Gene expression analysis may help the management of cancer patients, allowing the selection of subjects responding to treatment. The aim of this study was the characterization of expression pattern of genes involved in gemcitabine activity in pancreas tumor specimens and its correlation with treatment outcome. The role of drug transport and metabolism on gemcitabine cytotoxicity was examined with specific inhibitors, whereas transcription analysis of human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (dCK), 5V -nucleotidase (5V -NT), cytidine deaminase (CDA), and ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2) was done by quantitative reverse transcription-PCR in tumor tissue isolated by laser microdissection from surgical or biopsy samples of 102 patients. Association between clinical outcome and gene expression levels was estimated using Kaplan-Meier method and Cox's proportional hazards model. Transport and metabolism had a key role on gemcitabine sensitivity in vitro; moreover, hENT1, dCK, 5V -NT, CDA, RRM1, and RRM2 were detectable in most tumor specimens. hENT1 expression was significantly correlated with clinical outcome. Patients with high levels of hENT1 had a significantly longer overall survival [median, 25.7; 95% confidence interval (95% CI), 17.6-33.7 months in the higher expression tertile versus median, 8.5; 95% CI, 7.0-9.9 months in the lower expression tertile]. Similar results were obtained with disease-free survival and time to disease progression, and the multivariate analysis confirmed the prognostic significance of hENT1. This study suggests that the expression levels of hENT1 may allow the stratification of patients based on their likelihood of survival, thus offering a potential new tool for treatment optimization. (Cancer Res 2006; 66(7): 3928-35)

Purpose: Selecting patients according to key genetic characteristics may help to tailor chemotherapy and optimize the treatment in non^small cell lung cancer (NSCLC). Polymorphisms at the xeroderma pigmentosum group D (XPD), excision repair cross-complementing 1 (ERCC1), and cytidine deaminase (CDA) genes have been associated with alterations in enzymatic activity and may change sensitivity to the widely used cisplatin-gemcitabine regimen. Gln polymorphisms and response, clinical benefit, toxicity, time to progression (TTP), and overall survival (OS) was estimated using Pearson's m 2 tests, the Kaplan-Meier method, the log-rank test, and the Cox proportional hazards model. Results: The CDA Lys 27 Lys polymorphism significantly correlated with better clinical benefit (P = 0.04) and grade z3 neutropenia and thrombocytopenia, as well as with longer TTP and OS (P = 0.006 and P = 0.002, respectively), whereas no significant associations were found among ERCC1 and XPD polymorphisms and both response and clinical outcome. Finally, the enzymatic activity assay showed a significant lower mean in subjects harboring the CDA Lys 27 Lys polymorphism. Conclusions: Our data suggested the role of CDA Lys 27 Lys polymorphism as a possible predictive marker of activity, toxicity,TTP, and OS in advanced NSCLC patients treated with cisplatin and gemcitabine. These results may be explained by the lower enzymatic activity associated with the Lys 27 Lys CDA and offer a potential new tool for treatment optimization. Lung cancer is the leading cause of cancer-related deaths inWestern countries. Non -small cell lung cancer (NSCLC) accounts for >85% of primary lung cancers and approximately two thirds of NSCLC patients are diagnosed at an advanced stage (1).Pooled data from older randomized trials of cisplatin-based chemotherapy versus best supportive care showed that cisplatin-based chemotherapy was associated with a modest improvement in overall survival (OS; ref. 2). In more recent randomized trials, new cytotoxic drugs such as paclitaxel, docetaxel, vinorelbine, or gemcitabine in combination with a platinum compound have shown an absolute 15% to 20% improvement of survival in favor of chemotherapy versus best supportive care. In particular, the 1-year survival rate for best supportive care was 11% to 17% versus 30% to 35% for chemotherapy, which prolonged median survival by 3 to 4 months (3). However, none of the last-generation doublets was shown to be superior to the others and they all seemed to have reached the therapeutic plateau, with objective response rates of 30% to 40%, median survival time of 8 to 10 months, and 1-year survival rate of 30% to 40% (4). Indeed, a four-arm randomized phase III trial showed no substantial differences in response rate, time to progression (TTP), and OS among paclitaxel (24-hour infusion) -cisplatin, docetaxel-cisplatin, paclitaxel-carboplatin, and gemcitabine-cisplatin combination (4). Therefore, the cisplatin-gemcitabine combination is one of the standard regimens for the treatment of ...

