The biology of non-small-cell lung cancer: identifying new targets for rational therapy

Lung Cancer (2004) 46, 135—148

REVIEW

The biology of non-small-cell lung cancer: identifying new targets for rational therapy R. Rosell a,*, E. Felip b , R. Garcia-Campelo c , C. Balaña a a

Medical Oncology Service, Hospital Germans Trias i Pujol, Ctra Canyet, s/n 08916 Barcelona, Spain Medical Oncology Service, Hospital Vall d’Hebron, Barcelona, Spain c Hospital Juan Canalejo, La Coruña, Spain b

Received 25 August 2003 ; received in revised form 13 April 2004; accepted 29 April 2004

KEYWORDS Non-small-cell lung cancer; Pharmacogenetics; Targeted therapy; Biology of neoplasia

Summary Lung cancer, and in particular non-small-cell lung cancer (NSCLC), remains the leading cause of cancer death throughout the world. Almost three decades ago, the major concern was to identify whether cisplatin or cisplatin-based chemotherapy enhanced survival in metastatic NSCLC, and whether any survival benefit compensated for cisplatin-related toxicity. Over the last 10 years, significant advances have been achieved in molecular biology, including the identification of critical genes related to the pathogenesis of NSCLC, which have formed the basis for new targeted therapeutic approaches. These new approaches include novel agents against established chemotherapeutic targets such as thymidylate synthetase as well as agents that inhibit novel targets such as growth factor receptors and proteins important in angiogenesis. With the advent of genomic technologies that can identify patterns of gene expression, the hope is that therapy will be tailored to the genetic pattern of the patients’s tumor, and individualized treatments that minimize toxicity and maximize efficacy can be developed. © 2004 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Lung cancer is the leading cause of cancer death in the United States and throughout the world. Non-small-cell lung cancer (NSCLC) accounts for nearly 80% of cases [1,2]. From 1995 to 1999, cigarette smoking and exposure to secondhand smoke accounted for approximately 440,000 annual deaths in the United States. Worldwide, approximately 4 million people die annually from tobacco-attributable diseases, and this number is predicted to rise to 8.4 million by 2020 [1].

* Corresponding author. Tel.: +34 93 497 89 25; fax: +34 93 497 89 50. E-mail address: [email protected] (R. Rosell).

While traditionally, lung cancer has been classified according to its histologic morphology, and treatments selected solely on the basis of this histology, studies of large numbers of lung cancers have demonstrated different patterns of molecular alterations between the two major groups of lung carcinomas (small-cell lung cancer [SCLC] and NSCLC) and among the three major histologic types (small-cell, squamous cell, and adenocarcinoma) [3,4]. With the advent of techniques in molecular biology, our understanding of abnormalities in cancer cells at a molecular level has increased dramatically. Just as these early developments in identifying oncogenes and tumor suppressor genes have lead to a wide array of new targeted therapies, the development of sophisticated techniques such as high-throughput DNA sequencing and genome-wide

0169-5002/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2004.04.031

136 expression profiling will serve to accelerate the drug discovery and development process. Little more than a decade ago, the main thrust of discussion regarding the treatment of NSCLC focused on whether treatment beyond best supportive care offered any benefit at all. Now, with increased understanding of the molecular basis of this disease, the promise of targeted therapy–—that effective, non-toxic therapies will be developed–—is starting to come to fruition. This review outlines some of the molecular lesions linked to the pathogenesis, prognosis, and treatment of NSCLC to date and some of the targeted therapies currently being investigated. In addition, some of the genomic approaches to identifying cancer genes and examples of their use in drug development are discussed.

2. Biology of preneoplastic lesions Lung cancers are believed to arise after a series of progressive pathological changes in the respiratory mucosa. In the development of squamous cell carcinoma, oncogenic stimuli convert normal bronchial epithelium into hyperplastic, metaplastic, and dysplastic lesions. After these premalignant stages, lung cancer develops as a carcinoma in situ and then emerges as an overt carcinoma. While hyperplasia and squamous metaplasia are considered reactive and reversible changes, dysplasia and carcinoma in situ are the changes most frequently associated with the development of squamous cell lung carcinomas. Adenocarcinomas are also thought to develop at least in part from premalignant precursor lesions such as atypical adenomatous hyperplasia [3,5]. Molecular lesions can be identified at early stages of the pathogenesis of lung cancer. Myc upregulation, cyclin D1 overexpression, p53 protein accumulation, and DNA aneuploidy have been detected in dysplastic epithelium adjacent to invasive lung carcinomas. K-ras mutations have also been detected in atypical adenomatous hyperplasias. p53 mutations have been demonstrated in non-malignant epithelium of lung specimens obtained from lung cancer patients [3]. Other genetic and molecular alterations occur in the early carcinogenic process in the lungs of chronic smokers, and certain abnormalities may persist for many years after smoking cessation, including promoter hypermethylation of the p16 tumor suppressor gene and loss of heterozygosity (LOH) in multiple critical chromosome regions. Several of these alterations can be used as biomarkers for the early detection of lung cancer and risk assessment [6]. For example, the expression of telomerase catalytic subunit and retinoic acid re-

R. Rosell et al. ceptor ␤ has been related to an increased risk of lung cancer in heavy smokers [7]. Genetic alterations are frequently found in serum and plasma, including microsatellite alterations, LOH, aberrant methylation, and K-ras mutations [8—11]. Identifying these changes may lead to strategies for early detection of premalignant lesions in patients at risk and may form the basis of an effective surveillance and prevention strategy in the future.

3. Prognostic genetic markers Since the first article describing K-ras mutations in lung adenocarcinomas [12], a continual list of new genetic markers have been described, predicting disease-free survival and overall survival, mainly in surgically resected stage I NSCLC (Table 1). Tumors with K-ras mutations tend to be smaller and less differentiated than those without. In the most commonly identified mutation of K-ras seen in NSCLC, the normal DNA sequence -GGT- at codon 12 is commonly switched to -TGT- [13]. K-ras mutations in NSCLC cell lines were related to poor survival in stage IIIB-IV [14]. In addition, numerous reports have pointed out the prognostic value of K-ras codon 12 mutations in stage I NSCLC [13,15—17]. In a recent report, neither K-ras nor p53 mutations influenced survival in all patients; however, among patients receiving adjuvant chemotherapy, those without K-ras mutations had a median survival of almost 42 months, while among those with mutations, median survival bottomed out at nearly 25 months (P = 0.09; risk ratio = 0.59) [18]. Such differences were not seen in patients who did not receive adjuvant chemotherapy. In a randomized neoadjuvant trial, K-ras mutations were more often found in the surgery-alone arm than in the neoadjuvant treatment arm [19]. In a small neoadjuvant chemotherapy study of stage III NSCLC, the presence of K-ras mutations in the surgical specimens was a significant predictor of poor disease-free survival [20]. In several retrospectively analyzed surgical series, high retinoic acid receptor-␤ mRNA levels measured by in situ hybridization was correlated with poor survival in stage I NSCLC [21]. A significant inverse relationship between expression of cyclooxygenase 2 and survival has also been observed in stage I disease [22,23]. Hypermethylation of death-associated protein kinase (DAPK) was found in 44% of tumors and linked to significantly poorer survival [24]. Similarly, hypermethylation of RASSF1A, which codes for a Ras-binding protein with tumor suppressor properties, was linked to worse survival in one study [25]. How-

The biology of non-small-cell lung cancer: identifying new targets for rational therapy

