg/mL) ⫹ gemcitabine/cisplatin were administered to 18 patients with solid tumors; 9/17 evaluable patients had partial response and 7/17 had stable disease and 1 progressed.57 In the second trial, Iressa (250 mg/mL or 500 mg/ mL) ⫹ carboplatin/paclitaxel were administered to 25 chemotherapy-naı¨ve patients with NSCLC. Of 20 evaluable patients, 7 had partial responses, 10 had stabilization of disease and 3 progressed.54 Unfortunately, the encouraging results of these two phase I trials could not be confirmed in two larger phase III placebo-controlled trials (INTACT-1 and INTACT-2). These trials accrued over 2000 patients with previously untreated advanced NSCLC. The results showed that Iressa (250 or 500 mg/d) in combination with either carboplatin/paclitaxel or gemcitabine/cis-
platin provided no additional benefit over chemotherapy alone.58,59 Currently, clinical trials are evaluating the role of Iressa as third-line monotherapy for advanced NSCLC (NCI-Canada) as well as its postoperative adjuvant role in patients with completely resected early stage NSCLC (J. Dancey, NCI, personal communication)
Tarceva/OSI-774 Tarceva (Erlotinib, Genentech, Inc., San Francisco, CA) is an orally available, quinazolinebased EGFR-selective TKI which, similar to Iressa, has been shown to block EGFR phosphorylation and tumor cell proliferation and to promote apoptosis as well as chemosenitization in both in vitro and in vivo preclinical animal studies. A phase I trial of Tarceva (dose-escalation 25-200 mg/d) involving 40 patients with advanced solid tumors (4 NSCLC) identified 150 mg/d as the maximum tolerated dose with dose-limiting toxicities being diarrhea and skin rashes. Stable disease lasting more than 5 months was recorded in 1 patient with NSCLC.8 In a phase II trial, 57 patients with advanced NSCLC previously treated with cisplatin-based chemotherapy whose tumors expressed EGFR (⬎10% cells stained positive for EGFR by immunohistochemistry) were given Tarceva 150 mg/d. After 12 weeks of treatment, there was 1 complete response and 6 partial responses for an overall response rate of 12.3%. Stable disease was observed in 15 patients (26%).60,61 The treatment was well tolerated, but there was no correlation between tumor response and the magnitude of EGFR expression (% of positive cells or the intensity of EGFR expression). On the other hand, the appearance of skin rash, widely recognized as a predictable sideeffect of anti-EGFR therapy, has been shown to bear significant positive correlation with treatment-related survival in this trial (grade 2 or 3 rash: 19.6 months versus grade 1 rash: 8.5 months, P ⫽ 0.018 and versus no rash: 1.5 months, P ⬍ 0.0001).61 A phase III trial comparing Tarceva 150 mg/d monotherapy as second- or third-line treatment with best supportive care is ongoing.60 Phase I to III clinical trials combining Tarceva (150mg/d) with standard cytotoxic chemotherapeutics (gemcitabine/cisplatin- TALENT; carboplatin/paclitaxel-TRIBUTE) are being conducted and their results are eagerly awaited.60 As aberrant signaling via GFRs such as EGFR may
Growth Factor Receptors in Lung Cancer Therapy
play crucial roles in transformation, early tumor growth and development of metastasis, oral antiEGFR agents such as Tarceva or Iressa may be more useful if utilized in the clinical setting of adjuvant therapy or chemoprevention.
Cetuximab/IMC-225 Balsega and colleagues reported the results of three successive phase I trials of Cetuximab (Erbitux, ImClone Systems, New York, NY) given as monotherapy (single dose or weekly multiple dose) or in combination with cisplatin which involved a total of 52 patients with advanced cancers of different histologies.62 Cetuximab was dose-escalated from 5 to 100 mg/m2 in monotherapy trials. In the study combining Cetuximab with cisplatin (60mg/m2), limited to patients with EGFR-positive head/neck cancer (n ⫽ 16) or NSCLC (n ⫽ 6), the doses of Cetuximab were further escalated to 200 and 400 mg/m2. The doses of 200 to 400 mg/m2 showed complete saturation of systemic clearance of the antibody, which was hypothesized to correspond to saturation of binding of Cetuximab to tissue EGFR. Antibody against Cetuximab was detected in 1 patient and drug-related toxicity was minimal. In the monotherapy trial, 50% of patients achieved disease stabilization and received one or two additional courses. In the Cetuximab ⫹ cisplatin combination trial, partial responses was seen in 2 patients with head/neck cancer at 4 weeks; only one, however, showed continued partial response on subsequent evaluation. Disease stabilization was observed in 11/19 evaluable patients (58%); 9/13 patients (69%) treated at ⱖ50 mg/m2 completed 12 weeks of therapy. In a phase II trial, a loading dose of Cetuximab 400 mg/m2 followed by a maintenance dose of 250 mg/m2 was combined with Doxetacel 75 mg/m2 as a second-line therapy in NSCLC. After 6 weeks, 4/20 patients had a partial response and 6 had stable disease.63
Trastuzumab Inspired by the increased survival seen in patients with HER2/neu-overexpressing metastatic breast cancers treated with Trastuzumab (Herceptin, Genentech, San Francisco, CA) and cytotoxic chemotherapeutics, clinical trials using Trastuzumab ⫹ standard cytotoxics have been initiated for patients with metastatic NSCLC.10
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Currently, simultaneous phase II trials evaluating the efficacy of combinations of Trastuzumab with gemcitabine/cisplatin or with carboplatin/ paclitaxel or doxetaxel in metastatic NSCLC expressing at least 1⫹ HER2/neu are being conducted at MD Anderson Cancer Center, Memorial Sloan Kettering Cancer Center, by the ECOG and CALGB intergroups, and by Hoffman Laroche. Preliminary response data are available. In the Hoffman LaRoche-sponsored international study, overall response rate of 41% in the control arm (cisplatin ⫹ gemcitabine) was similar to the 36% response in the Trastuzumab ⫹ chemotherapy arm. In the MD Anderson study, a 40% partial response and 45% stable disease pattern were observed in 20 previously untreated evaluable patients treated with Trastuzumab ⫹ gemcitabine/cisplatin. The MSKCC trial, which is comprised of two arms, Trastuzumab plus either doxetaxel or paclitaxel, has enrolled 44 patients and thus far had an overall response rate of 26%. Preliminary data analysis from the ECOG study indicated that 21% of patients who received Trastuzumab and carboplatin/paclitaxel had partial response and 44% had stable disease as best response.