Gemcitabine and pemetrexed are effective agents in the treatment of non-small-cell lung cancer (NSCLC), and the present study investigates cellular and genetic aspects of their interaction against A549, Calu-1, and Calu-6 cells. Cells were treated with pemetrexed and gemcitabine, and their interaction was assessed using the combination index. The role of drug metabolism in gemcitabine cytotoxicity was examined with inhibitors of deoxycytidine kinase (dCK), 5Ј-nucleotidase, and cytidine deaminase, whereas the role of pemetrexed targets, thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) in drug chemosensitivity was analyzed in cytotoxicity rescue studies. The effect of gemcitabine and pemetrexed on Akt phosphorylation was investigated with enzyme-linked immunosorbent assay, whereas quantitative polymerase chain reaction (PCR) was used to study target gene-expression profiles and its modulation by each drug. Synergistic cytotoxicity was demonstrated, and pemetrexed significantly decreased the amount of phosphorylated Akt, enhanced apoptosis, and increased the expression of dCK in A549 and Calu-6 cells, as well as the expression of the human nucleoside equilibrative transporter 1 (hENT1) in all cell lines. PCR demonstrated a correlation between dCK expression and gemcitabine sensitivity, whereas expression of TS, DHFR, and GARFT was predictive of pemetrexed chemosensitivity. These data demonstrated that 1) gemcitabine and pemetrexed synergistically interact against NSCLC cells through the suppression of Akt phosphorylation and induction of apoptosis; 2) the gene expression profile of critical genes may predict for drug chemosensitivity; and 3) pemetrexed enhances dCK and hENT1 expression, thus suggesting the role of gene-expression modulation for rational development of chemotherapy combinations.Despite recent advances in early diagnosis and treatment, non-small-cell lung cancer (NSCLC) is a disease with a grim prognosis. Extensive clinical studies demonstrated that chemotherapy increases survival in the adjuvant setting (Arriagada et al., 2004) and in patients with advanced disease (Reck and Gatzemeier, 2004). Nonetheless, response rates remain lower than 15%, and median survival is less than 6 months, thus emphasizing the need for new effective drugs and combination regimens (Rosell and Crinò, 2002). However, the rationale for chemotherapy combinations has remained mostly empirical, determined from the antitumor activity of each agent and the lack of overlapping toxicities, despite many attempts to discover preclinical models for rational selection of drug interactions.Gemcitabine (2Ј,2Ј-difluorodeoxycytidine) is a deoxycytidine analog with a broad spectrum of anticancer activity against several solid tumors in preclinical models, and it is M.D.T. was supported by unrestricted research grants from Eli Lilly (Florence, Italy). R. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.doi:10.1124/...

Purpose: Gemcitabine is an inhibitor of ribonucleotide reductase (RR) and DNA synthesis and is an effective agent in the treatment of pancreas cancer. The present study investigates whether the multitargeted antifolate pemetrexed would be synergistic with gemcitabine against MIA PaCa-2, PANC-1, and Capan-1 pancreatic cancer cell lines.Experimental Design: Cells were treated with gemcitabine and pemetrexed, and the type of drug interaction was assessed using the combination index. Cytotoxicity of gemcitabine was examined with inhibitors of (a) deoxycytidine kinase (dCK), which activates gemcitabine by phosphorylation, and (b) 5-nucleotidase (drug dephosphorylation) and cytidine deaminase (drug deamination), the main inactivating enzymes. The effects of gemcitabine and pemetrexed on cell cycle were analyzed by flow cytometry, and apoptosis was examined by fluorescence microscopy. Finally, quantitative, real-time PCR was used to study the pharmacogenetics of the drug combination.Results: Synergistic cytotoxicity and enhancement of apoptosis was demonstrated, mostly with the sequence pemetrexed3gemcitabine. Pemetrexed increased cells in S phase, the most sensitive to gemcitabine, and a positive correlation was found between the expression ratio of dCK:RR and gemcitabine sensitivity. Indeed, pemetrexed significantly enhanced dCK gene expression (؉227.9, ؉86.0, and ؉135.5% in MIA PaCa-2, PANC-1, and Capan-1 cells, respectively), and the crucial role of this enzyme was confirmed by impairment of gemcitabine cytotoxicity after dCK saturation with 2-deoxycytidine.Conclusions: These data demonstrate that the gemcitabine and pemetrexed combination displays scheduledependent synergistic cytotoxic activity, favorably modulates cell cycle, induces apoptosis, and enhances dCK expression in pancreatic cancer cells.