137

Table 1 Biomarkers in resected non-small-cell lung cancer (NSCLC)a Biomarker

No. of patients

K-ras mut [13] wt

19 50

NSCLC

Survival†

P

Adenocarcinoma

∼ =18 months NR

0.002

RAR-␤ mRNA [21]

High Low

45 115

Stage I

Worse

0.045

COX-2 [22]

High Low

57 24

Stage I

66% 88%

0.034

DAPK [24]

Methyl Unmethyl

59 76

Stage I

46% 68%

0.007‡

RASSF1A [25]

Methyl Unmethyl

32 75

Stage I

37 months 49 months

0.046‡

CRMP-1 mRNA [26]

Low Normal

40 40

Stage I

28 months Leveled off 52%

IL-8 mRNA [27]

High Normal

61 61

Stage I

Shorter Longer

< 0.001

IL-10 [28]

Low High

44 94

SCC

40.9% 56%

0.080

MIF mRNA [29]

High Low

6 4

SCC

Shorter Longer

RANTES [30]

Low High

36 27

Stage I

Shorter Longer

0.002

IGFBP-3 [31]

Methyl Unmethyl

51 32

Stage I

53% 86%

0.006

11p15.5 LOH [32]

Present Absent

26 50

Stage I

24.9 months 36 months

0.038

ERCC1 mRNA [33]

Low High

NS

Stage I

35.5 months 94.6 months

0.010

128-gene set [34]

With

NS

Adenocarcinoma

Shorter

0.009

17-gene signature [34]

With Without

NS

Shorter Longer

0.010

0.016

Key: NS, not specified; SCC, squamous cell carcinoma; NR, not reached. a Several interesting studies have not been included due to space limitations. † Percentages indicate 5-year survival rates, months indicate median survival. ‡ Another study [11] found no survival differences according to methylation patterns.

ever, these results were not confirmed in a recent study, where neither DAPK nor RASSF1A methylation in tumor or serum influenced survival in surgically resected NSCLC patients [11]. Real-time (RT)-PCR of lung cancer specimens showed that reduced expression of collapsin response mediator protein-1 was associated with advanced disease, lymph node metastases, early post-operative relapse, and shorter survival [26]. Interleukin (IL)-8 mRNA overexpression was also associated with advanced disease, lymph node metastases, early relapse, and shorter survival [27]. Immunohistochemical analysis showed that patients whose tu-

mors lacked IL-10 had worse survival than those whose tumors retained IL-10 expression [28]. Along the same lines, using RT-PCR, overexpression of macrophage migration inhibitory factor was associated with poor survival [29]. Regulated upon activation, normal T-cell-expressed and -secreted overexpression was a predictor of significantly better survival in stage I lung adenocarcinoma [30]. Also in stage I disease, patients with hypermethylated insulin-like growth factor-binding protein-3 (IGFBP-3) had a significantly lower disease-free and overall survival than those without IGFBP-3 methylation [31]. LOH on chromosome segment 11p15.5,

138 which includes the gene for ribonucleotide reductase, confers poor survival in patients with stage I NSCLC [32]. Stage I patients with high ERCC1 mRNA expression also had significantly better survival than those with low ERCC1 expression [33]. However, tumors with efficient DNA repair mechanisms (high ERCC1 expression or without LOH on 11p15.5) could be chemoresistant and may not benefit from adjuvant chemotherapy. Using microarray platforms, a metastases-associated gene-expression signature has been identified [34]. There were no survival differences in lung adenocarcinoma classified according to 9248 highly varying genes; however, survival differences surfaced when using 128 metastases-associated genes. Interestingly, these differences were also evident with the use of only 17 genes (8 upregulated and 9 downregulated genes). This signature, taken as a whole, seems to contain predictive information. Of note, the gene-expression signature associated with metastases arises from both malignant and stromal elements in primary tumors, indicating that the large stromal component of the signature would have been missed had only malignant epithelial cells been isolated by laser capture microdissection [34]. Table 1 summarizes some genetic markers that have been identified as prognostic and predictive markers in stage I NSCLC.

4. Predicting response to chemotherapy The influence of the expression of DNA excision repair genes on cisplatin activity is a prototype for predicting response to chemotherapy on the basis of genetic profiles. The evidence that genetic polymorphisms implicated in tobacco-related cancers modify DNA adduct concentrations in human tissue has been the basis for testing the relationship between cisplatin DNA adducts and response to cisplatin treatment [35,36]. Schaake-Koning et al. [37] demonstrated that survival was significantly improved in stage III NSCLC patients treated with radiotherapy plus daily cisplatin; median survival was approximately 11.5 months. This approach demonstrated significant differences in survival in a small group of patients identified according to the cisplatin DNA adducts analyzed from buccal cells collected by wiping the inner cheek with a cotton swab 1 h after the fifth cisplatin administration. Median survival was 9 months overall, but patients with cisplatin DNA adducts had a median survival of 30 months, in contrast to only 5 months for those with undetectable or low cisplatin DNA adduct levels [38]. Cisplatin DNA adducts are considered to be the main lesions that cause cellular death and

R. Rosell et al. tumor response. The efficiency of removing the cisplatin DNA adducts by nucleotide excision repair is assumed to be central to cisplatin resistance. ERCC1, which is involved in DNA repair, is overexpressed in cisplatin-resistant cells [39]. We have examined the role of ERCC1 and RRM1 mRNA expression in paraffin-embedded pretreatment bronchial biopsies from gemcitabine/cisplatin-treated advanced NSCLC patients. There was a strong correlation between ERCC1 and RRM1 mRNA expression levels. Median survival was significantly longer among patients with low levels of both RRM1 and ERCC1 (not reached) than among those with high levels of both genes (6.8 months; P = 0.016) [40]. Genes involved in nucleotide excision repair could be useful discriminants for identifying patients who may not benefit from cisplatin-based chemotherapy thus, potentially sparing them the toxicity of this treatment. Knowledge of the influence of genetic profiles of NSCLC on drug activity has since been expanded to include agents other than cisplatin and has recently been extensively reviewed by Danesi et al. [41].

5. New approaches to treatment based on biologically defined targets It appears that a plateau has been reached with regard to response rates and survival outcomes using currently available therapies. The 5-year survival rate in lung cancer patients remains less than 15%, so new treatment strategies are clearly needed. As a direct result of an increasing awareness of the biology of NSCLC, novel agents that target-specific intracellular pathways related to the distinctive properties of cancer cells continue to be developed and tested in the clinic. Among these agents, angiogenesis inhibitors, epidermal growth factor receptor (EGFR) targeting agents, and protein kinase C (PKC) inhibitors have received particular attention.