Others Growth Factor-Targeted Agents Besides Tarceva and Iressa, there are other receptor TKIs currently completing phase I trials.64 PKI 166 is a selective EGFR TKI that inhibits HER2/neu TK activity at low nanomolar and micromolar concentrations. Similar to other EGFR TKIs, PKI 166 has been found to be safe with a toxicity profile that includes diarrhea, cutaneous rashes and transient elevation of liver enzymes. Dose-limiting toxicities were observed in doses exceeding 600 mg/d. Phase II trials are currently underway. GW570126 is a dual EGFR and HER2/neu TKI. In preclinical studies, this compound was found to be effective in inhibiting phosphorylation of EGFR or HER2/neu as well as ERK1/2 and AKT in tumor xenografts of cancer cells expressing either EGFR or HER2/neu, respectively. Toxicity studies were performed in healthy volunteers, and this drug was found to be safe with side effects including skin rash, headache, elevation of liver enzymes, and diarrhea. None of these toxicities were dose limiting.
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EKB 569 is an irreversible dual inhibitor of TK of EGFR and HER2/neu. Dose-limiting toxicity was observed at 125 mg/d on a 2-week on/2-week off schedule. The toxicity profile is similar to other TKIs as described above. CI-1033 is an irreversible erbB1 and erB2 TKI. Multiple phase I trials were conducted and the toxicity profile includes nausea, rash, diarrhea, and thrombocytopenia and hypersensitivity reaction (easily controlled by antihistamine premedication). Evaluation of tumor biopsies obtained before and 8 days after the onset of therapy indicated significant reduction of phosphorylated EGFR and Ki67 proliferation marker with a concomitant increase of p27 cyclin-dependent kinase inhibitor. There was also a correlation between the dose levels of CI1033 and modulation of biomarkers noted in tumor as well skin biopsies.
Conclusion GFR-targeted therapy for solid tumor exemplifies the paradigm of modern drug development that combines basic science knowledge with hypothesis-driven translational research and carefully designed and executed clinical trials. The results of clinical trials, while confirming the validity of the hypothesis, pose more questions than provide answers. Studies are currently underway to determine the molecular markers that may be predictive of the biological responses to anti-GFR therapeutic approaches. These will allow better selection of appropriate patients who are most likely benefit from this form of therapy.
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carcinoma and inhibition by anti-epidermal growth factor receptor treatments. Cancer Res 61:6500-6510, 2001 Wu X, Fan Z, Masui H, Rosen N, et al: Apoptosis induced by an anti-epidermal growth factor receptor monoclonal antibody in a human colorectal carcinoma cell line and its delay by insulin. J Clin Invest 95:1897-1905, 1995 Petit AM, Rak J, Hung MC, et al: Neutralizing antibodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors. Am J Pathol 151:1523-1530, 1997 Perrotte P, Matsumoto T, Inoue K, et al: Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice. Clin Cancer Res 5:257-265, 1999 Bruns CJ, Solorzano CC, Harbison MT, et al: Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma. Cancer Res 60:2926-2935, 2000 Ciardiello F, Caputo R, Bianco R, et al: Inhibition of growth factor production and angiogenesis in human cancer cells by ZD1839 (Iressa), a selective epidermal growth factor receptor tyrosine kinase inhibitor. Clin Cancer Res 7:1459-1465, 2001 Bruns CJ, Harbison MT, Davis DW, et al: Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms. Clin Cancer Res 6:1936-1948, 2000 Charoenrat P, Modjtahedi H, Rhys-Evans P, et al: Epidermal growth factor-like ligands differentially up-regulate matrix metalloproteinase 9 in head and neck squamous carcinoma cells. Cancer Res 60:1121-1128, 2000 Charoenrat P, Rhys-Evans P, Court WJ, et al: Differential modulation of proliferation, matrix metalloproteinase expression and invasion of human head and neck squamous carcinoma cells by c-erbB ligands. Clin Exp Metastasis 17:631-639, 1999 Baselga J, Norton L, Masui H, et al: Antitumor effects of doxorubicin in combination with anti-epidermal growth factor receptor monoclonal antibodies. J Natl Cancer Inst 85:1327-1333, 1993 Kern JA, Slebos RJ, Top B, et al: C-erbB-2 expression and codon 12 K-ras mutations both predict shortened survival for patients with pulmonary adenocarcinomas. J Clin Invest 93:516-520, 1994 Ciardiello F, Bianco R, Damiano V, et al: Antitumor activity of sequential treatment with topotecan and antiepidermal growth factor receptor monoclonal antibody C225. Clin Cancer Res 5:909-916, 1999 Ciardiello F, Caputo R, Bianco R, et al: Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor-selective tyrosine kinase inhibitor. Clin Cancer Res 6:2053-2063, 2000 Sirotnak FM, Zakowski MF, Miller VA, et al: Efficacy of cytotoxic agents against human tumor xenografts is markedly enhanced by coadministration of ZD1839 (Iressa), an inhibitor of EGFR tyrosine kinase. Clin Cancer Res 6:4885-4892, 2000