Chemotherapy has produced unsatisfactory results in pancreas cancer and novel approaches, including treatment tailoring by pharmacogenetic analysis and new molecular-targeted drugs, are required. The scarcity of effective therapies may reflect the lack of knowledge about the influence of tumor-related molecular abnormalities on responsiveness to drugs. Advances in the understanding of pancreas cancer biology have been made over the past decade, including the discovery of critical mutations in oncogenes (i.e., K-Ras) as well as the loss of tumor suppressor genes, such as TP53 and p16 INK4 . Other studies showed the dysregulation of the expression of proteins involved in the control of cell cycle, proliferation, apoptosis, and invasiveness, such as Bcl-2, Akt, mdm2, and epidermal growth factor receptor. These characteristics might contribute to the aggressive behavior of pancreatic cancer and influence response to treatment.

Purpose: Standard treatments have modest effect against pancreatic cancer, and current research focuses on agents targeting molecular pathways involved in tumor growth and angiogenesis. This study investigated the interactions between ZD6474, an inhibitor of tyrosine kinase activities of vascular endothelial growth factor receptor-2 and epidermal growth factor receptor (EGFR), gemcitabine, and ionizing radiation in human pancreatic cancer cells and analyzed the molecular mechanisms underlying this combination. Experimental Design: ZD6474, ionizing radiation, and gemcitabine, alone or in combination, were given in vitro to MIA PaCa-2, PANC-1, and Capan-1cells and in vivo to MIA PaCa-2 tumor xenografts. The effects of treatments were studied by the evaluation of cytotoxicity, apoptosis, cell cycle, EGFR and Akt phosphorylation, modulation of gene expression of enzymes related to gemcitabine activity (deoxycytidine kinase and ribonucleotide reductase), as well as vascular endothelial growth factor immunohistochemistry and microvessel count. Results: In vitro, ZD6474 dose dependently inhibited cell growth, induced apoptosis, and synergistically enhanced the cytotoxic activity of gemcitabine and ionizing radiation. Moreover, ZD6474 inhibited phosphorylation of EGFR and Akt and triggered cell apoptosis. PCR analysis showed that ZD6474 increased the ratio between gene expression of deoxycytidine kinase and ribonucleotide reductase. In vivo, ZD6474 showed significant antitumor activity alone and in combination with radiotherapy and gemcitabine, and the combination of all three modalities enhanced MIA PaCA-2 tumor growth inhibition compared with gemcitabine alone. Conclusions: ZD6474 decreases EGFR and Akt phosphorylation, enhances apoptosis, favorably modulates gene expression in cancer cells, and acts synergistically with gemcitabine and radiotherapy to inhibit tumor growth. These findings support the investigation of this combination in the clinical setting.Pancreatic cancer is a highly malignant illness that has steadily increased in incidence over recent decades, and it is now the fourth leading cause of death from cancer in the Western world. Despite this, there has been little improvement in prognosis over the past 20 years (1). Due to the delay of clinical symptoms, pancreatic cancer is usually detected at an advanced stage, and survival ranges between 4 and 6 months after diagnosis. Moreover, because of its aggressive biological behavior, this malignancy has a grim prognosis even following surgical resection, and the 5-year survival for all stages of the disease remains below 4% (2). Therefore, pancreatic cancer represents a clinical challenge and novel therapeutic approaches are warranted.Several studies showed that pancreatic cancer is characterized by dysregulation of molecular mechanisms involved in cell proliferation, invasiveness, and angiogenesis (3). Epidermal growth factor receptor (EGFR) is a key driver of cell proliferation, and

This study aimed to define the maximum-tolerated dose (MTD) of fixed dose rate (FDR) of gemcitabine (2 0 -2 0 -difluorodeoxycitidine) infusion with circulating haemopoietic progenitor support and to evaluate the activity of the treatment. Secondary end points were pharmacokinetic of gemcitabine and difluorodeoxyuridina (dFdU) measured at first course and the activity andexpression profile of cytidine deaminase (CdA) on circulating mononuclear cells. Patients with advanced pancreatic carcinoma received escalating dose of gemcitabine 10 mg m À2 min À1 every 2 weeks with circulating haemopoietic progenitor support. First dose level was 3000 mg m À2and the doses were increased by 500 mg m À2 until MTD. In all, 23 patients were enrolled. Toxicities were mild or moderate; the only patient treated at 7000 mg m À2 died because of toxicity; therefore; the MTD was established at 6500 mg m À2 . The overall response rate was 22.2%. The AUC of gemcitabine showed a dose-dependent increase, while the AUC of dFdU reached a plateau at 4500 mg m À2 . A significant relationship was found between the AUC of dFdU and CdA expression and activity (Po0.05). Moreover, progression rate and survival were significantly related to CdA expression and activity levels. The activity of high-dose gemcitabine is not superior to that reported with less intensive FDR schedules. The predictive role of CdA expression and activity on outcome deserves further investigation.

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