5.1. Pemetrexed Thymidylate synthase, although often viewed as the target of traditional cytotoxic agents such as 5-fluorouracil (5-FU), has been shown to have prognostic significance in NSCLC, and its expression has been correlated with proliferative activity in NSCLC. Thus, it may represent a rational therapeutic target for the treatment of NSCLC [42,43]. Fluoropyrimidines such as 5-FU, however, are metabolized by dihydropyrimidine dehydrogenase (DPD), which is often overexpressed in NSCLC, and

The biology of non-small-cell lung cancer: identifying new targets for rational therapy this may account for the general lack of activity of 5-FU in this malignancy [44]. Pemetrexed is a novel folate anti-metabolite that targets thymidylate synthase but is not metabolized by DPD. Pemetrexed gains entry into the cell via the reduced folate carrier and is an excellent substrate for folylpolyglutamate synthetase. The pentaglutamate form of pemetrexed is the predominant intracellular form and is more than 60-fold more potent in its inhibition of thymidylate synthase and 140-fold more potent toward glycinamide ribonucleotide formyl transferase than the monoglutamate [45]. Two phase II trials of pemetrexed attained response rates of 23% (using a dose of 600 mg/m2 in the first three patients, then 500 mg/m2 because of toxicity seen in this and another study) [46] and 16% (600 mg/m2 ) [47] and median survival of 9.6 and 7.2 months [47]. Toxicity was generally mild; myelotoxicity and skin toxicity were observed. Pemetrexed has been tested in patients with progressive disease within 3 months after first-line chemotherapy or progression while being treated with first-line chemotherapy [48]. Patients were stratified according to whether the first-line treatment included a platinum regimen. Pemetrexed was administered at 500 mg/m2 every 21 days. The response rate was 4.5% in the platinum-pretreated group and 14% in the non-platinum-pretreated group. Median survival was 6.4 and 4 months, respectively. Time to progression was 2.3 and 1.6 months, respectively. In this trial, no folic acid or Vitamin B12 was administered [48]. The largest phase III randomized trial in secondline treatment [49] yet reported compared docetaxel 75 mg/m2 every 3 weeks with pemetrexed 500 mg/m2 every 3 weeks. In the pemetrexed arm, folic acid (350—1000 ␮g daily) and vitamin B12 (1000 ␮g every 9 weeks) were administered. Patients were stratified according to pathologic stage, best response to prior chemotherapy, number of prior chemotherapy regimens, time since last chemotherapy, prior platinum- and taxane-based chemotherapy, and baseline homocysteine serum levels. Ninety percent of patients had received prior cisplatin treatment and 26% had received prior taxanes. The median number of cycles administered was four in both arms. Response rates were 8.8 and 9.1% for docetaxel and pemetrexed, respectively. Time to progression was 2.9 months in both arms. Median survival was 7.9 and 8.3 months for docetaxel and pemetrexed, respectively. One-year survival was 29.7% in both arms. While response, time to progression, and survival were similar in the two arms, in the pemetrexed arm, less severe neutropenia, fewer hospitaliza-

139

tions and less need for ancillary measures were observed [49]. This study opens the gates for new second-line chemotherapy combinations. A phase I study demonstrated the feasibility of pemetrexed plus cisplatin, and the recommended doses were 500 and 75 mg/m2 , respectively. Several responses were observed in mesothelioma patients [50]. Pemetrexed plus cisplatin was also examined in two phase II studies; response rates were 39 [51] and 45% [52]; median survival was 10.9 [51] and 8.9 [52] months. A randomized phase II study has compared pemetrexed 500 mg/m2 plus carboplatin (AUC 6) versus pemetrexed same dose plus oxaliplatin 120 mg/m2 . Both arms were administered every 3 weeks. Response rates were 31.6 and 26.8%, and median survivals were 9.9 and 9.3 months, respectively. Grades 3—4 hematologic and non-hematologic toxicities were very low [53]. A single-arm phase II study of pemetrexed plus carboplatin given identically as above was also recently reported [54]. The response rate was 28%, time to progression 4.8 months, and at the time of reporting, median survival was not yet attained. Pemetrexed toxicity has been related to folate levels and dietary folate supplementation. Methyltetrahydrofolate, which is the main circulating folate, is a substrate for the enzyme methionine synthase, which transfers the methyl group from methyltetrahydrofolate to convert homocysteine to methionine in a B12-dependent reaction. Hence, deficiency in folate or Vitamin B12 hinders the reaction and results in a rise in plasma homocysteine [55]. Niyikiza et al. [56] found that pretreatment plasma homocysteine levels significantly predicted severe thrombocytopenia and neutropenia with or without associated grades 3—4 diarrhea, mucositis, or infection. Decreasing homocysteine levels via vitamin supplementation led to a better safety profile for pemetrexed and possibly to an improved efficacy [57,58].

5.2. Angiogenesis inhibitors: rhu MAb vascular endothelial growth factor (VEGF) and matrix metalloproteinase inhibitors (MMPIs) The use of tumor angiogenesis as a therapeutic target is based on evidence showing the dependence of tumor on the process of angiogenesis for growth, invasion, and metastases. Increased angiogenesis in lung cancer is associated with poor prognosis, and inhibiting angiogenesis may inhibit tumor growth and metastasis [59]. VEGF and its receptors (VEGFR1 and 2) became logical targets for new therapeutic strategies that included the

140 development of monoclonal antibodies to VEGF, monoclonal antibodies to VEGFR, or VEGFR tyrosine kinase inhibitors. In a multicenter trial, 99 stage IIIB/IV NSCLC patients were randomized to standard carboplatin/paclitaxel therapy or one of the two experimental arms: carboplatin/paclitaxel with rhuMAb VEGF at a dose of 7.5 mg/kg, or carboplatin/paclitaxel with rhuMAb VEGF at a dose of 15 mg/kg [60]. Patients randomized to the control group were allowed to receive the antibody alone upon disease progression. The high-dose rhuMAb VEGF group had the highest response rate and the longest survival. The survival in all three groups was longer than in historical models. A worrying point, however, was that six patients treated with the combination of carboplatin/paclitaxel and rhuMAb VEGF developed severe hemoptysis (four episodes were fatal), and four of six episodes occurred in patients with centrally located squamous cell carcinomas. This drug is now being tested in phase III studies in non-squamous cell lung cancer patients. Preclinical studies have provided evidence that matrix metalloproteinases, a family of zinccontaining proteolytic enzymes, facilitate tumor invasion, metastases, and tumor-related angiogenesis. These observations suggest that reducing the activity of this family of enzymes may provide an opportunity to inhibit tumor progression. A variety of MMPIs have been developed: marimastat, prinomastat, Bay 12-9566, and BMS 275291. MMPIs have been shown to inhibit tumor growth and dissemination in preclinical models, and many of these agents have been or are being tested in lung cancer trials. Marimastat was tested in a phase III trial in SCLC patients after completion of chemotherapy treatment, and no survival differences were detected between the placebo and marimastat arm [61]. Marimastat has also been tested in stage III NSCLC patients whose disease was in remission or at least stable following induction therapy. In this study, marimastat did not improve survival. Prinomastat was tested in a phase III trial in stage IIIB and IV NSCLC patients. In contrast to the marimastat trials, prinomastat was given concurrently with paclitaxel/carboplatin. Comparison of survival on the prinomastat versus placebo regimens showed no significant differences. Bay 12-9566 was tested in SCLC and in stage III NSCLC. In each trial, eligibility was limited to patients who had achieved a partial or complete response after chemotherapy or chemoradiotherapy. Both trials were closed before reaching their full complement based on the results of an interim analysis. BMS-275291 is currently being tested in a phase II/III study in combination with standard chemotherapy, involving advanced NSCLC patients. In summary, at present,

R. Rosell et al. the results of trials involving MMPIs have been disappointing. It appears that MMPIs will probably not play a major role in the treatment of patients with advanced lung cancer [62].

