Combining Targeted Therapies and Drugs with Multiple Targets in the Treatment of NSCLC
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《肿瘤学家》
ABSTRACT
The first generation of clinical trials of targeted agents in non-small cell lung cancer (NSCLC) treatment has concluded. To date, only a few of these new agents can offer hope of a substantial impact on the natural history of the disease. Nevertheless, clinically meaningful advances have already been achieved. In chemotherapy-refractory advanced NSCLC patients, gefitinib and erlotinib, two epidermal growth factor receptor tyrosine kinase inhibitors, represent a further chance for tumor control and symptom palliation. In chemotherapy-naive, advanced, nonsquamous NSCLC patients, the combination of the anti–vascular endothelial growth factor monoclonal antibody bevacizumab with chemotherapy was demonstrated to produce better survival outcomes than with chemotherapy alone. The relative failure of first-generation targeted therapies in lung cancer may be a result of multilevel cross-stimulation among the targets of the new biological agents. Thus, blocking only one of these pathways allows others to act as salvage or escape mechanisms for cancer cells. Preclinical evidence of the synergistic antitumor activity achievable by combining targeted agents that block multiple signaling pathways has recently been emerging. Clinical trials of multitargeted therapy may represent the second generation of studies in this field, and some of these are already ongoing. In a recent phase I/II trial, the combination of erlotinib and bevacizumab demonstrated very promising activity in the treatment of advanced NSCLC pretreated with chemotherapy. Whether the multitargeted approach is best performed using combinations of selective agents or agents that intrinsically target various targets is a matter of debate.
INTRODUCTION
Lung cancer is the leading cause of cancer-related mortality in both men and women [1], with 1.2 million new cases diagnosed every year and 1 million deaths recorded worldwide in 2001 [2]. Non-small cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers. The majority of NSCLC patients present with advanced disease at diagnosis, and a large portion of those diagnosed with early-stage disease eventually recur, experiencing metastatic disease. For advanced disease, palliation and the patients’ quality of life are still the primary goals of therapy, with total cure remaining elusive.
Although chemotherapy has recently produced promising results as neoadjuvant and adjuvant strategies for early-stage patients [3, 4], and some progress has been made in the treatment of locally advanced and advanced disease [5, 6], treatment outcomes for NSCLC patients must still be considered disappointing. Thus, clinical research of new treatment strategies is warranted. Advances in the knowledge of tumor biology and mechanisms of oncogenesis have granted the singling out of several molecular targets for NSCLC treatment. Targeted therapies are designed to interfere with specific aberrant biological pathways involved in tumorigenesis. A large amount of preclinical in vivo and in vitro data has been gathered on the antitumor properties of a number of new biological agents, both as single agents and combined with other conventional treatment modalities, such as chemotherapy and radiotherapy. Consequently, several targeted agents have been introduced into clinical trials in NSCLC, with many phase I and phase II studies already completed and some phase III study results being recently made available. The first generation of clinical trials of targeted agents in NSCLC treatment has been concluded, and some conclusions can now be drawn. To date, only a few of these new agents can offer hope of a substantial impact on the natural history of the disease, and negative results are more commonly reported than positive ones. Nevertheless, clinically meaningful advances have already been achieved. In chemotherapy-refractory advanced NSCLC patients, gefitinib (Iressa®; Astra-Zeneca Pharmaceuticals, Wilmington, DE) and erlotinib (Tarceva®; OSI Pharmaceuticals, Inc., Melville, NY), two epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), represent a further chance for tumor control and symptom palliation for a subset of patients otherwise eligible only for supportive care [7–10]. In chemotherapy-naive advanced NSCLC patients (with non-squamous histology), the combination of the anti–vascular endothelial growth factor (anti-VEGF) monoclonal antibody bevacizumab (Avastin®; Genentech Inc., South San Francisco, CA) with chemotherapy has been demonstrated to produce better survival outcomes than chemotherapy alone [11].
Moreover, disappointing results should not be considered definitive, and important lessons can be learned from this first generation of clinical trials, which should be considered only the first step in clinical research in this field. In fact, one of the main reasons for the relative failure of the first generation of clinical trials of targeted therapy in lung cancer is that there is multilevel cross-stimulation among the targets of the new biological agents along several pathways of signal transduction that lead to neoplastic events, and blocking only one of these pathways allows others to act as salvage or escape mechanisms for cancer cells. Preclinical evidence of synergistic antitumor activity achievable by combining targeted agents that block multiple signaling pathways has recently been emerging [12,13]. Clinical trials of multitargeted therapy may represent the second generation of studies in this field, and some of these are already ongoing.
THE MAIN FIRST-GENERATION CLINICAL TRIALS OF TARGETED AGENTS IN NSCLC TREATMENT
Targeted Agents as Monotherapy
The main first-generation clinical trials of targeted therapies in NSCLC treatment are summarized in Table 1.
Gefitinib and erlotinib are two orally available EGFR-TKIs. Two large, phase II trials have been conducted on gefitinib monotherapy in patients with advanced NSCLC that was not controlled after one or more chemotherapy regimens. In those two studies, named Iressa Dose Evaluation in Advanced Lung Cancer (IDEAL)-1 and IDEAL-2, gefitinib was demonstrated to be active (with overall response rates in the range of 10%–20%), to improve disease symptoms in about 40% of patients, and to be well tolerated [7, 8]. On the basis of these clinical trials, gefitinib was licensed for the third-line treatment of platinum- and docetaxel-refractory advanced NSCLC in several countries, including Japan, Australia, and the U.S. However, very recently, Thatcher et al. [9] reported on a phase III trial named Iressa Survival Evaluation in Lung Cancer (ISEL), which compared gefitinib with best supportive care in patients with advanced NSCLC who had received one or two prior chemotherapy regimens. In 1,692 patients (ISEL is the largest study conducted in the refractory advanced NSCLC population), a difference between gefitinib and placebo was reported, although this did not reach statistical significance in the overall or adenocarcinoma histology populations. Preplanned subgroup analyses suggested a survival benefit in patients of Asian origin and in patients who never smoked. Specifically, at a median follow-up of 7 months, the median survival times were 5.6 months versus 5.1 months (hazard ratio [HR], 0.89; 95% confidence interval [CI], 0.78–1.03; p = .11), in the overall population, and 6.3 months versus 5.4 months, in patients with adenocarcinoma histology (HR, 0.83; 95% CI, 0.67–1.02; p = .07), for gefitinib and placebo, respectively. As stated before, preplanned subgroup analyses indicated statistically different survival outcomes in smokers compared with never-smokers and in patients of Asian versus non-Asian origin. In patients of Asian origin, gefitinib-treated patients survived longer than placebo-treated patients (HR, 0.66; 95% CI, 0.48–0.91; p = .01; median survival, 9.5 months vs. 5.5 months, respectively). A similar result was seen in never-smokers, where gefitinib-treated patients survived longer than placebo-treated patients (HR, 0.67; 95% CI, 0.49–0.91; p = .01; median survival, 8.9 months vs. 6.1 months, respectively). A statistically significant higher response rate was observed for gefitinib-treated patients compared with placebo-treated patients.
Erlotinib in a phase III, randomized, placebo-controlled trial has been proven to prolong survival (6.7 months vs. 4.7 months for erlotinib and placebo, respectively; p = .001) in NSCLC patients after first- or second-line chemotherapy [10]. The analysis of quality of life and time to deterioration in patient-reported symptoms showed statistically and clinically meaningful benefits for patients randomized to erlotinib. Moreover, erlotinib was active (response rate, 8.9%) and safe (only 5% of patients discontinued treatment for toxicity). Tsao et al. [14] used tumor biopsy samples from participants in the aforementioned trial to investigate whether responsiveness to erlotinib and its impact on survival were associated with tumor expression of EGFR and EGFR gene amplification and mutations. In multivariate analyses, adenocarcinoma (p = .01), never having smoked (p < .01), and EGFR expression (p = .03) were associated with an objective response. In contrast, survival after treatment with erlotinib was not influenced by the status of EGFR expression, the number of EGFR copies, or EGFR mutations. Thus, the presence of an EGFR mutation may increase responsiveness to the agent, but it is not indicative of a survival benefit.
