Epidermal growth factor receptor inhibitors: a new prospective in the treatment of lung cancer.

Lung cancer is the leading cause of death worldwide. Current treatment modalities, including chemotherapy, radiotherapy and surgery, provide only limited improvement in the natural course of this disease. Therefore, the development of new therapeutic strategies is highly awaited. This review focuses on recent achievements on a novel class of anticancer drugs targeting the EGFR (Epidermal Growth Factor Receptor). The EGFR family is a group of four structurally similar growth factor receptors with tyrosine-kinase activity (EGFR, HER2/neu, ErbB-3, ErbB-4), which dimerize upon binding with a number of ligands, including EGF (Epidermal Growth Factor) and TGF (Transforming Growth Factor), allowing downstream transduction of mitogenic signals. Overexpression of EGFR and HER2 is frequently found in non-small-cell lung cancer (NSCLC), which accounts for over 80% of all malignant lung tumors, and has been associated with a worse clinical outcome. New agents developed to inhibit EGFR function include monoclonal antibodies and small-molecule receptor tyrosine-kinase inhibitors. In this review, results of most recent clinical with EGFR inhibitors including monoclonal antibodies, such as Trastuzumab (Herceptin), IMC-C225 (Cetuximab) and others (ABX-EGF, EMD 72000), and tyrosine-kinase inhibitors, such as ZD1839 (Gefitinib, Iressa), OSI-774 (Erlotinib, Tarceva) and others (CI-1033, GW2016), are summarized. In particular, final results of phase II (IDEAL 1 and 2) and III (INTACT 1 and 2) studies of ZD1839 are reported. In IDEAL trials (ZD1839 single agent in patients pre-treated with chemotherapy) there was clear evidence of tumor regression, symptoms improvement and overall clinical benefit, whereas in the two INTACT trials (ZD1839 in combination with standard platinum-based chemotherapy in chemo-naive patients) ZD1839 did not improve either survival or other clinical endpoints. Possible explanations for these contradictory results and future perspectives are discussed.

Prognostic significance of K-ras, p53, bcl-2, PCNA, CD34 in radically resected non-small cell lung cancers.

The aim of this study was to investigate the prognostic significance of a panel of biological parameters in patients with radically resected non-small cell lung cancers (NSCLC). 269 cases with pathological stage I-IIIA NSCLC were retrospectively analysed. Immunohistochemistry was performed to detect protein expression of p53, bcl-2, proliferating cell nuclear antigen (PCNA) and CD34. Polymerase chain reaction (PCR)/direct nucleotide sequencing method was used to detect mutations in K-ras (codons 12, 13, 61, exons 1-2). The Kaplan-Meier estimates of survival were calculated for clinical and biological variables using the Cox model for multivariate analysis. Histological subtype and the pathologic tumour extension (pT) were the most powerful clinical-pathological prognostic factors for survival (P=0.030 and P=0.031, respectively), whereas among the biological parameters, p53 overexpression (P=0.032) and K-ras mutation (P=0.078) had a negative prognostic role, as demonstrated by multivariate analysis. Conversely, bcl-2, PCNA and CD34 expression were not correlated with survival. Statistically significant associations between p53 expression and the squamous cell carcinoma (SCC) subtype, bcl-2 expression and SCC subtype, K-ras mutation and p53 negative expression, p53 and bcl-2, bcl-2 and PCNA overexpression were observed. In conclusion, some biological characteristics such as the K-ras and p53 status may provide useful prognostic information in resected NSCLC patients, in addition to the classical clinico-pathological parameters. However, further studies are needed to clarify the value of adopting biological prognostic factor into clinical practice.

Interaction between novel anticancer agents and radiation in non-small cell lung cancer cell lines.

Integration of chemotherapy and radiation is the standard practice in the management of locally advanced inoperable NSCLC. To assess the biological interaction between third generation chemotherapeutic agents and radiation in non-small cell lung cancer (NSCLC) in vitro, we tested a number of different drugs (paclitaxel, docetaxel, gemcitabine, topotecan, SN-38 and cisplatin) combined with radiation, in lung cancer cell lines. Cellular chemosensitivity was determined, using the semi-automated colorimetric MTT assay, after 48, 72 and 96 h of exposure to increasing drug concentrations, (0.001-100 microM) and radiation doses (100-400 cGy). Cell lines used were the adenocarcinoma (ADK), A-549, and the squamous-cell carcinoma (SCC), LX-1. Cells were pre-treated with anticancer agents at 24, 12 and 0 h before irradiation. Cytofluorimetric cell cycle analysis was performed. A significant S-phase block or a G(2)/M block was seen with gemcitabine and topotecan or paclitaxel pre-treatment, respectively. Apoptosis was seen only after paclitaxel exposure in the A-549 cell line. Despite a similar pattern of cell-kinetic changes induced by chemotherapy pre-treatment in all cell lines, the adenocarcinoma A-549 cell line was not radiosensitized by any of the anticancer agents tested, whereas synergism was observed in the LX-1 squamous carcinoma cell line, when exposed to gemcitabine, SN-38, topotecan and cisplatin. Paclitaxel, despite a favourable cell cycle effect, was not found to be synergistic with radiotherapy in our experimental model. In conclusion, the observed synergism appears to be dose- and timing-independent and seems to be related to the histological subtype being present in SCC only. Favourable perturbation of the cell cycle is evident with all the new agents tested in both cell types, but was not sufficient to produce synergism with radiation.