5.3. EGFR as a target for cancer treatment: monoclonal antibodies and tyrosine kinase inhibitors (TKIs) Overexpression of EGFR, a 170-kd transmembrane protein that is encoded by the c-erbB-1 proto-oncogen, has been observed in a wide variety of human cancers and is associated with poor prognosis [63]. By the time of diagnosis, the majority of NSCLCs overexpress EGFR. Overexpression is most frequent in squamous carcinomas, less common in adenocarcinomas and large cell carcinomas, and least seen in small cell carcinomas. In NSCLC, EGFR is the target that has received the most clinical attention to date [64,65]. Numerous EGFR blockers have been evaluated, including monoclonal antibodies to the receptor and small-molecule TKIs. Preclinical studies have shown that both types of inhibitors blocked the in vitro growth of human NSCLC cell lines by inhibiting receptor phosphorylation and downstream protein phosphorylation, including mitogen-activated protein (MAP) kinases and serine/threanine protein kinase (AKT). Among the monoclonal antibodies to the receptor, two are being analyzed in clinical trials in lung cancer: the chimeric humanized antibody IMC-C225 and the human antibody ABX-EGF. Several studies using IMC-C225 in NSCLC are ongoing. Three trials are testing IMC-C225 in combination with paclitaxel/carboplatin, gemcitabine/carboplatin, and vinorelbine/cisplatin regimens in chemotherapy-na¨ıve patients, and a fourth trial is evaluating IMC-C225 in combination with docetaxel in the second-line setting. ABX-EGF, the fully human anti-EGFR antibody, blocks binding of both EGFR and transforming growth factor alpha to the EGFR, inhibits tyrosine phosphorylation of the EGFR, and inhibits cellular proliferation. This antibody is now being developed. Two small EGFR-TKI molecules, ZD1839 (gefitinib, Iressa® ) and OSI-774, are being analyzed in the treatment of NSCLC. ZD1839 was examined in four phase I trials. All four of these studies showed evidence for anti-tumor activity using ZD1839 in advanced NSCLC, with an acceptable tolerability profile [66]. Two large-scale, multicenter, double-blind phase II studies including 400 patients analyzed the efficacy and tolerability of ZD1839 at two dose levels (250 and 500 mg/day) in patients previously treated with at least one platinum-based combina-

The biology of non-small-cell lung cancer: identifying new targets for rational therapy tion (IDEAL 1 and 2). Preliminary results have been presented. In IDEAL 1, in which patients were allowed to receive ZD1839 as second-line therapy, an 18% response rate was achieved [67]. No differences were observed between the two doses in terms of response rate, median survival, progression-free survival, and symptom improvement, but the toxicity profile was lower for the 250 mg dose. In IDEAL 2, in which patients were required to have had at least two prior chemotherapy regimens, an 11% response rate was reported [68]. The toxicity results of this study also favored the 250 mg dose. Many patients in both trials presented a significant improvement in disease-related symptoms and quality of life. Two small pilot studies analyzed the effect of adding ZD1839 to cisplatin/gemcitabine and to carboplatin/paclitaxel. Both studies suggested that first-line therapy with ZD1839 and platinum-based chemotherapy is feasible and well tolerated. Two multinational, randomized, placebo-controlled phase III trials of ZD1839 in combination with chemotherapy were conducted with approximately 1100 patients in each trial. These two trials (INTACT 1 and 2) evaluated the role of ZD1839 (250 and 500 mg once daily) versus placebo in combination with cisplatin/gemcitabine and carboplatin/paclitaxel, respectively, in chemotherapy-na¨ıve advanced NSCLC patients. In both trials, the baseline characteristics of the patients were well-matched among the three treatment groups and were representative of the patient population with advanced NSCLC. The results clearly demonstrate that ZD1839 contributes no improvement in survival when added to standard platinum-based chemotherapy in patients with advanced NSCLC [69,70]. To evaluate the role of EGFR inhibitors in stage IIIB NSCLC, the Southwest Oncology Group is randomizing patients to receive induction treatment followed by oral ZD1839 or the same therapy followed by placebo. In a phase I study analyzing OSI-774, relatively long periods of stable disease were observed in NSCLC patients [71]. A phase II study using continuous oral OSI-774 was conducted in 56 patients with platinum-refractory advanced NSCLC [72]. OSI-774 was well tolerated, and a 12% response rate was achieved. These responses lasted from 2 to more than 8 months. Two large multicenter phase III studies of OSI-774 in combination with chemotherapy (cisplatin/gemcitabine or carboplatin/paclitaxel) as first-line treatment in stage IIIB/IV NSCLC have completed patient enrollment. Table 2 summarizes various clinical trials investigating EGFR inhibitors.

141

5.4. PKC inhibitors PKC was identified as a cytoplasmic, calciumactivated, phospholipid-dependent serine/threonine kinase from rat brain. The link between PKC and signal transduction was established by the demonstration that diacylglycerol can serve as the physiologic stimulator of PKC. When extracellular signals, such as hormones, growth factors, mitogens, and neurotransmitters, interact with their cell surface receptors, phospholipase C becomes activated and generates diacylglycerol and inositol triphosphate from inositol phospholipids. The latter, in turn, releases calcium, and these two second messengers, diacylglycerol and calcium, together with membrane phospholipids, activate PKC. Once activated, PKC phosphorylates proteins and triggers many cellular responses, including cell proliferation, differentiation, membrane transport, and gene expression [73]. High levels of PKC-␣ mRNA promote tumor formation, growth, and chemoresistance. It has been observed that pharmacological inhibitors of PKCs promote apoptosis in tumor cell lines and enhance chemosensitivity. A non-tumor-promoting PKC activator bryostatin 1 and the PKC inhibitors staurosporine and UCN-01 modulate ara-C-induced apoptosis in human myeloid leukemia cells overexpressing the anti-apoptotic protein Bcl-2 [74]. Antisense therapy is a new approach to pharmacology that causes highly selective inhibition of a molecular target. Antisense oligonucleotides are specific nucleotide sequences in antisense orientation that selectively hybridize to target mRNA. After hybridizing to target mRNA, antisense oligonucleotides reduce the expression of the target protein. This reduction of protein expression occurs most commonly by RNase H-mediated degradation of the hybridized mRNA. LY900003 (Affinitac, or ISIS 3521) is an antisense agent that downregulates PKC-␣ and increases chemosensitivity of cell lines. It has no effect on other proteins in the PKC family. This selective approach reduces the likelihood of non-specific toxicities [75]. A phase I study tested Affinitac as a 2 h infusion, three times per week for 3 weeks, every 4 weeks; no dose-limiting toxicity was reached [76]. In another phase I study, LY900003 was administered as a continuous IV infusion (0.5—3.0 mg/kg/day) over 21 days followed by a 7-day rest period. Dose-limiting toxicity was reached at 3 mg/kg/day [77]. In both phase I studies, responses were observed in patients with lymphoma, NSCLC [78], and ovarian cancer [79]. A phase II study was performed in NSCLC with LY900003 2 mg/kg/day as a 14-day continuous infusion, starting on day 1. On day 4, paclitaxel plus carboplatin was administered;

142

Table 2 Clinical trials with epidermal growth factor receptor inhibitors in non-small-cell lung cancer Agent

Setting

Regimen

Design

Response rate (%)

Comment

IMC-C225

First-line

Phase II

N/A

Ongoing trials

IMC-C225 ZD1839 (IDEAL 1) [68]

Second-line Previously treated with platinum combination Previously treated with platinum and docetaxel combination

IMC-C225 plus standard chemotherapy combinations IMC-C225 plus docetaxel Monotherapy

Phase II Phase II

N/A 18%

Ongoing trial Completed

Monotherapy

Phase II

12%

Completed

ZD1839 (IDEAL 2) [69]