Combination of Targeted Agents with Chemotherapy
In two large phase III trials named Iressa NSCLC Trial Assessing Combination Therapy (INTACT)-1 and INTACT-2, no survival benefit in favor of platinum-based polychemotherapy (cisplatin plus gemcitabine or carboplatin plus paclitaxel) plus gefitinib over chemotherapy alone was reported [15, 16]. As observed for the other EGFR-TKI gefitinib, the combination of erlotinib with platinum-based polychemotherapy (cisplatin plus gemcitabine or carboplatin plus paclitaxel) has been demonstrated to confer no survival advantage over chemotherapy alone in two large phase III randomized trials, named TALENT and TRIBUTE [17, 18].
Based on the promising results of a previous phase II randomized trial [19], a very recent randomized phase III trial compared the combination of bevacizumab (a recombinant humanized monoclonal antibody to VEGF) with chemotherapy (carboplatin and paclitaxel) versus chemotherapy alone in the treatment of advanced non-squamous NSCLC [11]. Patients with squamous histology were excluded because of the risk for grade 5 hemoptysis reported in previous studies. In more than 850 enrolled patients, a statistically significant advantage in median survival was reported in favor of the combination of bevacizumab plus chemotherapy (12.5 months vs. 10.2 months in the bevacizumab and chemotherapy-alone arms, respectively; p = .0075). In addition, the response rate (27% vs. 10%; p < .0001) and progression-free survival time (6.4 months vs. 4.5 months; p < .0001) favored the bevacizumab arm. The experimental regimen was well tolerated. In the bevacizumab and chemotherapy-alone arms, the following toxicities were reported: grade 4–5 neutropenia (24% vs. 16.4%), grade 3–4 thrombosis/embolism (3.8% vs. 3%), and grade 3–4 hemorrhage (4.1% vs. 1.0%). There were 11 treatment-related deaths, nine in the bevacizumab-containing arm and two in the chemotherapy-alone arm. Five deaths resulted from hemoptysis, all in the bevacizumab-plus-chemotherapy arm. That study represents the first evidence of superior efficacy of a targeted therapy combined with chemotherapy over chemotherapy alone in the treatment of NSCLC.
THE SECOND GENERATION OF CLINICAL TRIALS OF TARGETED THERAPIES: MULTITARGETED THERAPY
Multilevel cross-stimulation exists among the targets of the new biological anticancer agents. Molecular pathways involved in survival and replication of cancer cells are very complex (Fig. 1), and interfering with only single steps in these pathways may often be an insufficient therapeutic approach. An example of this complexity is what happens with EGFR signaling pathways [20]. Several ligands can bind to EGFR, including EGF and transforming growth factor alpha (TGF-). After the ligand binds to the receptor, the receptor dimerizes either as a homodimer or as heterodimer with other members of the EGFR family and undergoes autophosphorylation at specific tyrosine residues with in the intracellular domain. These autophosphorylation events in turn activate downstream signaling pathways, including the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3' kinase (PI3K)/Akt pathway. Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPKs. The MAPKs extracellular signal–regulated kinase (ERK)1 and ERK2 subsequently regulate gene transcription and have been linked to cell proliferation, survival, and transformation in laboratory studies. Akt also plays a critical role in controlling the balance between cell survival and apoptosis. Phosphorylation of Akt is required for its activation; once activated, Akt inactivates proapoptotic proteins, including the Bcl-2 family member Bad and caspase-9, and cell cycle regulatory molecules. This brief description makes intuitive sense because blocking only one molecular target allows others to act as salvage or escape mechanisms for cancer cells.
Another example of the cross-stimulation among different molecular pathways is the relationship between EGFR signaling and VEGF. In fact, activation of EGFR signaling by EGF or by TGF- can upregulate the production of VEGF in human cancer cells [21]. Some authors have provided evidence that EGFR blockade causes inhibition of the secretion of VEGF and of other angiogenic growth factors, including basic fibroblast growth factor (bFGF), interleukin-8, and TGF- [21]. In conclusion, this complexity has considerable potential to influence responsiveness and to provide resistance mechanisms for selective inhibitors of individual molecular targets, such as EGFR-TKIs (i.e., gefitinib and erlotinib).
Cancer cells have an inherent ability to harness diverse growth factor signaling pathways for growth advantage and cell survival, processes which may even be facilitated by the use of selective targeted agents. Because of these escape mechanisms, monotherapy with selective targeted agents is unlikely to prove fully effective, and de novo/acquired resistance comprises a significant problem.
Combining Targeted Agents
The main clinical trials of multitargeted therapy in NSCLC treatment are summarized in Table 2. Combinations of targeted agents and multiple targeted agents candidate to the treatment of NSCLC are summarized in Table 3.
HER-1/EGFR and VEGF share common downstream signaling pathways. They exert effects both directly and indirectly on tumor cells, and combining drugs that target these molecules may confer additional clinical benefit. VEGF is also downregulated by HER-1/EGFR inhibition [22], and a recent study suggested that blockade of VEGF may also inhibit HER-1/EGFR autocrine signaling [21]. Therefore, it is rational to suggest that dual blockade of these molecular targets may produce additive and even synergistic cytostatic effects. A number of preclinical studies have investigated the antitumor activity of combined anti-HER-1/EGFR and anti-VEGF agents [23]. A recent phase I/II study examined erlotinib and bevacizumab in patients with nonsquamous stage IIIB/IV NSCLC pretreated with one or more prior chemotherapy regimens [24]. In the phase I portion of the study, an erlotinib dose of 150 mg once daily orally plus a bevacizumab dose of 15 mg/kg i.v. every 21 days were established as the phase II dosages, although no dose-limiting toxicities were observed. Forty patients were enrolled in that trial (34 were treated at the phase II dosages); 21 were female, 30 had adenocarcinoma histology, and nine were never-smokers. The most common adverse events were mild-to-moderate rash, diarrhea, and proteinuria. Preliminary data showed no pharmacokinetic interaction between bevacizumab and erlotinib. Data on antitumor activity of this combination must be considered very promising. In fact, eight patients (20%; 95% CI, 7.6%–32.4%) had partial responses and 26 patients (65%; 95 CI, 50.2%–79.8%) had stable disease as their best response. The median overall survival time for the 34 patients treated at the phase II dosages was 12.6 months, with a progression-free survival time of 6.2 months.
The same authors, given recent evidence that a somatic mutation in the gene encoding the EGFR-TK domain correlated with response to EGFR-TKIs, analyzed available archival tissue from their study for the presence of this mutation. EGFR exons 18–21 and 23 were sequenced in tumors from nine patients [25]. Outcomes were correlated with EGFR mutation. By Response Evaluation Criteria in Solid Tumors (RECIST) criteria, three patients achieved partial responses, three had stable disease, and three had progressive disease. An EGFR-TK mutation in exon 20 was found in one tumor. Although a high degree of correlation between response to TKI therapy and the EGFR-TK mutation was previously noted [26, 27], thus far in this study, the mutation has been identified in only one of three tumors. Although these results are preliminary, they suggest that the combination of bevacizumab and erlotinib may provide benefit in a broader population than each agent individually.
Preclinical data indicate that the EGFR-TKI erlotinib demonstrates greater than additive cytotoxic effects in combination with the farnesyltransferase inhibitor (FTI) tipifarnib (ZarnestraTM; Ortho Biotech Products, L.P., Bridgewater, NJ) and blocks the PI3K/Akt pathway that has been implicated in tumor resistance to tipifarnib. In a phase I study, 12 patients with advanced solid tumors received escalating doses of erlotinib and tipifarnib [28]. No dose-limiting toxicities were observed. Grade 2 or higher treatment-related toxicities included rash (15% grade 2), diarrhea (5% grade 2, 5% grade 3), and fatigue (10% grade 2). No objective response was reported. Overall, the combination of erlotinib and tipifarnib appears well tolerated. The dosages recommended for phase II evaluation were 150 mg once daily for erlotinib and 300 mg twice daily for tipifarnib.
