From clinical specimens to human cancer preclinical models-a journey the NCI-cell line database-25 years later.

The intramural the National Cancer Institute (NCI) and more recently the University of Texas Southwestern Medical Center with many different collaborators comprised a complex, multi-disciplinary team that collaborated to generated large, comprehensively annotated, cell-line related research resources which includes associated clinical, and molecular characterization data. This material has been shared in an anonymized fashion to accelerate progress in overcoming lung cancer, the leading cause of cancer death across the world. However, this cell line collection also includes a range of other cancers derived from patient-donated specimens that have been remarkably valuable for other types of cancer and disease research. A comprehensive analysis conducted by the NCI Center for Research Strategy of the 278 cell lines reported in the original Journal of Cellular Biochemistry Supplement, documents that these cell lines and related products have since been used in more than 14 000 grants, and 33 207 published scientific reports. This has resulted in over 1.2 million citations using at least one cell line. Many publications involve the use of more than one cell line, to understand the value of the resource collectively rather than individually; this method has resulted in 2.9 million citations. In addition, these cell lines have been linked to 422 clinical trials and cited by 4700 patents through publications. For lung cancer alone, the cell lines have been used in the research cited in the development of over 70 National Comprehensive Cancer Network clinical guidelines. Finally, it must be underscored again, that patient altruism enabled the availability of this invaluable research resource.

Lung cancer in 2013: state of the art therapy for metastatic disease.

Lung cancer is the leading worldwide cause of cancer death and the majority of patients present with metastatic stage IV disease. At diagnosis, clinical, histologic, and molecular features must be considered in therapeutic decision-making for systemic therapy. Molecular testing for at least epidermal growth factor receptor (EGFR) and ALK should be performed in all patients before therapy. Platinum doublet chemotherapy may be considered for "fit" patients who do not have a molecular driver genetic abnormality. Bevacizumab can be considered for addition to the doublet in patients with nonsquamous cancers who have no contraindications. A pemetrexed combination is considered only in nonsquamous histology. Patients with EGFR mutations or ALK fusions should be treated with erlotinib or crizotinib, respectively, even in patients with tumor-related poor performance. The tyrosine-kinase inhibitors (TKIs) may be continued until multisite, symptomatic progression. For patients initially treated with a platinum doublet, maintenance chemotherapy with pemetrexed, erlotinib, gemcitabine, or possibly docetaxel is an option with selection based on clinical features, histology, type of initial therapy, and response to first-line therapy.

Clinical activity of pemetrexed: a multitargeted antifolate anticancer agent.

Pemetrexed is a new generation antifolate anticancer agent that inhibits several folate-dependent enzymes required for production of DNA and RNA intermediates. Early studies showed significant hematologic and nonhematologic toxicities with this agent. However it was found that many of these toxicities related to functional folate status and could be markedly reduced through routine supplementation with folic acid and vitamin B(12), without adversely affecting efficacy. Phase III studies with pemetrexed have established a clinical role for this drug as a single agent in the second-line treatment of non-small cell lung cancer and in combination with cisplatin for the frontline treatment of unresectable malignant pleural mesothelioma. Clinical trials of pemetrexed alone or in combination with other chemotherapeutic agents have shown considerable activity in many tumor types including colorectal, pancreatic and breast cancer, and urothelial tumors.

Adjuvant chemotherapy for resected non-small-cell lung cancer.

Because of the high rate of distant disease recurrence, the 5-year survival of patients who have undergone complete surgical resection of localized non-small-cell lung cancer (NSCLC) is approximately 50%. Initial results from early studies of adjuvant postoperative chemotherapy reported an adverse effect of alkylating agent and older chemotherapy regimens on survival. Cisplatin-based combinations were the first to show a survival advantage. A 1995 meta-analysis of these studies suggested a 13% reduction in the hazard ratio for death (HR = 0.87), leading to a 5% survival benefit at 5 years. Still, these trials involved limited numbers of patients (N = 1,394), and the results failed to reach statistical significance (P = .08). Of the five largest subsequent randomized trials of platinum-based adjuvant therapy, three showed a significant survival advantage. Although it is impossible to determine the reasons for the differing outcomes of these studies, several key features distinguish them, and the data suggest that medically fit patients with resected stage IB or II NSCLC should be offered chemotherapy with a platinum/new drug combination.