The role of systemic chemotherapy in the treatment of brain metastases from small-cell lung cancer.

Brain is the most common site of metastatic spread in small-cell lung cancer (SCLC). Approximately 10% of SCLC patients have brain metastases (BM) already at diagnosis and an additional 40% will develop central nervous system (CNS) involvement during their disease course. Although whole brain radiotherapy and corticosteroids is considered the treatment of choice, accumulating evidence suggests that systemic chemotherapy may also play an important role. The concept of the brain as a pharmacologic sanctuary site for established metastases is in contrast with recent clinical observations of frequent BM responses with systemic chemotherapy. During the last decade, several reports about the effect of systemic chemotherapy on BM from SCLC have been published. Pooled data from five studies report 66% response rate (RR) in 64 patients with initial BM. In addition, an average RR of 36% is derived from five studies including 135 patients with delayed BM treated with systemic single agent chemotherapy. Among new drugs with activity in patients with SCLC brain metastases, camptothecin analog topotecan is one of the most promising with a 52% RR. Although whole brain radiation remains the standard treatment of established BM in SCLC there is an emerging role for systemic chemotherapy, particularly with the use of new active drugs as part of combined modality treatments.

Pre-clinical evaluation of new antineoplastic agents in NSCLC cell lines: evidence of histological subtype-dependent cytotoxicity.

The antiproliferative effect of paclitaxel, docetaxel, gemcitabine, topotecan, SN-38 and cis-platin was studied on 5 non-small cell lung cancer (NSCLC) cell lines, 3 of which were adenocarcinoma (ADK) and 2 squamous cell carcinoma (SCC). Cellular chemosensitivity was determined using the MTT in vitro assay after 48, 72 and 96 h of exposure to drug in concentration ranging from 0.001 to 100 microM. A concentration-dependent cell growth inhibition was observed for paclitaxel, gemcitabine, topotecan, SN-38 and cis-platin in all cell lines tested. Docetaxel showed a concentration-independent cytotoxicity and was 104 times more potent than cis-platin (IC50 = 0. 001 vs. 10 microM). Paclitaxel, gemcitabine, topotecan and SN-38 were 102 times more potent than cis-platin, with median IC50 = 0.1 microM at 72 h. The level of drug-induced cell growth inhibition appeared to be correlated, for some of the six drugs tested, with the tumor histological subtype. In particular, topotecan and cis-platin were more active in squamous cell carcinoma than in adenocarcinoma cell lines (p=0.006 and 0.001 respectively at 0.1 microM concentration), while paclitaxel was more active in ADK than in SCC cell lines (p=0.004 at 0.01 microM concentration). Ca-Lu-6, a cell line that, contrary to most other lung cancer cell lines, is wild-type for most oncogenes/tumor suppressor genes, was by far the most sensitive cell line used (p=0.002, 0.003, 0.01 for paclitaxel, topotecan and cis-platin respectively, at 1 microM concentration), showing a >50% growth inhibition to new drugs at a concentration of 0.01 microM. In conclusion, all these new compounds tested were found to be more potent than cis-platin in affecting cellular proliferation of six NSCLC cell lines studied. We suggest that the specific histological subtype and molecular pattern of the cell line being treated could affect the antiproliferative effect of these drugs.

Analysis of K-ras, p53, bcl-2 and Rb expression in non-small cell lung cancer cell lines.

Six non-small cell lung cancer (NSCLC) cell lines (A-549, Ca-Lu-6, SK-Lu-1, Ca-Lu-1, SK-Mes-1 and LX-1) were studied to assess the presence of multiple concomitant alterations of different oncogenes (K-ras, bcl-2) and tumor suppressor genes (p53, Rb) in NSCLC. K-ras (exon 1) and p53 (exons 5-8) gene mutations were determined via a PCR-based-DGGE (Denaturing Gradient Gel Electro-phoresis) and by sequencing approach. Different mutations were found in the Ist exon of K-ras gene in 5 of 6 cell lines examined. Five of six cell lines contained K-ras mutations at codon 12 (A-549, SK-Lu-1, LX-1) or codon 13 (SK-Mes-1, Ca-Lu-1). In addition, 5 of 6 cell lines showed p53 mutations of exon 8 (SK-Mes-1, Ca-Lu-1 cod. 280; LX-1 cod. 273) or exon 6 (Ca-Lu-6 cod. 196; SK-Lu-1 cod. 193). In 4 of these cell lines, p53 protein nuclear expression was also confirmed with DO-7 mAb immunocytochemistry. Expression of cytoplasmic bcl-2 protein, by anti-bcl-2 mAb flow cytometric analysis, was found in A-549, Ca-Lu-1, SK-Lu-1, SK-Mes-1 cell lines. In contrast, RT-PCR analysis of Rb gene could not identify any change in the cell lines examined. In conclusion, most NSCLC cell lines tested displayed concomitant multiple oncogene/tumor suppressor gene alterations.