Nonclassic retinoids, such as the rexinoid bexarotene, repress both cyclin D1 and EGFR expression, and they are not dependent on the signalling of retinoic acid receptor beta (RAR-ß), which is often dysregulated in lung carcinogenesis. Combining the EGFR-TKI erlotinib with bexarotene induced at least additive suppression of growth and cyclin D1 expression in retinoic acid (RA)–resistant human bronchial epithelial cells that had silenced RAR-ß and in some lung cancer cell lines. A phase I trial of erlotinib and bexarotene was conducted in patients with advanced aerodigestive tract cancers, including NSCLC [29]. Twenty-four patients were enrolled, including 46% women and 79% with NSCLC. The regimen was demonstrated to be well tolerated. Toxicities were mild, with frequent hypertriglyceridemia and skin rash. Two dose-limiting toxicities were observed: creatine phosphokinase elevation and systemic pain. Four objective partial responses were seen in patients with NSCLC, and eight patients (including five with NSCLC) had stable disease. The median overall survival time was 14.1 months, and the 1-year survival rate was 59%. EGFR sequencing revealed no activating mutations in exons 18, 19, and 21 for two responding cases. Cyclin D1 protein expression was repressed in buccal swabs from five of six patients. The recommended phase II doses are 150 mg for erlotinib and 400 mg/m2 for bexarotene, once daily.
The mechanisms of resistance to EGFR-TKIs remain poorly understood but may be related, in part, to dysregulation of downstream apoptotic pathways, such as the PI3K/Akt/phosphatase and tensin homolog deleted on chromosome 10 (PTEN) axis. In preclinical models, inhibition of the PI3K/Akt pathway restores gefitinib sensitivity in resistant cell lines. The mammalian target of rapamycin (mTOR) has emerged as an important cancer therapeutic target. mTOR is a serine threonine kinase located down-stream of Akt that regulates cellular growth and G1/S cell-cycle progression. Rapamycin and its derivatives that specifically inhibit mTOR are now being actively evaluated in clinical trials. However, many cancer cells are resistant to rapamycin and its derivatives. The mechanism of this resistance remains a subject of major therapeutic significance.
Recently, Sun et al. [30] reported that the inhibition of mTOR by rapamycin triggers the activation of two survival signaling pathways that may contribute to drug resistance. In fact, treatment of human lung cancer cells with rapamycin not only suppressed the phosphorylation of p70S6 kinase and 4E-BP1, indicating an inhibition of mTOR signaling, but paradoxically also concurrently increased the phosphorylation of both Akt and eIF4E. In that study, the rapamycin-induced phosphorylation of Akt and eIF4E was suppressed by the PI3K inhibitor LY294002, suggesting the requirement of PI3K in this process. The activated Akt and eIF4E seem to attenuate rapamycin’s growth-inhibitory effects, serving as a negative feedback mechanism. In support of this model, rapamycin combined with LY294002 exhibited enhanced inhibitory effects on the growth and colony formation of cancer cells. Thus, this study provides a mechanistic basis for enhancing mTOR-targeted cancer therapy by combining an mTOR inhibitor with a PI3K or Akt inhibitor. RAD001 is an orally available mTOR inhibitor that inhibits growth of cancer cell lines, including A549. A phase I/II study is ongoing to evaluate the safety and activity of the combination of gefitinib and RAD001 in patients with advanced NSCLC with no prior history of EGFR-directed therapy [31]. Only six patients have been enrolled to date.
Crosstalk between EGFR and cyclooxygenase-2 (COX-2) signaling pathways results in regulation of tumorigenesis. Preclinical data have shown that [32] signaling through the COX-2 pathway activates the ERK/MAPK pathway in an EGFR-TKI–resistant manner. A phase I, dose-escalation trial to investigate the optimal biologic dose of the combination of the COX-2 inhibitor celecoxib and the EGFR-TKI erlotinib was performed in patients with refractory stage IIIB/IV NSCLC [33]. Primary end points of the study were an evaluation of the toxicity and biologically active dosages of the combination. The treatment was generally well tolerated and without unanticipated toxicities. To date, in 12 evaluable patients, four partial responses and three cases of stable disease have been reported in patients both with and without EGFR-activating mutations. A significant decline in urinary prostaglandin E-M (PGE-M) was demonstrated after 8 weeks of treatment with celecoxib at 400 mg twice daily, compared with doses of 200 mg and 300 mg twice daily. The addition of celecoxib at a dose of 400 mg twice daily may enhance erlotinib activity with an acceptable toxicity profile. A randomized phase II trial is planned comparing erlotinib plus celecoxib with erlotinib plus placebo in advanced NSCLC.
AZD2171 is a highly potent inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosinekinase activity. A phase I trial assessed the safety, tolerability, and pharmacokinetics of ascending once-daily oral doses of AZD2171 in combination with the EGFR-TKI gefitinib [34]. In the first 16 patients treated, the most frequently reported adverse events were diarrhea (14 of 16 patients), rash (9 of 16 patients), abdominal pain (9 of 16 patients), and hypertension (8 of 16 patients). Hypertension and diarrhea were the most commonly reported Common Toxicity Criteria grade 2 adverse events considered to be related to treatment. Causality for diarrhea and rash was assigned to gefitinib, while causality for hypertension was assigned to AZD2171. Dose-limiting toxicities were hypertension, anorexia, and dyspnea. The study has reached the maximum-tolerated dose (MTD) of AZD2171 (30 mg) in combination with gefitinib (250 mg).
Targeted Agents with Multiple Targets
Some targeted agents are active against a single target (as gefitinib and erlotinib) while others act against multiple molecular targets.
ZD6474 is an orally bioavailable aniliquinazoline derivative that has potent inhibitory activity against the VEGFR-2 (kinase domain region) on endothelial cells [35, 21]. Furthermore, it was recently shown that ZD6474 also inhibits EGFR-TK and ret tyrosine kinase [36]. Thus, ZD6474 is considered to be a multitargeted TKI. Inhibition of EGFR signaling has been shown to inhibit the secretion of VEGF, as well as other proangiogenic factors such as bFGF and TGF- [37]. The anti-EGFR activity of ZD6474 may therefore reduce the levels of VEGF and other growth factors released by tumor cells. Thus, treatment with ZD6474 could block neoangiogenesis more efficiently than treatment with a selective anti-VEGFR agent, because in addition to a direct inhibitory effect on VEGFR-2 signaling, it will also have an indirect effect on angiogenesis via blockade of EGFR-induced paracrine production of angiogenic growth factorssuch as VEGF, bFGF and TGF- by cancer cells.
Preclinical studies have demonstrated that the once-daily oral administration of ZD6474 results in dose-dependent inhibition of lung tumor growth in human xenograft models [35] and that its action consists of suppressing established tumor growth, invasion, and metastasis and induction of tumor-associated endothelial cell apoptosis [38]. Moreover, it has been demonstrated that combining ZD6474 with paclitaxel or radiation therapy [38, 21, 39] produces greater inhibition of tumor growth than with any of these treatments alone.