ZD1839 (INTACT 1) [70]

First-line

Gemcitabine/cisplatin plus: Placebo ZD1839 250 mg ZD1839 500 mg

Phase III

ZD1839 (INTACT 2) [71]

First-line

Paclitaxel/carboplatin plus: Placebo ZD1839 250 mg ZD1839 500 mg

Phase III

OSI-774 [72]

Previously treated with platinum

Monotherapy

Phase II

11%

Completed

OSI-774

First-line

Gemcitabine/cisplatin plus: Placebo OSI-774 150 mg

Phase III

N/A

Accrual completed Data not presented

OSI-774

First-line

Paclitaxel/carboplatin plus: Placebo OSI-774 150 mg

Phase III

N/A

Accrual completed Data not presented

Placebo: 43.9% 250 mg: 42.2% 500 mg: 47.6% Placebo: 32.5% 250 mg: 32.7% 500 mg: 31.5%

Completed; no survival differences

Completed; no survival differences

R. Rosell et al.

The biology of non-small-cell lung cancer: identifying new targets for rational therapy cycles were repeated every 3 weeks. Significant radiographic responses were observed, and median survival was 15.9 months; grade 3/4 toxicities were mainly neutropenia and thrombocytopenia [80]. Another phase II study combined LY900003 with gemcitabine plus cisplatin and obtained a response rate of 37% [81]. Yet another study combined LY900003 with docetaxel [82]. A large phase III randomized trial compared paclitaxel plus carboplatin with or without LY900003 in stage IV NSCLC has been completed. Also, an ongoing randomized trial is comparing gemcitabine plus cisplatin with or without LY900003. Preclinical data indicate that the PKC-␣ half-life is 7—24 h, and a 72 h pretreatment should decrease steady state protein levels prior to chemotherapy. A proapoptotic protein, IGFBP3, reached highest levels 72 h after starting LY900003 pretreatment [83].

5.5. Trastuzumab, gene therapy, and proapoptotic agents In NSCLC, there are other, less studied new treatments. The HER2/neu receptor, another EGFR family member, is also a target for drug development. Trastuzumab in combination with gemcitabine/cisplatin was studied in two phase II trials in stage IIIB/IV NSCLC patients [84,85]. In one study that was randomized, no differences were observed in response rate or time to progression with or without trastuzumab [84]. However, the study included very few patients with strong positive HER2/neu positive (3+) or HER2/neu amplification. In the other study, the addition of trastuzumab to cisplatin and gemcitabine was well tolerated, but further study is needed to see if the combination is superior to chemotherapy alone [85]. Another new approach is the use of gene therapy. The loss of tumor suppressor gene function is a common feature in lung cancer. Early preclinical trials showed that gene therapy with wild-type p53 could inhibit the growth of human lung cancer cells with mutated p53, while the growth of those with wild-type p53 was unaffected. Twenty-eight NSCLC patients who had progressed with conventional treatments received intratumoral injections of an adenovirus vector containing wild-type p53, and an 8% partial response rate was observed [86]. One major problem of this approach is the method of optimal gene administration and delivery. Exisulind is a novel oral anti-cancer proapoptotic agent that holds promise for the treatment of advanced NSCLC patients. At present, a phase I/II study evaluating the effects of exisulind plus docetaxel in advanced lung cancer patients is ongoing [87].

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5.6. Genomics in drug development The majority of oncogenes and tumor suppressor genes known to date have been identified using traditional molecular biology techniques such as positional cloning (e.g., erbB2), linkage analysis of families with a predisposition to cancer (e.g., BRCA-1) or as homologues of known viral oncogenes (e.g., RAS and MYC). New technologies allow for the rapid identification of both genotype and phenotype using microtechnology. Although a full discussion of microarray and other genomic technologies is beyond the scope of this review, Table 3 lists some of these emerging technologies and their potential applications to the identification of molecular abnormalities linked to the pathogenesis, prognosis, and treatment of lung cancer. These technologies can be applied to drug discovery and development in several ways. In addition to increasing our understanding of the gene expression abnormalities that contribute to the malignant phenotype, they can be used to discover new diagnostic and prognostic indicators and biomarkers of therapeutic response; identify new molecular targets for drug development; provide a better understanding of the molecular mode of action, including structure—activity relationships for on-target versus off-target effects; identify genes involved in conferring drug sensitivity and resistance; predict potential side-effects during preclinical development; and help to predict which patients are most likely to benefit from a given drug.

Table 3 Genomic technologies and their uses in pharmacogenomics [95] Technology

Application

Array—CGHa

Scanning for DNA deletion amplification Detection of DNA polymorphisms Quantitate mRNA expression

SNP analysis† Spotted cDNA microarrays Proteomics—2D gels Quantitate expression and mutation of proteins Proteomics—mass Quantitate expression and spectometry mutation of proteins Tissue arrays Rapid screening of antibodies or FISH‡ probes in paraffin sections of tissue Adapted with permission. a Comparative genomic hybridization. † Single nucleotide polymorphism. ‡ Fluorescence in situ hybridization.

144 An example of the power of these technologies to identify differences between cells types is their use in the potential molecular classification of NSCLC based on genetic profiles. Bhattacherjee et al. [88] used microarray technology and sophisticated computerized ‘‘cluster analysis’’ software to analyze 12,600 transcript sequences in 186 lung cancer samples. They were able to subdivide adenocarcinoma into subgroups defined on the basis of their molecular profiles and identify a subgroup of adenocarcinomas with a neuroendocrine gene expression profile and a poor overall prognosis. In addition, cluster analysis identified several samples that showed gene expression profiles of extrapulmonary origin, suggesting that these samples represented clinically unrecognized metastatic disease from adenocarcinomas of other origins (mainly colon and breast) and that genomic analysis could play a significant role in lung cancer diagnosis. Genomic technologies can also be used to understand the mechanism of action of a proposed therapeutic agent. Curcumin, a polyphenolic compound found in various spices, including turmeric, has been reported to exhibit anti-metastatic activity in various animal models; however, the mechanism of action is unclear [89,90]. Chen et al. [90] used microarray analysis of gene expression profiles to characterize the anti-invasive mechanisms of curcumin in a highly invasive lung adenocarcinoma cell line (CL1-5). Using microarray analysis, 81 genes were down-regulated and 71 genes were up-regulated following treatment with curcumin. Among the invasion-related genes that were suppressed were matrix metalloproteinase 14, neuronal cell adhesion molecule, and integrins 6 and ␤4. Moreover, heat-shock protein 27 (Hsp27), Hsp70, and Hsp40-like protein were induced. Curcumin also reduced the activity of MMP2, the down-stream gelatinase of MMP14, based on gelatin zymographic analysis. This study illustrates the utility of genomic profiling in studying the mechanism of action as well as potentially discovering genes important in sensitivity and resistance for use in preclinical development. While the study by Chen et al. represents an example where genomic analysis yielded a result that could be understood in the context of what is already known about the biology of metastasis and invasion, the results of such studies often are far less clear. For example, Zembutsu et al. [91] transplanted 85 cancer xenografts from nine human organs into nude mice, then analyzed the grafts for sensitivity to nine anti-cancer drugs. A cDNA microarray representing 23,040 genes was used to analyze expression profiles. The analy-