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37. Milas L, Mason K, Hunter N, et al: In vivo enhancement of tumor radioresponse by C225 antiepidermal growth factor receptor antibody. Clin Cancer Res 6:701-708, 2000 38. Magne N, Fischel JL, Dubreuil A, et al: Influence of epidermal growth factor receptor (EGFR), p53 and intrinsic MAP kinase pathway status of tumour cells on the antiproliferative effect of ZD1839 (“Iressa”). Br J Cancer 86:1518-1523, 2000 39. Suzuki T, Nakagawa T, Endo H, et al: The sensitivity of lung cancer cell lines to the EGFR-selective tyrosine kinase inhibitor ZD1839 (’Iressa’(1)) is not related to the expression of EGFR or HER-2 or to K-ras gene status. Lung Cancer 42:35-41, 2003 40. Wakeling AE, Guy SP, Woodburn JR, et al: ZD1839 (Iressa): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy. Cancer Res 62:5749-5754, 2002 41. Sirotnak FM, Zakowski MF, Miller VA, et al: Efficacy of cytotoxic agents against human tumor xenografts is markedly enhanced by coadministration of ZD1839 (Iressa), an inhibitor of EGFR tyrosine kinase. Clin Cancer Res 6:4885-4892, 2000 42. Solit DB, Basso AD, Olshen AB, et al: Inhibition of heat shock protein 90 function down-regulates Akt kinase and sensitizes tumors to Taxol. Cancer Res 63:2139-2144, 2000 43. An WG, Schnur RC, Neckers L, et al: Depletion of p185erbB2, Raf-1 and mutant p53 proteins by geldanamycin derivatives correlates with antiproliferative activity. Cancer Chemother Pharmacol 40:60-64, 1997 44. Xu W, Mimnaugh E, Rosser MF, et al: Sensitivity of mature Erbb2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90. J Biol Chem 276:3702-3708, 2001 45. Nguyen DM, Desai S, Chen A, et al: Modulation of metastasis phenotypes of non-small cell lung cancer cells by 17-allylamino 17-demethoxy geldanamycin. Ann Thorac Surg. 70:1853-60, 2000 46. Nguyen DM, Chen A, Mixon A, et al: Sequence-dependent enhancement of paclitaxel toxicity in non-small cell lung cancer by 17-allylamino 17-demethoxygeldanamycin. J Thorac Cardiovasc Surg 118:908-915, 1999 47. Nguyen DM, Lorang D, Chen GA, et al: Enhancement of paclitaxel-mediated cytotoxicity in lung cancer cells by 17-allylamino geldanamycin: in vitro and in vivo analysis. Ann Thorac Surg 72:371-378, 2001 48. Yu X, Guo ZS, Marcu MG, et al: Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228. J Natl Cancer Inst 94:504-513, 2002 49. Baselga J, Rischin D, Ranson M, et al: Phase I safety, pharmacokinetic, and pharmacodynamic trial of ZD1839, a selective oral epidermal growth factor receptor tyrosine kinase inhibitor, in patients with five selected solid tumor types. J Clin Oncol 20:4292-4302, 2002 50. Herbst RS, Maddox AM, Rothenberg ML, et al: Selective oral epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 is generally well-tolerated and has activity in non-small-cell lung cancer and other solid tumors: results of a phase I trial. J Clin Oncol 20:3815-3825, 2002
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51. Ranson M, Hammond LA, Ferry D, et al: ZD1839, a selective oral epidermal growth factor receptor-tyrosine kinase inhibitor, is well tolerated and active in patients with solid, malignant tumors: results of a phase I trial. J Clin Oncol 20:2240-2250, 2002 52. Nakagawa K, Tamura T, Negoro S, et al: Phase I pharmacokinetic trial of the selective oral epidermal growth factor receptor tyrosine kinase inhibitor gifitinib (Iressa, ZD1839) in Japanese patients with solid malignant tumors. Ann Oncol 14:922-930, 2003 53. Fukuoka M, Yano S, Giaccone G, et al: Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer. J Clin Oncol 21:2237-2246, 2003 54. Miller VA, Johnson DH, Krug LM, et al: Pilot trial of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib plus carboplatin and paclitaxel in patients with stage IIIB or IV non-small-cell lung cancer. J Clin Oncol 21:2094-2100, 2003 55. Cella D. Impact of ZD1839 on non-small cell lung cancerrelated symptoms as measured by the functional assessment of cancer therapy-lung scale. Semin Oncol 30 39-48, 2003 (suppl 1) 56. Dancey JE, Freidlin B: Targeting epidermal growth factor receptor–are we missing the mark? Lancet 362:62-64, 2003 57. Gonzalez-Larriba JL, Giaccone G, van Oosterom A, et al: ZD1839 (Iressa) in combination with gemcitabine and cisplatin in chemo-naive patients with advanced solid tumors: final results of a phase I trial. Proc ASCO 21:95a, 2002
58. Giaccone G, Johnson DH, Manegold C, et al: A phase III clinical trial of ZD1839 (Iressa) in combination with gemcitabine and cisplatin in chemotherapy-naive patients with advanced non-small cell lung cancer (INTACT 1). Ann Oncol 13:40a, 2002 (suppl 5) 59. Johnson DH, Herbst R, Giaccone G, et al. ZD1839 (Iressa) in combination with paclitaxel and carboplatin in chemotherapy-naive patients with advanced non-small cell lung cancer (NSCLC): results from a phase III clinical trial (INTACT 2). Ann Oncol13:468a, 2002 (suppl 5) 60. Herbst RS: Erlotinib (Tarceva): An update on the clinical trial program. Semin Oncol 30:34-46, 2003 (suppl 7) 61. Perez–Soler, R, Chachoua, A, Huberman, M, et al Determinants of tumor response and survival in patients with relapsing NSCLV treated with Tarceva (erlotinib HCl, OSI-774). Final report of a phase II study. Presented at the American Society of Clinical Oncolcogy Molecular Therapy Symposium, November 8-10, San Diego, CA 62. Baselga J, Pfister D, Cooper MR, et al: Phase I studies of anti-epidermal growth factor receptor chimeric antibody C225 alone and in combination with cisplatin. J Clin Oncol 18:904-914, 2000 63. Kim, ES, Mauer, AM, Fossella, FV, et al A phase II study of Erbitux (IMC-c225), an epidermal growth factor receptor (EGFR) blocking antibody, in combination with docetaxel in chemotherapy refractory/resistant patients with advanced non-small cell lung cancer (NSCLC). Proc ASCO 21: 293a, 2002 64. Bonomi P: Clinical studies with non-iressa EGFR tyrosine kinase inhibitors. Lung Cancer 41:S43-S48, 2003