The safety and tolerability of ZD6474 was evaluated in two phase I, open-label studies (Western and Japanese populations) of patients with refractory tumors, including NSCLC [40, 41]. ZD6474 was administered orally at doses up to 300 mg once daily and showed a very favorable toxicity profile. Reported drug-related side effects were minimal and include facial flushing and facial rash, fatigue, diarrhea, and asymptomatic Q-T interval prolongation. All these adverse events were manageable by dose interruption or reduction. No grade 3 or 4 side effects have been reported. Over 40% of patients in the Western study had disease stabilization of at least 8 weeks, and in the Japanese study, four of nine patients with refractory NSCLC had partial responses. A very recent phase II randomized trial compared ZD6474 with the EGFR-TKI gefitinib in the treatment of chemotherapy-pretreated advanced NSCLC patients [42]. About 160 patients were randomized to receive either a daily oral dose of ZD6474 (300 mg) or gefitinib (250 mg), and treatment continued until disease progression or evidence of toxicity. ZD6474 produced a statistically significant longer time to progression (TTP) than gefitinib (11.9 weeks vs. 8.1 weeks, respectively; i.e., a 58% longer TTP favor of ZD6474; p = .011). The adverse event profile of ZD6474 was similar to that seen in previous trials and included rash (grade 1–2, 25.3%; grade 3, 2.4%), diarrhea (grade 1–2, 48.2%; grade 3, 4.8%; grade 4, 2.4%), and asymptomatic QTc prolongation (grade 1, 21.7%). ZD6474 was also recently assessed in combination with docetaxel versus docetaxel alone in pretreated advanced NSCLC patients [43]. A total of 127 patients was randomized to receive once-daily oral treatment with ZD6474 (100 mg or 300 mg) or placebo, in combination with docetaxel. Treatment with ZD6474 resulted in a longer TTP (the primary end point of the study) than with docetaxel alone. In fact, the estimated HR for TTP was 0.635 for the comparison of the 100-mg dose of ZD6474 plus docetaxel with docetaxel alone, that is, a 57% longer TTP with the addition of ZD6474 at a dose of 100 mg (p = .074). The estimated HR for TTP was 0.829 for the comparison of the 300-mg dose of ZD6474 plus docetaxel with docetaxel alone, that is, a 21% longer TTP with the addition of ZD6474 at a dose of 300 mg (p = .416). The estimated median TTPs were 12.0 weeks for docetaxel alone, 18.7 weeks for the 100-mg dose of ZD6474 plus docetaxel, and 17.0 weeks for 300-mg dose of ZD6474 plus docetaxel. In this combination study with docetaxel, the adverse event profile for ZD6474 was similar to that seen in earlier studies. ZD6474 is also currently being investigated in a phase II randomized trial in combination with carboplatin/paclitaxel versus chemotherapy alone, in the first-line treatment of NSCLC. Moreover, ZD6474 is among the best candidates for being tested in the first-line treatment of advanced NSCLC of the elderly, because of its favorable toxicity profile, suitable to a unique population like elderly NSCLC patients. Very recently, preclinical data indicated that the growth of EGFR-TKI–resistant tumors can be inhibited by ZD6474 [44, 45]. The combination of ZD6474 and SC-236, a selective COX-2 inhibitor, has been demonstrated to display supra-additive growth inhibition of some cancer cell lines in vitro. Moreover, the combination in nude mice bearing established lung adenocarcinoma cancer xenografts produced a longer tumor growth inhibition than with each agent alone [46]. These data suggest that the multitargeted TKI ZD6474 should also be tested in clinical trials in combination with COX-2 inhibitors.
AEE788 is an orally active, small-molecule, multitargeted TKI with potent inhibitory activity against several tyrosine kinases, including EGFR, HER-2, and VEGFR-2. A phase I study is being conducted to assess its safety, pharmacokinetics, pharmacodynamics, MTD, and optimal biological dose in patients with advanced solid tumors [47, 48]. Sixty-nine patients have been treated. The most common adverse events (>20%) were diarrhea (67%), fatigue/asthenia (51%), anorexia (49%), rash (43%), nausea (42%), and vomiting (28%). Most adverse events were mild to moderate. Dose-limiting toxicities were observed at the 500-mg and 550-mg dose levels. A dose-dependent inhibition of EGFR signaling in skin and tumor was observed. As expected, inhibition of endothelial pMAPK and Ki67 occurred at higher doses than EGFR inhibition. The study is ongoing, and correlation of clinical response with pharmacokinetics and pharmacodynamics will suggest the optimal schedule of this drug to test in phase II studies.
Sorafenib (BAY 43-9006) is a potent inhibitor of Raf-1 and also active against VEGFR-2, VEGFR-3, and platelet-derived growth factor receptor (PDGFR)-ß [49]. As the Ras/Raf signaling pathway is an important mediator of angiogenesis, sorafenib has the potential to inhibit tumor cell proliferation and angiogenesis through blockade of the Raf/MEK/ERK pathway at the level of Raf kinase and the receptor tyrosine kinases VEGFR-2 and PDGFR-ß. Sorafenib has demonstrated in vivo antitumor activity by inhibiting tumor cell proliferation and angiogenesis in a range of tumor lines that express a mutation in K-Ras or B-Raf, including the A549 NSCLC xenograft [49]. Very recently, a phase I study of sorafenib was performed in Japanese cancer patients to determine a recommended dose and to clarify profiles of toxicities and pharmacokinetics [50]. Thirty-one patients, including 10 NSCLC patients, were treated. Common drug-related adverse events were skin toxicities, laboratory data abnormalities, diarrhea, and anorexia. Partial responses according to RECIST criteria were observed in an NSCLC patient (at the 200-mg twice-daily dosage). Two other NSCLC patients, treated with 200 mg or 400 mg twice daily, had stable disease for 24 weeks or more. The recommended dosage of sorafenib for phase II studies, based on safety and activity data, is 400 mg twice daily.
Preclinical data in the A549 NSCLC xenograft model showed that sorafenib does not antagonize the effect of gefitinib. A phase I study was conducted to assess the safety and efficacy of this combination in patients with refractory or recurrent NSCLC [51]. Twelve patients were treated. The most common drug-related adverse events were fatigue (75% of patients), diarrhea (75%), alanine aminotransferase (ALT) (58%) and aspartate aminotransferase (42%) alterations, rash/desquamation (42%), and anorexia (42%), and were predominantly grade 1–2. Drug-related serious adverse events were seen in three patients and consisted of diarrhea, ALT alteration, and dyspnea. Overall, the combination was well tolerated and led to tumor regression or stable disease in 9 of 12 patients (one partial response and eight stable disease). The dosages recommended for further evaluation were 400 mg twice daily for sorafenib combined with 250 mg once daily for gefitinib.
BIBF 1120 is a potent orally available inhibitor of VEGF, PDGF, and FGF receptor kinases. It also inhibits members of the src family of tyrosine kinases (Src, LcK, Lyn). Thirty-nine patients with a variety of advanced solid malignancies, including NSCLC, were enrolled in a phase I study [52]. The most common toxicities were nausea, vomiting, diarrhea, abdominal pain, fatigue, and asymptomatic, reversible liver enzyme elevations. Ten patients had stable disease. Overall, BIBF 1120 was well tolerated, and 400 mg once daily was defined as the MTD.
CONCLUSION
Lung cancer is a heterogeneous disease with several mutations, and it is unlikely that any one signaling pathway is driving the oncogenic behavior of all tumors. With the exception of some rare cancers in which growth can be dependent on a single factor, selective targeted agents seem to have limited single-agent activity. This is fully in line with the concept that, for most tumors, there are multiple factors driving tumor growth. Perhaps future drug development should focus on somewhat less selectivity. The complexity of the signaling process, in general, further supports the need to interfere at different stages to avoid an escape mechanism for the cell.
Whether the multitargeted approach should best be performed using combinations of selective agents or agents that intrinsically target various targets is a matter of debate. However, the latter approach would lead most easily to drug registration once activity is noted. Combination trials are relatively easy only when the compounds are all owned or licensed by one company. However, the promising results achieved with the combination of erlotinib and bevacizumab in the treatment of chemotherapy-refractory advanced NSCLC may stimulate other similar clinical trials on combinations of multitargeted agents. The second generation of clinical trials on multitargeted therapy for NSCLC has begun and offers great hope of impacting the dismal prognosis of this disease.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Cesare Gridelli has acted as a consultant for AstraZaneca, Eli Lilly, and Roche. Fortunto Ciardiello has acted as a consultant for AstraZeneca and sanofi-aventis.
