Single-agent chemotherapy has shown limited activity as second-line treatment in metastatic non-small cell lung cancer (NSCLC), with short-lived responses and modest survival benefit over best supportive care or placebo. There are multiple ways to improve the poor outcome of patients whose disease progresses after first-line chemotherapy. First, individualizing second-line chemotherapy could optimize its effect; the discovery of dramatic responses and significant improvement in survival in patients with epidermal growth factor receptor (EGFR) gene mutations who are treated with EGFR tyrosine kinase inhibitors may lead to the application of other novel therapeutic approaches. Cancer vaccines, using autologous tumor cells genetically modified with granulocyte-macrophage colony-stimulating factor, constitute a new therapeutic option for patients with chemoresistant advanced NSCLC. Vaccines based on lymphocyte-defined tumor antigens, such as melanoma-associated antigen-3, toll-like receptor 9, and mucin 1, are also in the first stages of testing and have shown promising preliminary results. New approaches in gene therapy, including a p53-based method, are currently being investigated. The ultimate goal of gene therapy is to target cancerous stem cells, the importance of which is beginning to be recognized in NSCLC through the study of abnormalities in the wingless (Wnt) pathway. At the preclinical level, small interfering RNA sequences have been used successfully to neutralize multiple abnormal components of the Wnt pathway.