L
ung cancer is the second-most common malignant disease in both men and women in the United States. It is estimated that lung cancer [(20% small cell lung cancer (SCLC), 80% nonsmall cell lung cancer (NSCLC)] will account for 171,900 new cases and 157,200 deaths in 20031 with case-to-mortality ratio of almost one, which underscores of the lack of an effective therapy for this disease. The overwhelming majority of SCLC presents as stages III or IV for which multidrug chemotherapy regimens and thoracic/cranial radiotherapy is the only available treatment modality with an overall 5-year survival rate of less than 25%.2 Surgical resection offers the best chance for cure in patients with early stages (I and II) NSCLC. About 70% of newly diagnosed cases of NSCLC, unfortunately, present as either locally advanced (stage III) or metastatic disease (stage IV), for which combinations of chemotherapy and radiation are the standard-of-care treatment strategy. Overall, the rate of meaningful tumor response that can be translated to increased survival by standard cytoFrom the Section of Thoracic Oncology, Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD Address reprint requests to Dao M. Nguyen, Room 2B07, Building 10, 10 Center Drive, Bethesda, MD 20892. 1043-0679/04/1601-0003$30.00/0 doi:10.1053/j.semtcvs.2003.12.002
toxic chemotherapy regimens administered to patients with stage IV NSCLC remains low, with an overall 2-year survival rate of only 11%.3 It is clear that a new paradigm/direction for lung cancer therapeutics is needed. Growth factors and their receptors, particularly those of the epidermal growth factor receptor (EGFR) superfamily, have been shown to play crucial roles in carcinogenesis. More importantly, a large percentage of solid tumors express high levels of growth factor receptors (GFRs). This observation provides the rationale for the research efforts designed to develop anti-GFR compounds as part of a novel anticancer therapeutic strategy for a variety of solid malignancies including lung cancer.
Molecular Biology of Growth Signaling Pathways Of all the GFRs expressed on lung cancer cells, those belonging to the EGFR superfamily (EGFR/erbB1 and HER2/neu/erbB2) are the best studied and most extensively exploited as targets for development of novel therapeutics for this disease.4 EGFR-mediated signal transduction, serving as a model for molecular signaling by other tyrosine kinase membrane receptors, will be described in detail in this review. The EGFR superfamily consists of four members: EGFR (erbB1), HER2/neu (erbB2), erbB3,
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Figure 1. Tyrosine kinase GFR-dependent signal transduction pathways, biological response and relevant targeted pharmacologic inhibitors
and erbB4. These surface receptors are composed of a ligand-binding extracellular domain, a transmembrane segment, and an intracellular domain that has tyrosine kinase (TK) activity. Binding of ligands (EGF, TGF-␣, amphiregulin, and others) to the extracellular domain of EGFR results in formation of homodimers (EGFR-EGFR) or heterodimers (EGFR and other members) that result in the stimulation of intrinsic receptor tyrosine kinase activity. This leads to autophosphorylation of multiple tyrosine residues located in the intracellular domain of the receptors. The phosphorylated intracellular domain then serves as a docking site for various adaptor molecules (such as SOS, Grb2) as well as functional proteins (PI3K p110 subunit). These in turn activate multiple parallel intracellular signaling pathways (such as those mediated by ERK1/2, JNK, PI3K/ AKT, STAT, PKC). The biological responses from such diverse intracellular signalings include cell proliferation, survival, motility, invasion, adhesion, and angiogenesis (Fig 1). Overexpression
of EGFR or HE2/neu and dysregulation of the downstream signal transduction pathways play a critical role in tumorigenesis.5 While normal bronchial mucosa weakly expresses EGFR mainly confined to the basal layer, bronchial squamous metaplasia and dysplasia overexpress EGFR.6-8 EGFR overexpression (higher than levels of receptor expression in surrounding normal tissues) is very frequent in NSCLC (84% of squamous cell carcinoma, 65% of adenocarcinoma and large cell carcinoma), while none is detected in small cell lung cancer.6 In addition to NSCLC, high levels of EGFR expression have been documented in other solid tumor malignancies including colon, prostate, head/ neck, ovary, and breast. In contrast to the HER2/ neu overexpression identified in breast cancer that is most frequently caused by gene amplification, the molecular mechanism of EGFR overexpression in lung cancer cells is unclear. Coexpression of EGFR ligand TGF-␣, frequently seen in NSCLC and prostate cancer, can mediate a
Growth Factor Receptors in Lung Cancer Therapy