R. Rosell et al. sis yielded 1578 genes whose expression levels correlated significantly with chemosensitivity. There was a significant correlation between 333 of these genes and two or more drugs, while 32 correlated with six or seven drugs. Similar analyses have also yielded a large number of genes correlated with chemosensitivity and resistance [92,93]. Genomics represent a powerful tool for understanding the biology of NSCLC and identifying putative diagnostic and therapeutic targets. However, given the large number of putative target genes generally identified using genomic technologies, target validation remains an important component of the target identification and drug discovery process. A study by Scherf et al. [94] illustrates the difficulty in interpreting the results of genomic profiling. They used cDNA microarrays to assess gene expression profiles in the 60 stable human cancer cell lines used by the National Cancer Institute drug discovery screening program. The sensitivity of these cell lines to over 70,000 agents is well characterized, and for this study, data from 1400 compounds, including the majority of currently used chemotherapeutic agents, was used. Relationships seen when the cell lines were clustered according to gene expression were very different from those observed when the cell lines were clustered according to their response to various drugs. While some of the gene expression profiles could be understood in the context of their correlation to drug sensitivity, the authors concluded that only a few of the relationships could be interpreted. They also noted that perhaps the most interesting correlations are those that cannot be understood in the context of our current understanding of cell biology, suggesting that these correlations represent an opportunity to discover previously unknown biological pathways and relationships.

6. Conclusion With the increasingly sophisticated tools of molecular biology, the pathogenesis of NSCLC is at the same time becoming better understood and revealing itself to be more complex than previously imagined. Our greater understanding of this biology has already improved our understanding of currently available therapies and has lead to novel targeted therapies, including improved traditional chemotherapeutic agents such as pemetrexed, small molecule inhibitors of important signal transduction pathways such as ZD1839, and inhibitors

The biology of non-small-cell lung cancer: identifying new targets for rational therapy of angiogenesis. Genomic and proteomic technologies represent new tools for the next step towards improved therapy and outcomes for patients with NSCLC. Combining these new tools with the insights gained from traditional cell biology research will certainly yield new treatment approaches and more tailored therapies that will in turn lead us further towards the goal of effective, low toxicity therapies for patients with NSCLC.

[13] [14]

[15] [16]

Acknowledgements The research reported in this manuscript was partially supported by a grant from Redes Temáticas de Investigación Cooperativa de Centros de Cáncer (CO-010).

[17]

[18]

References [1] Giovino GA. Epidemiology of tobacco use in the United States. Oncogene 2002;21:7326—40. [2] Ries LAG, Eisner MP, Kosary CL, et al. SEER Cancer Statistics Review, 1973–1999, National Cancer Institute, Bethesda, MD. Available at: http://www.seer.cancer. gov/csr/1973 1999/, 2002. Accessed February 2003. [3] Wistuba II, Mao L, Gazdar AF. Smoking molecular damage in bronchial epithelium. Oncogene 2002;21:7298— 306. [4] Nacht M, Dracheva T, Gao Y, et al. Molecular characteristics of non-small cell lung cancer. Proc Natl Acad Sci USA 2001;98:15203—8. [5] Osada H, Takahashi T. Genetic alterations of multiple tumor suppressors and oncogenes in the carcinogenesis and progression of lung cancer. Oncogene 2002;21:7421— 34. [6] Mao L. Recent advances in the molecular diagnosis of lung cancer. Oncogene 2002;21:6960—9. [7] Soria JC, Xu X, Liu DD, et al. Retinoic acid receptor beta and telomerase catalytic subunit expression in bronchial epithelium of heavy smokers. J Natl Cancer Inst 2003;95:165—8. [8] Sanchez-Cespedes M, Monzo M, Rosell R. Detection of chromosome 3p alterations in serum DNA of non-small-cell lung cancer patients. Ann Oncol 1998;9:113—6. [9] Esteller M, Sanchez-Cespedes M, Rosell R, Sidransky D, Baylin SB, Herman JG. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer Res 1999;59:67—70. [10] Sozzi G, Conte D, Mariani L, et al. Analysis of circulating tumor DNA in plasma at diagnosis and during follow-up of lung cancer patients. Cancer Res 2001;61: 4675—8. [11] Ramirez JL, Sarries C, Lopez de Castro P, et al. Methylation patterns and K-ras mutations in tumor and paired serum of resected non-small-cell lung cancer patients. Cancer Lett 2003;193:207—16. [12] Rodenhuis S, van de Wetering ML, Mooi WJ, Evers SG, Van Zandwijk N, Bos JL. Mutational activation of the K-ras

[19]

[20]

[21] [22]

[23] [24]

[25]

[26] [27]

[28]

[29]

145

oncogene. A possible pathogenic factor in adenocarcinoma of the lung. N Engl J Med 1987;317:929—35. Slebos RJ, Kibbelaar RE, Dalesio O, et al. K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N Engl J Med 1990;323:561—5. Mitsudomi T, Steinberg SM, Oie HK, et al. ras gene mutations in non-small cell lung cancers are associated with shortened survival irrespective of treatment intent. Cancer Res 1991;51:4999—5002. Rosell R, Li S, Skacel Z, et al. Prognostic impact of mutated K-ras gene in surgically resected non-small cell lung cancer patients. Oncogene 1993;8:2407—12. Nelson HH, Christiani DC, Mark EJ, Wiencke JK, Wain JC, Kelsey KT. Implications and prognostic value of K-ras mutation for early-stage lung cancer in women. J Natl Cancer Inst 1999;91:2032—8. Graziano SL, Gamble GP, Newman NB, et al. Prognostic significance of K-ras codon 12 mutations in patients with resected stage I and II non-small-cell lung cancer. J Clin Oncol 1999;17:668—75. Schiller JH, Adak S, Feins RH, et al. Lack of prognostic significance of p53 and K-ras mutations in primary resected non-small-cell lung cancer on E4592: a laboratory ancillary study on an Eastern Cooperative Oncology Group prospective randomized trial of postoperative adjuvant therapy. J Clin Oncol 2001;19:448—57. Rosell R, Gomez-Codina J, Camps C, et al. A randomized trial comparing preoperative chemotherapy plus surgery with surgery alone in patients with non-small-cell lung cancer. N Eng J Med 1994;330:153—8. Broermann P, Junker K, Brandt BH, et al. Trimodality treatment in stage III nonsmall cell lung carcinoma: prognostic impact of K-ras mutations after neoadjuvant therapy. Cancer 2002;94:2055—62. Khuri FR, Lotan R, Kemp BL, et al. Retinoic acid receptorbeta as a prognostic indicator in stage I non-small-cell lung cancer. J Clin Oncol 2000;18:2798—804. Achiwa H, Yatabe Y, Hida T, et al. Prognostic significance of elevated cyclooxygenase 2 expression in primary, resected lung adenocarcinomas. Clin Cancer Res 1999;5: 1001—5. Khuri FR, Wu H, Lee JJ, et al. Cyclooxygenase-2 overexpression is a marker of poor prognosis in stage I non-small cell lung cancer. Clin Cancer Res 2001;7:861—7. Tang X, Khuri FR, Lee JJ, et al. Hypermethylation of the death-associated protein (DAP) kinase promoter and aggressiveness in stage I non-small-cell lung cancer. J Natl Cancer Inst 2000;92:1511—6. Burbee DG, Forgacs E, Zöchbauer-Müller S, et al. Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J Natl Cancer Inst 2001;93:691—9. Shih JY, Yang SC, Hong TM, et al. Collapsin response mediator protein-1 and the invasion and metastasis of cancer cells. J Natl Cancer Inst 2001;93:1392—400. Yuan A, Yang PC, Yu CJ, et al. Interleukin-8 messenger ribonucleic acid expression correlates with tumor progression, tumor angiogenesis, patient survival, and timing of relapse in non-small-cell lung cancer. Am J Respir Crit Care Med 2000;162:1957—63. Soria JC, Moon C, Kemp BL, et al. Lack of interleukin10 could predict poor outcome in patients with stage I non-small-cell lung cancer. Clin Cancer Res 2003;9: 1785—91. Tomiyasu M, Yoshino I, Suemitsu R, Okamato T, Sugimachi K. Quantification of macrophage migration inhibitory factor mRNA expression in non-small cell lung cancer tis-