Early-stage non-small-cell lung cancer: current perspectives in combined-modality therapy.

The most effective treatment for patients with early-stage non-small-cell lung cancer (NSCLC) remains complete surgical resection, providing the disease is medically operable and adequately staged. The effectiveness of surgical resection, however, is limited by high rates of distant recurrence caused by the presence of metastatic disease that is not apparent at the time of surgery. Thus, induction, adjuvant chemotherapy, and radiation therapy, as well as a combination of both, have been studied for their ability to reduce local and distant recurrence rates and to improve survival. Adjuvant chest radiation therapy following resection decreases local relapse rates but also decreases overall patient survival, with an increase in the hazard ratio of death. A previous metaanalysis of cisplatin-based adjuvant chemotherapy showed a 13% reduction in the hazard ratio of death and a 5% improvement in 5-year survival, but the differences in the small sample failed to reach statistical significance. Newer 2-drug combinations were shown to reduce the hazard ratio of death by 14%, with a 4.3% improvement in 5-year survival in the largest trial recently reported. These newer 2-drug combinations also have the benefits of reduced toxicity and improved delivery. Induction chemotherapy offers several potential advantages compared with adjuvant chemotherapy, such as improved delivery, early control of micrometastatic disease, and reduction of the primary tumor size prior to surgery, thus allowing for more conservative and possibly complete resection of the tumor. A number of clinical trials have shown that induction chemotherapy is safe and feasible, with no significant increase in surgical complications, and results in favorable survival rates in patients with resectable NSCLC. A number of phase III randomized trials are currently under way to confirm the benefits of induction chemotherapy in patients with stage IB-IIIA NSCLC and to compare induction chemotherapy versus adjuvant chemotherapy following surgery versus surgery alone. In addition, biologically targeted agents are currently under study for patients with advanced NSCLC.

New investigative regimens and cytotoxic agents in thoracic cancers: gemcitabine and pemetrexed.

Several new antimetabolites, administered alone or in combination, are changing the therapeutic landscape for thoracic cancer. Two-drug combinations involving these newer drugs are becoming the standard of care for non-small-cell lung cancer (NSCLC), largely due to improvements in survival rates, time to disease progression, and response rates as well as an improved safety profile. Gemcitabine (Gemzar) has elicited considerable interest in this disease, as a combination partner in chemotherapeutic regimens. Another promising agent is pemetrexed (Alimta), a folate-based inhibitor of thymidylate synthase. In preclinical development, pemetrexed both alone and in combination with other cytotoxic agents has exhibited activity across a broad range of tumor models, including NSCLC and mesothelioma. In clinical trials of patients with NSCLC, pemetrexed has been an effective, well-tolerated agent that can be used as monotherapy or in combination with other agents at full dose. In clinical trials of patients with mesothelioma, the combination of pemetrexed and cisplatin demonstrated a significant improvement in survival, response, and patient quality-of-life parameters. The principle toxicities of pemetrexed can be minimized by folate and vitamin B12 supplements.

Antitumor activity of ZD6126, a novel vascular-targeting agent, is enhanced when combined with ZD1839, an epidermal growth factor receptor tyrosine kinase inhibitor, and potentiates the effects of radiation in a human non-small cell lung cancer xenograft model.