constitutive activation of the receptor via an autocrine/paracrine loop.5 The association between EGFR overexpression and poor prognosis has been extensively investigated in multiple studies with inconclusive results. Recent meta-analysis, however, has indicated that EGFR expression might be a poor prognostic factor for survival in NSCLC, but the amplitude of the impact is small and subjected to publication bias.9 About 33% of NSCLC (adenocarcinoma and large cell carcinoma but not squamous cell carcinoma) express high levels of HER2/neu. While gene amplification is the common mechanism responsible for high levels of HER2/neu expression (3⫹/4⫹) in 30% of breast cancers that are susceptible to Herceptin-based therapy,10 HER2/neu overexpression in lung cancer is much less intense (2⫹) and gene amplification is rare. Immunohistochemical staining of tumor NSCLC using the FDA-approved Herceptest indicated that ⱖ2⫹ HER2/neu expression was observed in 20% to 30% of cases.10 The predictive value of HER2/neu overexpression and poor clinical outcome in patients with NSCLC has been investigated but the results remain controversial.6 Despite the lack of a clear understanding of the prognostic relevance of EGFR family members in NCSLC, their overexpression patterns do make them suitable for molecularlybased therapy. In addition to members of the EGFR superfamily, other GFRs have been described in cultured lung cancer cell lines as well as tumor tissues. These include platelet-derived growth factor receptor (PDGFR),11 the hepatocyte growth factor/scatter factor (HGF/SF) receptor (c-Met)12,13 and the insulin-like growth factor (IGF-R)14 as well as c-kit.15 In-depth description of the molecular biology and the therapeutic potentials of these receptors and their cognate growth factors are beyond the scope of this review. Preclinical studies have been published describing the expression of these receptors in both NSCLC and SCLC and their putative roles in promoting cancer development.11,15,16 Of note, is the expression of PDGFRs and c-kit on lung cancer cells for which there exist several potent TKIs for this group of GFRs, particularly the wellstudied drug Imatinib mesylate (Gleevec, formerly STI571, Novartis Pharma AG, Basel, Switzerland).17,18
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Molecular Therapy for Lung Cancer by Targeting the Growth Signaling Pathways The signal transduction activity of GFRs can be abrogated by: 1) inhibition of receptor expression using gene therapy (antisense approach); 2) antagonistic monoclonal antibodies that prevent binding of ligands to receptors or; or 3) pharmacologic (low-molecular weight) receptor-selective tyrosine kinase inhibitors. The later two approaches are the preferred strategies currently under intense clinical development. Some of these anti-EGFR agents (tyrosine kinase inhibitors Iressa, Tarceva and monoclonal antibody IMC-225) are now in phase III clinical trials for patients with advanced-staged NSCLC. The underlying rationale for developing EGFR-targeted therapy is that differential expression of EGFR in tumor cells versus normal tissues can provide an exploitable therapeutic window in cancers known to overexpress EGFR. Both treatment strategies have been shown to be safe in clinical trials involving hundreds of patients. Tyrosine kinase inhibitors are administered orally, making them ideal for chronic maintenance therapy. Functionally, these agents can indirectly block erbB2 signaling via inhibition of erbB1/erbB2 heterodimers as well as other lateral signaling pathways that converge with the erbB1 pathway.7,19 Monoclonal antibodies, on the other hand, have to be administered intravenously. They, however, have a very long half-life. Functionally, these antibodies, in addition to blocking ligand binding to receptors, induce downregulation of surface receptors as well as incite antibody-dependent cell-mediated cytotoxicity.20 It is not possible at this point to predict which of these two well-tolerated approach will be more effective. In addition to membrane receptors, all of the intracellular intermediaries of EGFR-dependent signaling pathways have been selected as potential molecular targets for clinical development.21 Some of the pharmacologic antagonists are in early phase clinical trials (Table 1).
Preclinical Studies Growth Factor Receptor Antagonists Antireceptor mouse monoclonal antibodies (mAb) have been developed to bind to the easily accessible extracellular domain of the receptor
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Table 1. Selected Agents Targeting GFR-Dependent Signal Transduction Pathways Currently in Clinical Trials for Solid Tumors Including NSCLC Class
Agent
Target
Antibodies
Cetuximab ABX-EGF EMD-72000 RH3 MDX-447 Trastuzumab Gefitinib Erlotinib GW0126 CI-1033 EKB-569 PI-166 ISIS-2503⫹ R 115777⫹⫹ SCH 66336 BMS-214662 ISIS-5132 L-779-450 BAY-43-9006 PD 184352 17-AAG FK228
EGFR EGFR EGFR EGFR EGFR EGFR EGFR EGFR EGFR, HER2/neu EGFR, HER2/neu, HER4 EGFR EGFR, HER2/neu Ras Ras Ras Ras Raf Raf Raf MEK Multiple (HSP90)* Multiple (HDACI)**
GFR-TKI
Others
Phase of Development Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase
2, 2 1 2 1 2 3 3 1, 1, 1 1, 2 2, 2 2, 2 1 2 1 1, 1,
3
2 2 2 3 3
2 2
⫹, Antisense oligonucleotide. ⫹⫹, Farnesyl transferase inhibitor. *HSP90: heat shock protein 90. **HDACI: histone deacetylase inhibitor.