146

[30] [31]

[32]

[33]

[34] [35]

[36] [37]

[38]

[39]

[40]

[41] [42]

[43]

[44]

[45]

[46]

R. Rosell et al. sues and its clinical significance. Clin Cancer Res 2002;8: 3755—60. Moran CJ, Arenberg DA, Huang CC, et al. RANTES expression is a predictor of survival in stage I lung adenocarcinoma. Clin Cancer Res 2002;8:3803—12. Chang YS, Wang L, Liu D, et al. Correlation between insulinlike growth factor-binding protein-3 promoter methylation and prognosis of patients with stage I nonsmall cell lung cancer. Clin Cancer Res 2002;8:3669— 75. Bepler G, Gautam A, McIntyre LM, et al. Prognostic significance of molecular genetic aberrations on chromosome segment 11p15.5 in non-small-cell lung cancer. J Clin Oncol 2002;20:1353—60. Simon G, Sharma S, Smith P, Bepler G. Increased ERCC1 expression predicts for improved survival in resected patients with non-small-cell lung cancer (NSCLC). Eur J Cancer 2002;38(Suppl):S15. Ramaswamy S, Ross KN, Lander ES, Golub TR. A molecular signature of metastasis in primary solid tumors. Nat Genet 2003;33:49—54. Blommaert FA, Michael C, Terheggen PM, et al. Druginduced DNA modification in buccal cells of cancer patients receiving carboplatin and cisplatin combination chemotherapy. Cancer Res 1993;53:5669—75. Schellens JH, Ma J, Planting AS, et al. Relationship between the exposure to cisplatin. Br J Cancer 1996;73:1569— 675. Schaake-Koning C, Van den Boagert W, Dalesio O, et al. Effects of concomitant cisplatin and radiotherapy on inoperable non-small-cell lung cancer. N Engl J Med 1992;326:524— 30. van de Vaart PJ, Belderbos J, de Jong D, et al. DNAadduct levels as a predictor of outcome for NSCLC patients receiving daily cisplatin and radiotherapy. Int J Cancer 2000;89:160—6. Furuta T, Ueda T, Aune G, Sarasin A, Kraemer KH, Pommier Y. Transcription-coupled nucleotide excision repair as a determinant of cisplatin sensitivity of human cells. Cancer Res 2002;62:4899—902. Rosell R, Danenberg KD, Alberola V, et al. Ribonucleotide reductase messenger RNA expression and survival in gemcitabine/cisplatin-treated advanced non-small cell lung cancer patients. Clin Cancer Res 2004;10:1318— 25. Danesi R, De Braud F, Fogli S, et al. Pharmacogenetics of anticancer drug sensitivity in non-small cell lung cancer. Pharmacol Rev 2003;55:57—103. Nakagawa T, Otake Y, Yanagihara K, et al. Expression of thymidylate synthase is correlated with proliferative activity in non-small cell lung cancer (NSCLC). Lung Cancer 2004;43:145—9. Shintani Y, Ohta M, Hirabayashi H, et al. New prognostic indicator for non-small-cell lung cancer, quantitation of thymidylate synthase by real-time reverse transcription polymerase chain reaction. Int J Cancer 2003;104: 790—5. Fukushima M, Morita M, Ikeda K, Nagayama S. Population study of expression of thymidylate synthase and dihydropyrimidine dehydrogenase in patients with solid tumors. Int J Mol Med 2003;12:839—44. Hughes A, Calvert P, Azzabi A, et al. Phase I clinical and pharmacokinetic study of pemetrexed and carboplatin in patients with malignant pleural mesothelioma. J Clin Oncol 2002;20:3533—44. Rusthoven JJ, Eisenhauer E, Butts C, et al. Multitargeted antifolate LY231514 as first-line chemotherapy for patients

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55] [56]

[57]

[58] [59]

[60]

[61]

with advanced non-small-cell lung cancer: a phase II study. National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 1999;17:1194—9. Clarke SJ, Abratt R, Goedhals L, Boyer MJ, Millward MJ, Ackland SP. Phase II trial of pemetrexed disodium (ALIMTA, LY231514) in chemotherapy-na¨ıve patients with advanced non-small-cell lung cancer. Ann Oncol 2002;13:737— 41. Smit EF, Mattson K, Von Pawel J, et al. Alimta (pemetrexed disodium) a second line treatment of non-smallcell lung cancer: a phase II study. Ann Oncol 2003;14:455— 60. Hanna NH, Shepherd FA, Fossella FV, et al. Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy. J Clin Oncol 2004;22:1589— 97. Thödtmann R, Depenbrock H. Dumez and pharmacokinetic phase I study of multitargeted antifolate (LY231514) in combination with cisplatin. J Clin Oncol 1999;10: 3009—16. Manegold C, Gatzemeier U, von Pawel J, et al. Front-line treatment of advanced non-small-cell lung cancer with MTA (LY231514, pemetrexed disodium, ALIMTA) and cisplatin: a multicenter phase II trial. Ann Oncol 2000;11:435— 40. Shepherd FA, Dancey J, Arnold A, et al. Phase II study of pemetrexed disodium, a multitargeted antifolate, and cisplatin as first-line therapy in patients with advanced non-small cell lung carcinoma: a study of the National Cancer Institute of Canada Clinical Trials Group. Cancer 2001;92:595—600. Scagliotti G, Kortsik C, Castellano D, et al. Phase II randomized study of pemetrexed + carboplatin or oxaliplatin, as front-line chemotherapy in patients with locally advanced or metastatic non-small-cell lung cancer. Proc Am Soc Clin Oncol 2003;22:625 (abstract 2513). Zinner R, Obasaju CK, Fossella FV, et al. Alimta plus carboplatin (AC) in patients (pts) with advanced non-smallcell lung cancer (NSCLC): a phase II trial. Proc Am Soc Clin Oncol 2003;22:642 (abstract 2580). Curtin NJ, Hughes AN. Pemetrexed disodium, a novel antifolate with multiple targets. Lancet Oncol 2001;2:298— 306. Niyikiza C, Baker SD, Seitz DE, et al. Homocysteine and methylmalonic acid: markers to predict and avoid toxicity from pemetrexed therapy. Mol Cancer Therap 2002;1:545— 52. Sarries C, Haura EB, Roig B, et al. Pharmacogenomic strategies for developing customized chemotherapy in nonsmall cell lung cancer. Pharmacogenomics 2002;3:763— 80. Rosell R, Crino L. Pemetrexed combination therapy in the treatment of non-small cell lung cancer. Semin Oncol 2002;29(2 Suppl 5):23—9. O’Byrne KJ, Koukourakis MI, Giatromanolaki A. Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small cell lung cancer. Br J Cancer 2000;82:1427—32. DeVore RF, Fehrenbacher L, Herbst RS, et al. A randomized phase II trial comparing RhuMab VEGF (recombinant humanized monoclonal antibody to vascular endothelial cell growth factor) plus carboplatin/paclitaxel (CP) to CP alone in patients with stage IIIB/IV NSCLC. Proc Am Soc Clin Oncol 2000;19:485a (abstract 1896.). Shepherd FA, Giaccone G, Seymour L, et al. Prospective, randomized, double-blind, placebo-controlled trial