OBJECTIVE: Targeting the tumor vasculature may offer an alternative or complementary therapeutic approach to targeting growth factor signaling in lung cancer. The aim of these studies was to evaluate the antitumor effects in vivo of the combination of ZD6126, a tumor-selective vascular-targeting agent; ZD1839 (gefitinib, Iressa), an epidermal growth factor receptor tyrosine kinase inhibitor; and ionizing radiation in the treatment of non-small cell lung cancer xenograft model. METHODS: Athymic nude mice with established flank A549 human non-small cell lung cancer xenograft model xenografts were treated with fractionated radiation therapy, ZD6126, ZD1839, or combinations of each treatment. ZD6126 (150 mg/kg) was given i.p. the day after each course of radiation. Animals treated with ZD1839 received 100 mg/kg per dose per animal, 5 or 7 days/wk for 2 weeks. Immunohistochemistry was done to evaluate the effects on tumor growth using an anti-Ki67 monoclonal antibody. Effects on tumor-induced vascularization were quantified using an anti-factor VIII-related antigen monoclonal antibody. RESULTS: ZD6126 attenuated the growth of human A549 flank xenografts compared with untreated animals. Marked antitumor effects were observed when animals were treated with a combination of ZD6126 and fractionated radiation therapy with protracted tumor regression. ZD6126 + ZD1839 resulted in a greater tumor growth delay than either agent alone. Similar additive effects were seen with ZD1839 + fractionated radiation. Finally, the addition of ZD6126 to ZD1839 and radiation therapy seemed to further improve tumor growth control, with a significant tumor growth delay compared with animals treated with single agent or with double combinations. Immunohistochemistry showed that ZD1839 induced a marked reduction in A549 tumor cell proliferation. Both ZD1839 and ZD6126 treatment substantially reduced tumor-induced angiogenesis. ZD6126 caused marked vessel destruction through loss of endothelial cells and thrombosis, substantially increasing the level of necrosis seen when combined with radiation therapy. The combination of radiation therapy, ZD6126, and ZD1839 induced the greatest effects on tumor growth and angiogenesis. CONCLUSION: This first report shows that a selective vascular-targeting agent (ZD6126) + an anti-epidermal growth factor receptor agent (ZD1839) and radiation have additive in vivo effects in a human cancer model. Targeting the tumor vasculature offers an excellent strategy to enhance radiation cytotoxicity. Polytargeted therapy with agents that interfere with both growth factor and angiogenic signaling warrants further investigation.

Phase II study of pemetrexed-gemcitabine combination in patients with advanced-stage non-small cell lung cancer.

PURPOSE: Cisplatin is one of the most active agents for the treatment of non-small cell lung cancer (NSCLC). It is also known for significant toxicity, which makes it unsuitable for certain patients. Our purpose was to evaluate the efficacy and toxicity of a promising cisplatin-free combination, gemcitabine plus pemetrexed, in NSCLC. EXPERIMENTAL DESIGN: Chemo-naive patients with inoperable NSCLC were eligible for this study. Gemcitabine (1250 mg/m2) was given intravenously on days 1 and 8, followed by intravenous pemetrexed (500 mg/m2) on day 8. After inclusion of 13 patients, folic acid and vitamin B12 supplementation was added to lower pemetrexed-induced toxicity. Quality of life was assessed with the Lung Cancer Symptom Scale. RESULTS: Sixty patients enrolled; 58 were evaluable for response. All patients had a World Health Organization performance status of 0 or 1. Eighty-seven percent had stage IV disease. Nine patients had a confirmed partial response [overall response rate, 15.5%; 95% confidence interval (CI), 7.3-27.4%]. Twenty-nine (50.0%) patients had stable disease. Median overall survival was 10.1 months (95% CI, 7.9-13.0 months), with a 1- and 2-year overall survival of 42.6% (95% CI, 30.0-55.3%) and 18.5% (95% CI, 7.9-29.1%). Median progression-free survival was 5.0 months. Median response duration was 3.3 months. There were no deaths attributed to treatment. Common Toxicity Criteria grade 3/4 toxicities were neutropenia (61.7%), febrile neutropenia (16.7%), fatigue (23.3%), and elevations of aspartate aminotransferase (15.0%) and alanine aminotransferase (20.0%). CONCLUSIONS: This combination had good tolerance and achieved promising overall survival with extended 1- and 2-year survival rates. This cisplatin-free regimen warrants further evaluation in randomized trials.