and compete with ligand binding to the receptor. To eliminate immunologic response to xeno-immunoglobulins, humanized (chimeric human: murine) antibodies have been produced. The two prime examples of monoclonal antibodies are antiHER2/neu Herceptin (Trastuzumab) and anti-EGFR IMC-225 (Cetuximab). In addition to preventing ligand binding and activation of receptor phosphorylation, these antibodies also induce antibody-mediated cell-dependent cytotoxicity as well as antibody-mediated receptor dimerization and downregulation. Receptor tyrosine kinase inhibitors (RTKI) are low molecular weight compounds, identified by highthroughput screenings of natural and synthetic chemicals that compete, reversibly or irreversibly, with ATP for binding to the tyrosine kinase portion of the receptor thereby inhibiting the receptor’s catalytic activity. Extensive preclinical in vitro and in vivo studies have been performed to evaluate the antitumor properties of these agents. Treating cancer cells of diverse histology with either EGFR TKIs or MAbs results in dose-de-
pendent reduction of phosphorylated (activated) EGFR, AKT, ERK1/2 without effecting overall protein levels. This indicates that these compounds functionally inhibit the kinase activity as well as the downstream signalings of EGFR. This was paralleled with elevated levels of the cdk 2 inhibitor p27, hypophosphorylation of the Rb protein, reduction of cdk4 and cell arrest at G1 phase of the cell cycle.19,22,23 Induction of apoptosis has been reported with anti-EGFR mAb treatment in vitro24 but this is the exception rather than the rule with this treatment modality. GFR antagonists also result in a significant decrease in tumor cell-derived production of angiogenic growth factors such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and interleukin-8.25-28 This correlates with a reduction of microvessel density and increase of apoptosis of endothelial cells in human tumor xenografts.27-29 GFR blockade also results in significant reduction of the expression and activity of several matrix metalloproteinases (MMPs), most notably MMP-9, and a decrease of cancer cell motility and invasive-
Growth Factor Receptors in Lung Cancer Therapy
ness through an in vitro Matrigel matrix.30,31 This corresponds with inhibition of tumor growth and metastasis formation in vivo. The inhibitory effects on invasion, metastasis, angiogenesis may explain why anti-GFR treatments are often more effective in vivo and in vitro. GFR blockade has been shown to enhance tumor cell response to cytotoxic agents of different classes (cisplatin, doxorubicin, paclitaxel, topotecan) in an additive or supraadditive (synergistic) fashion both in tissue culture as well as in animal models of human cancer xenografts.32-37 Even though considered an EGFR-targeted therapy, it is peculiar to observe that the growth inhibitory response of cancer cells to Iressa, both in vitro and in vivo preclinical studies, and as monotherapy or in combination with cytotoxic agents, can not be reliably predicted by the levels of EGFR expression on the cell membrane.38-41 EGFR levels may not be the most accurate biomarker predictive of cellular responsiveness to EGFR blockade. Phosphorylation of EGFR and/or downstream effectors such as AKT or ERK1/2 (indicative of activation) in target cells may be of more value in determining their susceptibility to GFR inhibition regardless of the total levels of EGFR.
Other Agents In addition to specific growth factor receptor antagonists described above, there are other pharmacologic agents that exert potent anticancer property by inhibiting the expression of critical components of the signaling pathways. One example of such a compound is the HSP-antagonist 17-AAG. 17-AAG, at nanomolar concentrations, inhibits the chaperone function of heat shock protein 90 (HSP90), thus mediating a rapid and profound degradation of EGFR, ErbB2, c-Met, Raf, total and phosphorylated AKT as well as other oncoproteins in malignant cells.42-44 Treating cultured NSCLC cells expressing high levels of EGFR and/or HER2/neu with 17-AAG resulted in profound growth inhibition, significant sensitization of cancer cells to paclitaxel, drastic reduction of VEGF and MMP-9 production as well as invasion to Matrigel extracellular matrix in vitro.45 The antiangiogenic property as well as the chemosensitization effect of 17-AAG was confirmed in an animal models of NSCLC xenografts.42,46,47 More recently, our laboratory also described the link between down-regulation of
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EGFR, erbB2, Raf-1 expression and inhibition of signal transduction via Raf-1 and ERK pathway to the growth inhibitory property of the histone deacetylase inhibitor FK228 in NSCLC cells.48
Clinical Studies Iressa/ZD1839 Iressa (Gefitinib, AstraZeneca, London, UK) is an EGFR-TKI that has been studied in four phase I dose-escalation clinical trials in patients with solid tumors. These include 100 patients with advanced NSCLC who failed multiple regimens of cytotoxic chemotherapies.49-52 Patients received Iressa 50-1000 mg/d (14-days on/14-days off or 28-days continuous regimens). The maximum tolerated dose was 700 mg/d, with acneiform rash and diarrhea being the most commonly cited dose-limiting toxicities. Other side effects included fatigue, nausea, anorexia, and elevated liver enzymes. These were, however, mild (grade 1,2) and reversible on cessation of drug administration. Partial response or stable disease lasting for 1 to ⬎ 6 months were observed in 22% to 25% of patients with NSCLC. Pharmacodynamic study of the effect of Iressa on EGFR signaling in skin biopsies of patients undergoing treatment with this EGFR-TKI indicated significant reduction of phosphorylated EGFR, MAPK ERK1/2, proliferation antigen Ki67, and elevation of p27/KIP1 similar to those observed in preclinical studies. Similar pharmacodynamic studies evaluating pre- and on-therapy tumor biopsies are currently underway. A possible correlation between EGFR-TKI biologic effects in skin versus tumor may exist which allows accurate prediction of potential benefits early in the course of therapy from skin biopsy.22 The encouraging results from these phase I studies led to two phase II studies (IDEAL-1 and IDEAL-2) of Iressa 250 mg/d and 500 mg/d involving 426 patients with advanced, pretreated NSCLC at two different sites (United States and Japan).53,54 EGFR expression on tumor cells was not an inclusion criterion for enrollment to these trials. It is important to note that the IDEAL trials included prospective evaluation of quality of life/improvement of disease-related symptoms as endpoints of the study. These were measured using the Functional Assessment of Cancer Therapy–Lung (FACT-L) questionnaire and the asso-