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The first generation of clinical trials of targeted agents in non-small cell lung cancer (NSCLC) treatment has concluded. To date, only a few of these new agents can offer hope of a substantial impact on the natural history of the disease. Nevertheless, clinically meaningful advances have already been achieved. In chemotherapy-refractory advanced NSCLC patients, gefitinib and erlotinib, two epidermal growth factor receptor tyrosine kinase inhibitors, represent a further chance for tumor control and symptom palliation. In chemotherapy-naive, advanced, nonsquamous NSCLC patients, the combination of the anti–vascular endothelial growth factor monoclonal antibody bevacizumab with chemotherapy was demonstrated to produce better survival outcomes than with chemotherapy alone. The relative failure of first-generation targeted therapies in lung cancer may be a result of multilevel cross-stimulation among the targets of the new biological agents. Thus, blocking only one of these pathways allows others to act as salvage or escape mechanisms for cancer cells. Preclinical evidence of the synergistic antitumor activity achievable by combining targeted agents that block multiple signaling pathways has recently been emerging. Clinical trials of multitargeted therapy may represent the second generation of studies in this field, and some of these are already ongoing. In a recent phase I/II trial, the combination of erlotinib and bevacizumab demonstrated very promising activity in the treatment of advanced NSCLC pretreated with chemotherapy. Whether the multitargeted approach is best performed using combinations of selective agents or agents that intrinsically target various targets is a matter of debate.
INTRODUCTION
Lung cancer is the leading cause of cancer-related mortality in both men and women [1], with 1.2 million new cases diagnosed every year and 1 million deaths recorded worldwide in 2001 [2]. Non-small cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers. The majority of NSCLC patients present with advanced disease at diagnosis, and a large portion of those diagnosed with early-stage disease eventually recur, experiencing metastatic disease. For advanced disease, palliation and the patients’ quality of life are still the primary goals of therapy, with total cure remaining elusive.
Although chemotherapy has recently produced promising results as neoadjuvant and adjuvant strategies for early-stage patients [3, 4], and some progress has been made in the treatment of locally advanced and advanced disease [5, 6], treatment outcomes for NSCLC patients must still be considered disappointing. Thus, clinical research of new treatment strategies is warranted. Advances in the knowledge of tumor biology and mechanisms of oncogenesis have granted the singling out of several molecular targets for NSCLC treatment. Targeted therapies are designed to interfere with specific aberrant biological pathways involved in tumorigenesis. A large amount of preclinical in vivo and in vitro data has been gathered on the antitumor properties of a number of new biological agents, both as single agents and combined with other conventional treatment modalities, such as chemotherapy and radiotherapy. Consequently, several targeted agents have been introduced into clinical trials in NSCLC, with many phase I and phase II studies already completed and some phase III study results being recently made available. The first generation of clinical trials of targeted agents in NSCLC treatment has been concluded, and some conclusions can now be drawn. To date, only a few of these new agents can offer hope of a substantial impact on the natural history of the disease, and negative results are more commonly reported than positive ones. Nevertheless, clinically meaningful advances have already been achieved. In chemotherapy-refractory advanced NSCLC patients, gefitinib (Iressa®; Astra-Zeneca Pharmaceuticals, Wilmington, DE) and erlotinib (Tarceva®; OSI Pharmaceuticals, Inc., Melville, NY), two epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), represent a further chance for tumor control and symptom palliation for a subset of patients otherwise eligible only for supportive care [7–10]. In chemotherapy-naive advanced NSCLC patients (with non-squamous histology), the combination of the anti–vascular endothelial growth factor (anti-VEGF) monoclonal antibody bevacizumab (Avastin®; Genentech Inc., South San Francisco, CA) with chemotherapy has been demonstrated to produce better survival outcomes than chemotherapy alone [11].
Moreover, disappointing results should not be considered definitive, and important lessons can be learned from this first generation of clinical trials, which should be considered only the first step in clinical research in this field. In fact, one of the main reasons for the relative failure of the first generation of clinical trials of targeted therapy in lung cancer is that there is multilevel cross-stimulation among the targets of the new biological agents along several pathways of signal transduction that lead to neoplastic events, and blocking only one of these pathways allows others to act as salvage or escape mechanisms for cancer cells. Preclinical evidence of synergistic antitumor activity achievable by combining targeted agents that block multiple signaling pathways has recently been emerging [12,13]. Clinical trials of multitargeted therapy may represent the second generation of studies in this field, and some of these are already ongoing.
THE MAIN FIRST-GENERATION CLINICAL TRIALS OF TARGETED AGENTS IN NSCLC TREATMENT
Targeted Agents as Monotherapy
The main first-generation clinical trials of targeted therapies in NSCLC treatment are summarized in Table 1.
Gefitinib and erlotinib are two orally available EGFR-TKIs. Two large, phase II trials have been conducted on gefitinib monotherapy in patients with advanced NSCLC that was not controlled after one or more chemotherapy regimens. In those two studies, named Iressa Dose Evaluation in Advanced Lung Cancer (IDEAL)-1 and IDEAL-2, gefitinib was demonstrated to be active (with overall response rates in the range of 10%–20%), to improve disease symptoms in about 40% of patients, and to be well tolerated [7, 8]. On the basis of these clinical trials, gefitinib was licensed for the third-line treatment of platinum- and docetaxel-refractory advanced NSCLC in several countries, including Japan, Australia, and the U.S. However, very recently, Thatcher et al. [9] reported on a phase III trial named Iressa Survival Evaluation in Lung Cancer (ISEL), which compared gefitinib with best supportive care in patients with advanced NSCLC who had received one or two prior chemotherapy regimens. In 1,692 patients (ISEL is the largest study conducted in the refractory advanced NSCLC population), a difference between gefitinib and placebo was reported, although this did not reach statistical significance in the overall or adenocarcinoma histology populations. Preplanned subgroup analyses suggested a survival benefit in patients of Asian origin and in patients who never smoked. Specifically, at a median follow-up of 7 months, the median survival times were 5.6 months versus 5.1 months (hazard ratio [HR], 0.89; 95% confidence interval [CI], 0.78–1.03; p = .11), in the overall population, and 6.3 months versus 5.4 months, in patients with adenocarcinoma histology (HR, 0.83; 95% CI, 0.67–1.02; p = .07), for gefitinib and placebo, respectively. As stated before, preplanned subgroup analyses indicated statistically different survival outcomes in smokers compared with never-smokers and in patients of Asian versus non-Asian origin. In patients of Asian origin, gefitinib-treated patients survived longer than placebo-treated patients (HR, 0.66; 95% CI, 0.48–0.91; p = .01; median survival, 9.5 months vs. 5.5 months, respectively). A similar result was seen in never-smokers, where gefitinib-treated patients survived longer than placebo-treated patients (HR, 0.67; 95% CI, 0.49–0.91; p = .01; median survival, 8.9 months vs. 6.1 months, respectively). A statistically significant higher response rate was observed for gefitinib-treated patients compared with placebo-treated patients.
Erlotinib in a phase III, randomized, placebo-controlled trial has been proven to prolong survival (6.7 months vs. 4.7 months for erlotinib and placebo, respectively; p = .001) in NSCLC patients after first- or second-line chemotherapy [10]. The analysis of quality of life and time to deterioration in patient-reported symptoms showed statistically and clinically meaningful benefits for patients randomized to erlotinib. Moreover, erlotinib was active (response rate, 8.9%) and safe (only 5% of patients discontinued treatment for toxicity). Tsao et al. [14] used tumor biopsy samples from participants in the aforementioned trial to investigate whether responsiveness to erlotinib and its impact on survival were associated with tumor expression of EGFR and EGFR gene amplification and mutations. In multivariate analyses, adenocarcinoma (p = .01), never having smoked (p < .01), and EGFR expression (p = .03) were associated with an objective response. In contrast, survival after treatment with erlotinib was not influenced by the status of EGFR expression, the number of EGFR copies, or EGFR mutations. Thus, the presence of an EGFR mutation may increase responsiveness to the agent, but it is not indicative of a survival benefit.