Sputum cytological atypia as a predictor of incident lung cancer in a cohort of heavy smokers with airflow obstruction.

Individuals with cytological atypia in sputum may be at increased risk for lung cancer. We conducted a longitudinal analysis of the association between lung cancer incidence and cytological atypia in sputum samples collected prospectively from an ongoing cohort of adults at high risk for lung cancer. Cohort members had a smoking history of > or = 30 pack-years and chronic obstructive pulmonary disease documented by pulmonary airflow testing. Sputum samples collected at baseline and periodically thereafter were examined by standard cytological methods. From the cohort of 2,006 people, there were 83 incident lung cancers over 4,469 person-years of observation. At baseline, the association between personal and behavioral characteristics, and sputum cytological atypia was assessed by multiple logistic regression. The association between sputum cytological atypia and incident lung cancer was then assessed by hazard ratios using proportional hazards regression analysis, adjusting for potential confounding factors. Cytological atypia graded as moderate or worse was associated with continuing cigarette smoking (adjusted odds ratio, 2.5; 95% confidence interval, 1.5-4.1), and with lower levels of intake of fruits and vegetables (P for trend = 0.04). Atypia was not associated with several other factors, including the degree of airflow obstruction, the use of vitamin supplements, nonsteroidal anti-inflammatory drugs, or metered-dose steroid inhalers. Incident lung cancer was increased among those with moderate or worse cytological atypia (adjusted hazards ratio, 2.8; 95% confidence interval, 1.4-5.5). This association was not confounded by other risk factors. We conclude that in this high-risk cohort, cytological atypia is associated with continuing smoking and low intake of fruits and vegetables, but that independent of these and other factors, the risk of incident lung cancer is increased among those with moderate or worse grades of cytological atypia in their sputum.

Incorporation of pemetrexed (Alimta) into the treatment of non-small cell lung cancer (thoracic tumors).

Lung cancer is the leading cause of cancer death in the United States and throughout the world. The overall 5-year survival rate for lung cancer is dismal: 14% in the United States and even lower in other parts of the world. Recent developments in the armamentarium of chemotherapeutic agents for lung cancer have shown that two-drug combinations improve survival, relieve symptoms, and improve quality of life; however, complete response rates are still approximately 1% in stage IV disease and less than 20% of advanced stage patients survive 2 years. Therefore, improved therapeutic agents that increase efficacy are sorely needed. Most lung cancers overexpress thymidylate synthase and a variety of genes involved in cell cycle regulation. Previous studies have shown that some inhibitors of DNA synthesis (eg, gemcitabine) can improve the survival of advanced lung cancer patients, especially when combined with other agents such as cisplatin. The multitargeted antifolate, pemetrexed (Alimta; Eli Lilly and Co, Indianapolis, IN) was developed because it inhibits multiple enzymes involved in DNA synthesis including thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyl transferase. The early studies of pemetrexed showed that the important dose-limiting toxicities were myelosuppression, mucositis, and diarrhea, all of which are common with any antimetabolite. Subsequent studies described in this article will show that these toxicities can be significantly reduced by the use of vitamin supplementation with folate and B12, and that pemetrexed has considerable activity in non-small cell lung cancer and mesothelioma.

Preclinical studies of gemcitabine and trastuzumab in breast and lung cancer cell lines.