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ciated Lung Cancer Subscale (LCS). There was no significant difference in efficacy between 250 mg/d and 500 mg/d in both trials. In IDEAL-1, Iressa 250 mg/d produced an objective response in 18%, stable disease in 36%, an overall symptom improvement rate of 40% (69% in patients with a tumor response), and improvement of quality of life in 24%. Multivariate analysis identified the following as positive prognostic indicators for response to Iressa: female sex, Japanese ethnic background, and adenocarcinoma. It is not yet known if EGFR status of tumor influences the efficacy of EGFR-TKI. In the more heavily pretreated IDEAL-2 population, similar treatment yielded 12% objective response rate, 31% stabilization of disease, as well as 43% and 34% improvement of symptoms and quality of life respectively. These phase II studies indicated a correlation between improvements of NSCLCrelated symptoms and quality of life with tumor response with symptom response uniquely predictive of progression-free and overall survival.55 The lower-dose cohorts reported better tolerance with mild diarrhea and skin rash as the most common side effects. The positive data obtained from these phase II trials has led to the approval of Iressa in Japan, the USA and Australia for use in patients with advanced NSCLC previously treated with chemotherapy.56 Based on these favorable phase II results and preclinical data demonstrating supraadditive antitumor effects of the combination of EGFR TKI and cytotoxic chemotherapeutics35,41 in NSCLC, two phase I trials of Iressa ⫹ chemotherapy were performed. In the first trial, Iressa (250 mg/mL or 500 mg/mL) ⫹ gemcitabine/cisplatin were administered to 18 patients with solid tumors; 9/17 evaluable patients had partial response and 7/17 had stable disease and 1 progressed.57 In the second trial, Iressa (250 mg/mL or 500 mg/ mL) ⫹ carboplatin/paclitaxel were administered to 25 chemotherapy-naı¨ve patients with NSCLC. Of 20 evaluable patients, 7 had partial responses, 10 had stabilization of disease and 3 progressed.54 Unfortunately, the encouraging results of these two phase I trials could not be confirmed in two larger phase III placebo-controlled trials (INTACT-1 and INTACT-2). These trials accrued over 2000 patients with previously untreated advanced NSCLC. The results showed that Iressa (250 or 500 mg/d) in combination with either carboplatin/paclitaxel or gemcitabine/cis-
platin provided no additional benefit over chemotherapy alone.58,59 Currently, clinical trials are evaluating the role of Iressa as third-line monotherapy for advanced NSCLC (NCI-Canada) as well as its postoperative adjuvant role in patients with completely resected early stage NSCLC (J. Dancey, NCI, personal communication)
Tarceva/OSI-774 Tarceva (Erlotinib, Genentech, Inc., San Francisco, CA) is an orally available, quinazolinebased EGFR-selective TKI which, similar to Iressa, has been shown to block EGFR phosphorylation and tumor cell proliferation and to promote apoptosis as well as chemosenitization in both in vitro and in vivo preclinical animal studies. A phase I trial of Tarceva (dose-escalation 25-200 mg/d) involving 40 patients with advanced solid tumors (4 NSCLC) identified 150 mg/d as the maximum tolerated dose with dose-limiting toxicities being diarrhea and skin rashes. Stable disease lasting more than 5 months was recorded in 1 patient with NSCLC.8 In a phase II trial, 57 patients with advanced NSCLC previously treated with cisplatin-based chemotherapy whose tumors expressed EGFR (⬎10% cells stained positive for EGFR by immunohistochemistry) were given Tarceva 150 mg/d. After 12 weeks of treatment, there was 1 complete response and 6 partial responses for an overall response rate of 12.3%. Stable disease was observed in 15 patients (26%).60,61 The treatment was well tolerated, but there was no correlation between tumor response and the magnitude of EGFR expression (% of positive cells or the intensity of EGFR expression). On the other hand, the appearance of skin rash, widely recognized as a predictable sideeffect of anti-EGFR therapy, has been shown to bear significant positive correlation with treatment-related survival in this trial (grade 2 or 3 rash: 19.6 months versus grade 1 rash: 8.5 months, P ⫽ 0.018 and versus no rash: 1.5 months, P ⬍ 0.0001).61 A phase III trial comparing Tarceva 150 mg/d monotherapy as second- or third-line treatment with best supportive care is ongoing.60 Phase I to III clinical trials combining Tarceva (150mg/d) with standard cytotoxic chemotherapeutics (gemcitabine/cisplatin- TALENT; carboplatin/paclitaxel-TRIBUTE) are being conducted and their results are eagerly awaited.60 As aberrant signaling via GFRs such as EGFR may
Growth Factor Receptors in Lung Cancer Therapy
play crucial roles in transformation, early tumor growth and development of metastasis, oral antiEGFR agents such as Tarceva or Iressa may be more useful if utilized in the clinical setting of adjuvant therapy or chemoprevention.