Combination of Targeted Agents with Chemotherapy
In two large phase III trials named Iressa NSCLC Trial Assessing Combination Therapy (INTACT)-1 and INTACT-2, no survival benefit in favor of platinum-based polychemotherapy (cisplatin plus gemcitabine or carboplatin plus paclitaxel) plus gefitinib over chemotherapy alone was reported [15, 16]. As observed for the other EGFR-TKI gefitinib, the combination of erlotinib with platinum-based polychemotherapy (cisplatin plus gemcitabine or carboplatin plus paclitaxel) has been demonstrated to confer no survival advantage over chemotherapy alone in two large phase III randomized trials, named TALENT and TRIBUTE [17, 18].
Based on the promising results of a previous phase II randomized trial [19], a very recent randomized phase III trial compared the combination of bevacizumab (a recombinant humanized monoclonal antibody to VEGF) with chemotherapy (carboplatin and paclitaxel) versus chemotherapy alone in the treatment of advanced non-squamous NSCLC [11]. Patients with squamous histology were excluded because of the risk for grade 5 hemoptysis reported in previous studies. In more than 850 enrolled patients, a statistically significant advantage in median survival was reported in favor of the combination of bevacizumab plus chemotherapy (12.5 months vs. 10.2 months in the bevacizumab and chemotherapy-alone arms, respectively; p = .0075). In addition, the response rate (27% vs. 10%; p < .0001) and progression-free survival time (6.4 months vs. 4.5 months; p < .0001) favored the bevacizumab arm. The experimental regimen was well tolerated. In the bevacizumab and chemotherapy-alone arms, the following toxicities were reported: grade 4–5 neutropenia (24% vs. 16.4%), grade 3–4 thrombosis/embolism (3.8% vs. 3%), and grade 3–4 hemorrhage (4.1% vs. 1.0%). There were 11 treatment-related deaths, nine in the bevacizumab-containing arm and two in the chemotherapy-alone arm. Five deaths resulted from hemoptysis, all in the bevacizumab-plus-chemotherapy arm. That study represents the first evidence of superior efficacy of a targeted therapy combined with chemotherapy over chemotherapy alone in the treatment of NSCLC.
THE SECOND GENERATION OF CLINICAL TRIALS OF TARGETED THERAPIES: MULTITARGETED THERAPY
Multilevel cross-stimulation exists among the targets of the new biological anticancer agents. Molecular pathways involved in survival and replication of cancer cells are very complex (Fig. 1), and interfering with only single steps in these pathways may often be an insufficient therapeutic approach. An example of this complexity is what happens with EGFR signaling pathways [20]. Several ligands can bind to EGFR, including EGF and transforming growth factor alpha (TGF-). After the ligand binds to the receptor, the receptor dimerizes either as a homodimer or as heterodimer with other members of the EGFR family and undergoes autophosphorylation at specific tyrosine residues with in the intracellular domain. These autophosphorylation events in turn activate downstream signaling pathways, including the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3' kinase (PI3K)/Akt pathway. Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPKs. The MAPKs extracellular signal–regulated kinase (ERK)1 and ERK2 subsequently regulate gene transcription and have been linked to cell proliferation, survival, and transformation in laboratory studies. Akt also plays a critical role in controlling the balance between cell survival and apoptosis. Phosphorylation of Akt is required for its activation; once activated, Akt inactivates proapoptotic proteins, including the Bcl-2 family member Bad and caspase-9, and cell cycle regulatory molecules. This brief description makes intuitive sense because blocking only one molecular target allows others to act as salvage or escape mechanisms for cancer cells.
Another example of the cross-stimulation among different molecular pathways is the relationship between EGFR signaling and VEGF. In fact, activation of EGFR signaling by EGF or by TGF- can upregulate the production of VEGF in human cancer cells [21]. Some authors have provided evidence that EGFR blockade causes inhibition of the secretion of VEGF and of other angiogenic growth factors, including basic fibroblast growth factor (bFGF), interleukin-8, and TGF- [21]. In conclusion, this complexity has considerable potential to influence responsiveness and to provide resistance mechanisms for selective inhibitors of individual molecular targets, such as EGFR-TKIs (i.e., gefitinib and erlotinib).
Cancer cells have an inherent ability to harness diverse growth factor signaling pathways for growth advantage and cell survival, processes which may even be facilitated by the use of selective targeted agents. Because of these escape mechanisms, monotherapy with selective targeted agents is unlikely to prove fully effective, and de novo/acquired resistance comprises a significant problem.
Combining Targeted Agents
The main clinical trials of multitargeted therapy in NSCLC treatment are summarized in Table 2. Combinations of targeted agents and multiple targeted agents candidate to the treatment of NSCLC are summarized in Table 3.
HER-1/EGFR and VEGF share common downstream signaling pathways. They exert effects both directly and indirectly on tumor cells, and combining drugs that target these molecules may confer additional clinical benefit. VEGF is also downregulated by HER-1/EGFR inhibition [22], and a recent study suggested that blockade of VEGF may also inhibit HER-1/EGFR autocrine signaling [21]. Therefore, it is rational to suggest that dual blockade of these molecular targets may produce additive and even synergistic cytostatic effects. A number of preclinical studies have investigated the antitumor activity of combined anti-HER-1/EGFR and anti-VEGF agents [23]. A recent phase I/II study examined erlotinib and bevacizumab in patients with nonsquamous stage IIIB/IV NSCLC pretreated with one or more prior chemotherapy regimens [24]. In the phase I portion of the study, an erlotinib dose of 150 mg once daily orally plus a bevacizumab dose of 15 mg/kg i.v. every 21 days were established as the phase II dosages, although no dose-limiting toxicities were observed. Forty patients were enrolled in that trial (34 were treated at the phase II dosages); 21 were female, 30 had adenocarcinoma histology, and nine were never-smokers. The most common adverse events were mild-to-moderate rash, diarrhea, and proteinuria. Preliminary data showed no pharmacokinetic interaction between bevacizumab and erlotinib. Data on antitumor activity of this combination must be considered very promising. In fact, eight patients (20%; 95% CI, 7.6%–32.4%) had partial responses and 26 patients (65%; 95 CI, 50.2%–79.8%) had stable disease as their best response. The median overall survival time for the 34 patients treated at the phase II dosages was 12.6 months, with a progression-free survival time of 6.2 months.
The same authors, given recent evidence that a somatic mutation in the gene encoding the EGFR-TK domain correlated with response to EGFR-TKIs, analyzed available archival tissue from their study for the presence of this mutation. EGFR exons 18–21 and 23 were sequenced in tumors from nine patients [25]. Outcomes were correlated with EGFR mutation. By Response Evaluation Criteria in Solid Tumors (RECIST) criteria, three patients achieved partial responses, three had stable disease, and three had progressive disease. An EGFR-TK mutation in exon 20 was found in one tumor. Although a high degree of correlation between response to TKI therapy and the EGFR-TK mutation was previously noted [26, 27], thus far in this study, the mutation has been identified in only one of three tumors. Although these results are preliminary, they suggest that the combination of bevacizumab and erlotinib may provide benefit in a broader population than each agent individually.
Preclinical data indicate that the EGFR-TKI erlotinib demonstrates greater than additive cytotoxic effects in combination with the farnesyltransferase inhibitor (FTI) tipifarnib (ZarnestraTM; Ortho Biotech Products, L.P., Bridgewater, NJ) and blocks the PI3K/Akt pathway that has been implicated in tumor resistance to tipifarnib. In a phase I study, 12 patients with advanced solid tumors received escalating doses of erlotinib and tipifarnib [28]. No dose-limiting toxicities were observed. Grade 2 or higher treatment-related toxicities included rash (15% grade 2), diarrhea (5% grade 2, 5% grade 3), and fatigue (10% grade 2). No objective response was reported. Overall, the combination of erlotinib and tipifarnib appears well tolerated. The dosages recommended for phase II evaluation were 150 mg once daily for erlotinib and 300 mg twice daily for tipifarnib.