Overexpression of the HER2/neu oncogene and receptor protein has been reported in 20%-30% of patients with breast cancer and is associated with a poor prognosis. HER2/neu expression in breast cancer patients assessed by fluorescence in situ hybridization or immunohistochemistry is a predictor for response to trastuzumab, a humanized monoclonal antibody against the HER2/neu cell-surface protein. Data regarding HER2/neu expression in lung cancer are more limited, and there is little information regarding HER2/neu expression and response to trastuzumab alone or in combination with chemotherapeutic agents. Gemcitabine is an active agent against non-small-cell lung cancer (NSCLC) and has demonstrated activity in breast cancer as well. In vitro modified tetrazolium salt growth assays were performed to determine whether the combination of trastuzumab/gemcitabine produced synergistic or additive effects on breast and lung cancer cell lines. The effects of trastuzumab alone, gemcitabine alone, and the trastuzumab/gemcitabine combination was evaluated on 4 NSCLC cell lines, 1 small-cell lung cancer (SCLC) cell line, and 2 breast cancer cell lines. HER2/neu surface protein expression was assessed by fluorescence flow cytometry and immunohistochemistry. Fluorescence in situ hybridization analysis was used to study gene expression. Trastuzumab treatment alone resulted in growth inhibition in all cell lines expressing HER2/neu and the inhibitive effect correlated with the level of cell surface HER2/neu protein expression. Treatment with gemcitabine alone resulted in growth inhibition in both breast and NSCLC cell lines. A synergistic growth inhibition effect was seen with the trastuzumab/ gemcitabine combination as indicated by combination index values < 1. The degree of synergy observed did not directly correlate with the level of surface protein expression, as synergy was seen even in cancer cell lines expressing low levels of HER2/neu. No treatment effect was seen in the SCLC cell line, which did not express HER2/neu. These preclinical studies indicate a need to study the clinical synergistic effects of the gemcitabine/trastuzumab combination in breast cancer and NSCLC patients whose tumors overexpress HER2/ neu.

Pemetrexed as a single agent in the therapy of advanced lung cancer.

Pemetrexed is a multitargeted antifolate with activity as a single agent against non-small cell lung cancer at a recommended dose of 500 mg/m(2) as a 10-minute infusion every 21 days. The response rate in untreated patients with stage IIIB/IV disease is from 18% to 23%. It is also active in the second-line setting in patients progressing after pretreatment with a number of drugs or after a very short treatment-free interval. The response rate in these difficult-to-treat patients is approximately 9%. Toxicity in both situations is mild; myelotoxicity is primarily seen. This review describes phase I and II studies performed before folic acid and vitamin B(12) supplementation.

Future directions in the development of pemetrexed.

Pemetrexed is a novel antifolate that inhibits several folate-dependent enzymes in addition to thymidylate synthase. Such a drug may have theoretical advantages over fluoropyrimidines and specifically acting antifolates. Phase I studies identified a preferred schedule of an intravenous dose once every 3 weeks. Phase II studies showed a broad spectrum of activity in solid tumors (ie, non-small cell lung, colorectal, and pancreatic cancers) usually considered refractory to most antimetabolites. Binary combinations with cisplatin, carboplatin, and gemcitabine have proven feasible and show encouraging activity in lung cancer and mesothelioma. Problems of sporadic life-threatening toxicity identified in early studies of this and other antifolates have been resolved by the discovery that they were caused by functional folate deficiency and may be avoided by a low-dose nutritional supplement of folic acid and vitamin B(12). Pemetrexed is a promising new drug that should make a contribution to solid tumor oncology.

Pemetrexed safety and dosing strategy.

Pemetrexed is a novel antifolate/antimetabolite that inhibits several folate-dependent enzymes, including thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide transformylase. As a class, antifolates have been associated with sporadic severe myelosuppression with gastrointestinal toxicity. Although infrequent, a combination of such toxicities carries a high risk of potentially life-threatening complications. Severe toxicity from pemetrexed-based therapy has become more predictable using the vitamin deficiency marker homocysteine and, to a lesser extent, methylmalonic acid. Evidence now suggests that reducing total plasma homocysteine levels by supplementation with folic acid and vitamin B(12) leads to a better safety profile for pemetrexed, while not adversely affecting its efficacy.