Cetuximab/IMC-225 Balsega and colleagues reported the results of three successive phase I trials of Cetuximab (Erbitux, ImClone Systems, New York, NY) given as monotherapy (single dose or weekly multiple dose) or in combination with cisplatin which involved a total of 52 patients with advanced cancers of different histologies.62 Cetuximab was dose-escalated from 5 to 100 mg/m2 in monotherapy trials. In the study combining Cetuximab with cisplatin (60mg/m2), limited to patients with EGFR-positive head/neck cancer (n ⫽ 16) or NSCLC (n ⫽ 6), the doses of Cetuximab were further escalated to 200 and 400 mg/m2. The doses of 200 to 400 mg/m2 showed complete saturation of systemic clearance of the antibody, which was hypothesized to correspond to saturation of binding of Cetuximab to tissue EGFR. Antibody against Cetuximab was detected in 1 patient and drug-related toxicity was minimal. In the monotherapy trial, 50% of patients achieved disease stabilization and received one or two additional courses. In the Cetuximab ⫹ cisplatin combination trial, partial responses was seen in 2 patients with head/neck cancer at 4 weeks; only one, however, showed continued partial response on subsequent evaluation. Disease stabilization was observed in 11/19 evaluable patients (58%); 9/13 patients (69%) treated at ⱖ50 mg/m2 completed 12 weeks of therapy. In a phase II trial, a loading dose of Cetuximab 400 mg/m2 followed by a maintenance dose of 250 mg/m2 was combined with Doxetacel 75 mg/m2 as a second-line therapy in NSCLC. After 6 weeks, 4/20 patients had a partial response and 6 had stable disease.63
Trastuzumab Inspired by the increased survival seen in patients with HER2/neu-overexpressing metastatic breast cancers treated with Trastuzumab (Herceptin, Genentech, San Francisco, CA) and cytotoxic chemotherapeutics, clinical trials using Trastuzumab ⫹ standard cytotoxics have been initiated for patients with metastatic NSCLC.10
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Currently, simultaneous phase II trials evaluating the efficacy of combinations of Trastuzumab with gemcitabine/cisplatin or with carboplatin/ paclitaxel or doxetaxel in metastatic NSCLC expressing at least 1⫹ HER2/neu are being conducted at MD Anderson Cancer Center, Memorial Sloan Kettering Cancer Center, by the ECOG and CALGB intergroups, and by Hoffman Laroche. Preliminary response data are available. In the Hoffman LaRoche-sponsored international study, overall response rate of 41% in the control arm (cisplatin ⫹ gemcitabine) was similar to the 36% response in the Trastuzumab ⫹ chemotherapy arm. In the MD Anderson study, a 40% partial response and 45% stable disease pattern were observed in 20 previously untreated evaluable patients treated with Trastuzumab ⫹ gemcitabine/cisplatin. The MSKCC trial, which is comprised of two arms, Trastuzumab plus either doxetaxel or paclitaxel, has enrolled 44 patients and thus far had an overall response rate of 26%. Preliminary data analysis from the ECOG study indicated that 21% of patients who received Trastuzumab and carboplatin/paclitaxel had partial response and 44% had stable disease as best response.
Others Growth Factor-Targeted Agents Besides Tarceva and Iressa, there are other receptor TKIs currently completing phase I trials.64 PKI 166 is a selective EGFR TKI that inhibits HER2/neu TK activity at low nanomolar and micromolar concentrations. Similar to other EGFR TKIs, PKI 166 has been found to be safe with a toxicity profile that includes diarrhea, cutaneous rashes and transient elevation of liver enzymes. Dose-limiting toxicities were observed in doses exceeding 600 mg/d. Phase II trials are currently underway. GW570126 is a dual EGFR and HER2/neu TKI. In preclinical studies, this compound was found to be effective in inhibiting phosphorylation of EGFR or HER2/neu as well as ERK1/2 and AKT in tumor xenografts of cancer cells expressing either EGFR or HER2/neu, respectively. Toxicity studies were performed in healthy volunteers, and this drug was found to be safe with side effects including skin rash, headache, elevation of liver enzymes, and diarrhea. None of these toxicities were dose limiting.
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EKB 569 is an irreversible dual inhibitor of TK of EGFR and HER2/neu. Dose-limiting toxicity was observed at 125 mg/d on a 2-week on/2-week off schedule. The toxicity profile is similar to other TKIs as described above. CI-1033 is an irreversible erbB1 and erB2 TKI. Multiple phase I trials were conducted and the toxicity profile includes nausea, rash, diarrhea, and thrombocytopenia and hypersensitivity reaction (easily controlled by antihistamine premedication). Evaluation of tumor biopsies obtained before and 8 days after the onset of therapy indicated significant reduction of phosphorylated EGFR and Ki67 proliferation marker with a concomitant increase of p27 cyclin-dependent kinase inhibitor. There was also a correlation between the dose levels of CI1033 and modulation of biomarkers noted in tumor as well skin biopsies.
Conclusion GFR-targeted therapy for solid tumor exemplifies the paradigm of modern drug development that combines basic science knowledge with hypothesis-driven translational research and carefully designed and executed clinical trials. The results of clinical trials, while confirming the validity of the hypothesis, pose more questions than provide answers. Studies are currently underway to determine the molecular markers that may be predictive of the biological responses to anti-GFR therapeutic approaches. These will allow better selection of appropriate patients who are most likely benefit from this form of therapy.
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