The biology of non-small-cell lung cancer: identifying new targets for rational therapy

[62] [63]

[64] [65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73] [74]

[75]

[76]

of marimastat after response to first-line chemotherapy in patients with small-cell lung cancer: a trial of the National Cancer Institute of Canada-Clinical Trials Group and the European Organization for Research and Treatment of Cancer. J Clin Oncol 2002;20: 4434—9. Coussens LM, Finglenton B, Matrisian LM. Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 2002;295:2387—92. Salomon DS, Brandt R, Ciardello F, Normanno N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Haematol 1995;19:183— 232. Mendelsohn J. Blockade of receptors from growth factors: an anticancer therapy. Clin Cancer Res 2000;6:747—53. Rosell R, Fosella F, Milas L. Molecular markers and targeted therapy with novel agents: prospects in the treatment of non-small cell lung cancer. Lung Cancer 2002;38(1Suppl):43—9. 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 2002;20:2240—50. 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 2002;21:2237—46. Kris MG, Natale RB, Herbst RS, et al. A phase II trial of XD1839 (‘‘Iressa’’) in advanced non-small cell lung cancer (NSCLC) patients who had failed platinum- and docetaxel-based regimens (IDEAL 2). Proc Am Soc Clin Oncol 2002;21:292a (abstract 1166). Giaccone G, Herbst RS, Manegold C, et al. Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: a phase III trial: INTACT 1. J Clin Oncol 2004;22:777—84. Herbst R, Giaccone G, Schiller JH, et al. Gefitinib in combination with paclitaxel and carboplatin in advanced nonsmall-cell lung cancer: a phase III trial-INTACT 2. J Clin Oncol 2004;22:785—94. Hidalgo M, Siu LL, Nemunaitis J, et al. Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies. J Clin Oncol 2001;19:3267— 79. Perez-Soler R, Chachoua A, Huberman M, et al. A phase II trial of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor OSI-774, following platinum-based chemotherapy in patients (pts) with advanced, EGFR-expressing, non-small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2001;20:310a (abstract 1235). Basu A. The potential of protein kinase C as a target for anticancer treatment. Pharmacol Ther 1993;59:257—80. Wang S, Vrana JA, Bartimole TM, et al. Agents that downregulate or inhibit protein kinase C circumvent resistance to 1-beta-d-arabinofuranosylcytosine-induced apoptosis in human leukemia cells that overexpress Bcl-2. Mol Pharmacol 1997;52:1000—9. Dean NM, McKay R, Condon TP, Bennett CF. Inhibition of protein kinase C-alpha expression in human A549 cells by antisense oligonucleotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters. J Biol Chem 1994;269:16416—24. Nemunaitis J, Holmlund JT, Kraynak M, et al. Phase I evaluation of ISIS 3521, an antisense oligodeoxynucleotide to

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86] [87]

[88]

[89] [90]

[91]

147

protein kinase C-alpha, in patients with advanced cancer. J Clin Oncol 1999;17:3586—95. Yuen AR, Halsey J, Fisher GA, et al. Phase I study of an antisense oligonucleotide to protein kinase C-alpha (ISIS 3521/CGP 64128A) in patients with cancer. Clin Cancer Res 1999;5:3357—63. Yuen A, Halsey J, Fisher G, et al. Phase I/II trial of ISIS 3521, an antisense inhibitor of PKCalpha, with carboplatin and paclitaxel in non-smallcell lung cancer. Proc Am Soc Clin Oncol 2001;20: 309a. Ritch PS, Belt R, George S, et al. Phase I/II trial of ISIS 3512/LY900003, an antisense inhibitor of PKC-alpha with cisplatin and gemcitabine in advanced non-small-cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2002;21:309a. Moore MR, Saleh M, Jones CM, et al. Phase II trial of ISIS 3512/LY900003, an antisense inhibitor of PKC-alpha, with docetaxel in non-small-cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2002;21:297a. Shen L NM, Glazer RI. Induction of p53-dependent, insulinlike growth factor-binding protein-3-mediated apoptosis in glioblastoma multiforme cells by a protein kinase C alpha antisense oligonucleotide. Mol Pharmacol 1999;55:396— 402. Hidalgo M LL, Nemunaitis J, et al. Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies. J Clin Oncol 2001;19:3267— 79. Perez-Soler R, Chachoua A, Huberman M, et al. A phase II trial of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor OSI-774, following platinum-based chemotherapy in patients (pts) with advanced, EGFRexpressing, non-small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2001;20:310a (abstract 1235). Gatzemeier U, Groth G, Butts C, et al. Randomized phase II trial of gemcitabine-cisplatin with or without trastuzumab in HER2-positive non-small-cell lung cancer. Ann Oncol 2004;15:19—27. Zinner RG, Glisson BS, Fossella FV, et al. Trastuzumab in combination with cisplatin and gemcitabine in patients with Her2-overexpressing, untreated, advanced nonsmall cell lung cancer: report of a phase II trial and findings regarding optimal identification of patients with Her2-overexpressing disease. Lung Cancer 2004;44:99— 110. Swisher SG, Roth JA, Nemunaitis J, et al. Adenovirusmediated p53 gene transfer in advanced non-small-cell lung cancer. J Natl Cancer Inst 1999;91:763—71. Chan DC, Earle KA, Zhao TL, et al. Exisulind in combination with docetaxel inhibits growth and metastasis of human lung cancer and prolongs survival in athymic nude rats with orthotopic lung tumors. Clin Cancer Res 2002;8: 904—12. Bhattacharjee A, Richards WG, Staunton J, et al. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenoma subclasses. Proc Natl Acad Sci USA 2001;98:13790—5. Menon LG, Kuttan G. Inhibition of lung metastasis in mice induced by B16F10 melanoma cells by polyphenolic compounds. Cancer Lett 1995;95:221—5. Chen HW, Yu SL, Chen JJ, et al. Anti-invasive gene expression profile of curcumin in lung adenocarcinoma based on a high throughput microarray analysis. Mol Pharmacol 2004;65:99—110. Zembutsu H, Ohnishi Y, Tsunoda T, et al. Genomewide cDNA microarray screening to correlate gene ex-

148 pression profiles with sensitivity of 85 human cancer xenografts to anticancer drugs. Cancer Res 2002;62:518— 27. [92] Dan S, Tsunoda T, Kitahara O, et al. An integrated database of chemosensitivity to 55 anticancer drugs and gene expression profiles of 39 human cancer cell lines. Cancer Res 2002;62:1139—47. [93] Kikuchi T, Daigo Y, Katagiri T, et al. Expression profiles of non-small cell lung cancers on cDNA microar-

R. Rosell et al. rays: identification of genes for prediction of lymph-node metastasis and sensitivity to anti-cancer drugs. Oncogene 2003;22:2192—205. [94] Scherf U, Ross DT, Waltham M, et al. A gene expression database for the molecular pharmacology of cancer. Nat Genet 2000;24:236—44. [95] Franklin WA, Carbone DP. Molecular staging and pharmacogenomics. Clinical implications: from lab to patients and back. Lung Cancer 2003;41(Suppl 1):S147—54.