Nonclassic retinoids, such as the rexinoid bexarotene, repress both cyclin D1 and EGFR expression, and they are not dependent on the signalling of retinoic acid receptor beta (RAR-ß), which is often dysregulated in lung carcinogenesis. Combining the EGFR-TKI erlotinib with bexarotene induced at least additive suppression of growth and cyclin D1 expression in retinoic acid (RA)–resistant human bronchial epithelial cells that had silenced RAR-ß and in some lung cancer cell lines. A phase I trial of erlotinib and bexarotene was conducted in patients with advanced aerodigestive tract cancers, including NSCLC [29]. Twenty-four patients were enrolled, including 46% women and 79% with NSCLC. The regimen was demonstrated to be well tolerated. Toxicities were mild, with frequent hypertriglyceridemia and skin rash. Two dose-limiting toxicities were observed: creatine phosphokinase elevation and systemic pain. Four objective partial responses were seen in patients with NSCLC, and eight patients (including five with NSCLC) had stable disease. The median overall survival time was 14.1 months, and the 1-year survival rate was 59%. EGFR sequencing revealed no activating mutations in exons 18, 19, and 21 for two responding cases. Cyclin D1 protein expression was repressed in buccal swabs from five of six patients. The recommended phase II doses are 150 mg for erlotinib and 400 mg/m2 for bexarotene, once daily.
The mechanisms of resistance to EGFR-TKIs remain poorly understood but may be related, in part, to dysregulation of downstream apoptotic pathways, such as the PI3K/Akt/phosphatase and tensin homolog deleted on chromosome 10 (PTEN) axis. In preclinical models, inhibition of the PI3K/Akt pathway restores gefitinib sensitivity in resistant cell lines. The mammalian target of rapamycin (mTOR) has emerged as an important cancer therapeutic target. mTOR is a serine threonine kinase located down-stream of Akt that regulates cellular growth and G1/S cell-cycle progression. Rapamycin and its derivatives that specifically inhibit mTOR are now being actively evaluated in clinical trials. However, many cancer cells are resistant to rapamycin and its derivatives. The mechanism of this resistance remains a subject of major therapeutic significance.
Recently, Sun et al. [30] reported that the inhibition of mTOR by rapamycin triggers the activation of two survival signaling pathways that may contribute to drug resistance. In fact, treatment of human lung cancer cells with rapamycin not only suppressed the phosphorylation of p70S6 kinase and 4E-BP1, indicating an inhibition of mTOR signaling, but paradoxically also concurrently increased the phosphorylation of both Akt and eIF4E. In that study, the rapamycin-induced phosphorylation of Akt and eIF4E was suppressed by the PI3K inhibitor LY294002, suggesting the requirement of PI3K in this process. The activated Akt and eIF4E seem to attenuate rapamycin’s growth-inhibitory effects, serving as a negative feedback mechanism. In support of this model, rapamycin combined with LY294002 exhibited enhanced inhibitory effects on the growth and colony formation of cancer cells. Thus, this study provides a mechanistic basis for enhancing mTOR-targeted cancer therapy by combining an mTOR inhibitor with a PI3K or Akt inhibitor. RAD001 is an orally available mTOR inhibitor that inhibits growth of cancer cell lines, including A549. A phase I/II study is ongoing to evaluate the safety and activity of the combination of gefitinib and RAD001 in patients with advanced NSCLC with no prior history of EGFR-directed therapy [31]. Only six patients have been enrolled to date.
Crosstalk between EGFR and cyclooxygenase-2 (COX-2) signaling pathways results in regulation of tumorigenesis. Preclinical data have shown that [32] signaling through the COX-2 pathway activates the ERK/MAPK pathway in an EGFR-TKI–resistant manner. A phase I, dose-escalation trial to investigate the optimal biologic dose of the combination of the COX-2 inhibitor celecoxib and the EGFR-TKI erlotinib was performed in patients with refractory stage IIIB/IV NSCLC [33]. Primary end points of the study were an evaluation of the toxicity and biologically active dosages of the combination. The treatment was generally well tolerated and without unanticipated toxicities. To date, in 12 evaluable patients, four partial responses and three cases of stable disease have been reported in patients both with and without EGFR-activating mutations. A significant decline in urinary prostaglandin E-M (PGE-M) was demonstrated after 8 weeks of treatment with celecoxib at 400 mg twice daily, compared with doses of 200 mg and 300 mg twice daily. The addition of celecoxib at a dose of 400 mg twice daily may enhance erlotinib activity with an acceptable toxicity profile. A randomized phase II trial is planned comparing erlotinib plus celecoxib with erlotinib plus placebo in advanced NSCLC.
AZD2171 is a highly potent inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosinekinase activity. A phase I trial assessed the safety, tolerability, and pharmacokinetics of ascending once-daily oral doses of AZD2171 in combination with the EGFR-TKI gefitinib [34]. In the first 16 patients treated, the most frequently reported adverse events were diarrhea (14 of 16 patients), rash (9 of 16 patients), abdominal pain (9 of 16 patients), and hypertension (8 of 16 patients). Hypertension and diarrhea were the most commonly reported Common Toxicity Criteria grade 2 adverse events considered to be related to treatment. Causality for diarrhea and rash was assigned to gefitinib, while causality for hypertension was assigned to AZD2171. Dose-limiting toxicities were hypertension, anorexia, and dyspnea. The study has reached the maximum-tolerated dose (MTD) of AZD2171 (30 mg) in combination with gefitinib (250 mg).
Targeted Agents with Multiple Targets
Some targeted agents are active against a single target (as gefitinib and erlotinib) while others act against multiple molecular targets.
ZD6474 is an orally bioavailable aniliquinazoline derivative that has potent inhibitory activity against the VEGFR-2 (kinase domain region) on endothelial cells [35, 21]. Furthermore, it was recently shown that ZD6474 also inhibits EGFR-TK and ret tyrosine kinase [36]. Thus, ZD6474 is considered to be a multitargeted TKI. Inhibition of EGFR signaling has been shown to inhibit the secretion of VEGF, as well as other proangiogenic factors such as bFGF and TGF- [37]. The anti-EGFR activity of ZD6474 may therefore reduce the levels of VEGF and other growth factors released by tumor cells. Thus, treatment with ZD6474 could block neoangiogenesis more efficiently than treatment with a selective anti-VEGFR agent, because in addition to a direct inhibitory effect on VEGFR-2 signaling, it will also have an indirect effect on angiogenesis via blockade of EGFR-induced paracrine production of angiogenic growth factorssuch as VEGF, bFGF and TGF- by cancer cells.
Preclinical studies have demonstrated that the once-daily oral administration of ZD6474 results in dose-dependent inhibition of lung tumor growth in human xenograft models [35] and that its action consists of suppressing established tumor growth, invasion, and metastasis and induction of tumor-associated endothelial cell apoptosis [38]. Moreover, it has been demonstrated that combining ZD6474 with paclitaxel or radiation therapy [38, 21, 39] produces greater inhibition of tumor growth than with any of these treatments alone.
The safety and tolerability of ZD6474 was evaluated in two phase I, open-label studies (Western and Japanese populations) of patients with refractory tumors, including NSCLC [40, 41]. ZD6474 was administered orally at doses up to 300 mg once daily and showed a very favorable toxicity profile. Reported drug-related side effects were minimal and include facial flushing and facial rash, fatigue, diarrhea, and asymptomatic Q-T interval prolongation. All these adverse events were manageable by dose interruption or reduction. No grade 3 or 4 side effects have been reported. Over 40% of patients in the Western study had disease stabilization of at least 8 weeks, and in the Japanese study, four of nine patients with refractory NSCLC had partial responses. A very recent phase II randomized trial compared ZD6474 with the EGFR-TKI gefitinib in the treatment of chemotherapy-pretreated advanced NSCLC patients [42]. About 160 patients were randomized to receive either a daily oral dose of ZD6474 (300 mg) or gefitinib (250 mg), and treatment continued until disease progression or evidence of toxicity. ZD6474 produced a statistically significant longer time to progression (TTP) than gefitinib (11.9 weeks vs. 8.1 weeks, respectively; i.e., a 58% longer TTP favor of ZD6474; p = .011). The adverse event profile of ZD6474 was similar to that seen in previous trials and included rash (grade 1–2, 25.3%; grade 3, 2.4%), diarrhea (grade 1–2, 48.2%; grade 3, 4.8%; grade 4, 2.4%), and asymptomatic QTc prolongation (grade 1, 21.7%). ZD6474 was also recently assessed in combination with docetaxel versus docetaxel alone in pretreated advanced NSCLC patients [43]. A total of 127 patients was randomized to receive once-daily oral treatment with ZD6474 (100 mg or 300 mg) or placebo, in combination with docetaxel. Treatment with ZD6474 resulted in a longer TTP (the primary end point of the study) than with docetaxel alone. In fact, the estimated HR for TTP was 0.635 for the comparison of the 100-mg dose of ZD6474 plus docetaxel with docetaxel alone, that is, a 57% longer TTP with the addition of ZD6474 at a dose of 100 mg (p = .074). The estimated HR for TTP was 0.829 for the comparison of the 300-mg dose of ZD6474 plus docetaxel with docetaxel alone, that is, a 21% longer TTP with the addition of ZD6474 at a dose of 300 mg (p = .416). The estimated median TTPs were 12.0 weeks for docetaxel alone, 18.7 weeks for the 100-mg dose of ZD6474 plus docetaxel, and 17.0 weeks for 300-mg dose of ZD6474 plus docetaxel. In this combination study with docetaxel, the adverse event profile for ZD6474 was similar to that seen in earlier studies. ZD6474 is also currently being investigated in a phase II randomized trial in combination with carboplatin/paclitaxel versus chemotherapy alone, in the first-line treatment of NSCLC. Moreover, ZD6474 is among the best candidates for being tested in the first-line treatment of advanced NSCLC of the elderly, because of its favorable toxicity profile, suitable to a unique population like elderly NSCLC patients. Very recently, preclinical data indicated that the growth of EGFR-TKI–resistant tumors can be inhibited by ZD6474 [44, 45]. The combination of ZD6474 and SC-236, a selective COX-2 inhibitor, has been demonstrated to display supra-additive growth inhibition of some cancer cell lines in vitro. Moreover, the combination in nude mice bearing established lung adenocarcinoma cancer xenografts produced a longer tumor growth inhibition than with each agent alone [46]. These data suggest that the multitargeted TKI ZD6474 should also be tested in clinical trials in combination with COX-2 inhibitors.
AEE788 is an orally active, small-molecule, multitargeted TKI with potent inhibitory activity against several tyrosine kinases, including EGFR, HER-2, and VEGFR-2. A phase I study is being conducted to assess its safety, pharmacokinetics, pharmacodynamics, MTD, and optimal biological dose in patients with advanced solid tumors [47, 48]. Sixty-nine patients have been treated. The most common adverse events (>20%) were diarrhea (67%), fatigue/asthenia (51%), anorexia (49%), rash (43%), nausea (42%), and vomiting (28%). Most adverse events were mild to moderate. Dose-limiting toxicities were observed at the 500-mg and 550-mg dose levels. A dose-dependent inhibition of EGFR signaling in skin and tumor was observed. As expected, inhibition of endothelial pMAPK and Ki67 occurred at higher doses than EGFR inhibition. The study is ongoing, and correlation of clinical response with pharmacokinetics and pharmacodynamics will suggest the optimal schedule of this drug to test in phase II studies.
Sorafenib (BAY 43-9006) is a potent inhibitor of Raf-1 and also active against VEGFR-2, VEGFR-3, and platelet-derived growth factor receptor (PDGFR)-ß [49]. As the Ras/Raf signaling pathway is an important mediator of angiogenesis, sorafenib has the potential to inhibit tumor cell proliferation and angiogenesis through blockade of the Raf/MEK/ERK pathway at the level of Raf kinase and the receptor tyrosine kinases VEGFR-2 and PDGFR-ß. Sorafenib has demonstrated in vivo antitumor activity by inhibiting tumor cell proliferation and angiogenesis in a range of tumor lines that express a mutation in K-Ras or B-Raf, including the A549 NSCLC xenograft [49]. Very recently, a phase I study of sorafenib was performed in Japanese cancer patients to determine a recommended dose and to clarify profiles of toxicities and pharmacokinetics [50]. Thirty-one patients, including 10 NSCLC patients, were treated. Common drug-related adverse events were skin toxicities, laboratory data abnormalities, diarrhea, and anorexia. Partial responses according to RECIST criteria were observed in an NSCLC patient (at the 200-mg twice-daily dosage). Two other NSCLC patients, treated with 200 mg or 400 mg twice daily, had stable disease for 24 weeks or more. The recommended dosage of sorafenib for phase II studies, based on safety and activity data, is 400 mg twice daily.
Preclinical data in the A549 NSCLC xenograft model showed that sorafenib does not antagonize the effect of gefitinib. A phase I study was conducted to assess the safety and efficacy of this combination in patients with refractory or recurrent NSCLC [51]. Twelve patients were treated. The most common drug-related adverse events were fatigue (75% of patients), diarrhea (75%), alanine aminotransferase (ALT) (58%) and aspartate aminotransferase (42%) alterations, rash/desquamation (42%), and anorexia (42%), and were predominantly grade 1–2. Drug-related serious adverse events were seen in three patients and consisted of diarrhea, ALT alteration, and dyspnea. Overall, the combination was well tolerated and led to tumor regression or stable disease in 9 of 12 patients (one partial response and eight stable disease). The dosages recommended for further evaluation were 400 mg twice daily for sorafenib combined with 250 mg once daily for gefitinib.
BIBF 1120 is a potent orally available inhibitor of VEGF, PDGF, and FGF receptor kinases. It also inhibits members of the src family of tyrosine kinases (Src, LcK, Lyn). Thirty-nine patients with a variety of advanced solid malignancies, including NSCLC, were enrolled in a phase I study [52]. The most common toxicities were nausea, vomiting, diarrhea, abdominal pain, fatigue, and asymptomatic, reversible liver enzyme elevations. Ten patients had stable disease. Overall, BIBF 1120 was well tolerated, and 400 mg once daily was defined as the MTD.
CONCLUSION
Lung cancer is a heterogeneous disease with several mutations, and it is unlikely that any one signaling pathway is driving the oncogenic behavior of all tumors. With the exception of some rare cancers in which growth can be dependent on a single factor, selective targeted agents seem to have limited single-agent activity. This is fully in line with the concept that, for most tumors, there are multiple factors driving tumor growth. Perhaps future drug development should focus on somewhat less selectivity. The complexity of the signaling process, in general, further supports the need to interfere at different stages to avoid an escape mechanism for the cell.
Whether the multitargeted approach should best be performed using combinations of selective agents or agents that intrinsically target various targets is a matter of debate. However, the latter approach would lead most easily to drug registration once activity is noted. Combination trials are relatively easy only when the compounds are all owned or licensed by one company. However, the promising results achieved with the combination of erlotinib and bevacizumab in the treatment of chemotherapy-refractory advanced NSCLC may stimulate other similar clinical trials on combinations of multitargeted agents. The second generation of clinical trials on multitargeted therapy for NSCLC has begun and offers great hope of impacting the dismal prognosis of this disease.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Cesare Gridelli has acted as a consultant for AstraZaneca, Eli Lilly, and Roche. Fortunto Ciardiello has acted as a consultant for AstraZeneca and sanofi-aventis.
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