Prognostic significance of initial platelet counts and fibrinogen level in advanced non-small cell lung cancer.

Thrombocytosis and coagulation systems activation are commonly associated with disease progression and are suggested poor prognostic factors in patients with malignancies. This study aimed to investigate the prevalence and prognostic significance of thrombocytosis and elevated fibrinogen levels in patients with advanced non-small cell lung cancer (NSCLC). Initial platelet counts and fibrinogen levels were reviewed in 854 patients with histologically proven NSCLC. Thrombocytosis was defined as platelet counts > 450 × 10(9)/L. A serum fibrinogen level > 4.5 g/L was considered high. At the time of diagnosis, initial platelet counts and serum fibrinogen levels were evaluated before treatment. Clinicopathologic data including histological type, tumor, node, metastasis (TNM) stage, performance status, treatment method, and survival time were evaluated. Initial thrombocytosis was found in 6.9% of patients, and elevated fibrinogen levels were found in 55.1% of patients. Patients with thrombocytosis had a significantly poorer prognosis than patients with normal platelet counts (P < 0.001). In multivariate survival analysis, thrombocytosis was an independent prognostic factor (P < 0.001). An elevated serum fibrinogen level was associated with poor prognosis (P < 0.001). In conclusion, initial thrombocytosis and a high fibrinogen level are independent factors for predicting poor prognosis in patients with advanced NSCLC.

Clinical implication of microscopic anthracotic pigment in mediastinal staging of non-small cell lung cancer by endobronchial ultrasound-guided transbronchial needle aspiration.

Microscopic anthracotic pigment (MAP) is frequently observed in endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) specimen in non-small cell lung cancer, but its clinical interpretation is not well-known. The aim of this study was to evaluate the clinical implication of MAP in mediastinal staging of non-small cell lung cancer. From May 2010 to July 2011, consecutive potentially operable non-small cell lung cancer patients who underwent EBUS-TBNA for mediastinal staging were recruited. Of the total 133 patients, 102 (76.7%) were male patients. Median age was 68 yr. Total 279 mediastinal lymph nodes were sampled by EBUS-TBNA; station 4R (100, 35.8%) and station 7 (86, 30.8%) were the most common sites. Malignant lymph nodes were 100 (35.8%). MAP was observed in 61 (21.7%) lymph nodes, and among them only 3 were malignant lymph nodes (P < 0.001). The lymph nodes with MAP were smaller (9.0 vs 10.8 mm, P = 0.001) and showed low standard uptake values on FDG-PET (4.4 vs 4.7, P = 0.256). In multivariate analysis, MAP was negatively associated with malignant lymph node (adjusted OR, 0.12; 95% CI, 0.03-0.42; P < 0.001). In potentially operable non-small cell lung cancer patients, MAP in endobronchial ultrasound-guided transbronchial needle aspiration specimens is strongly associated with benign mediastinal and hilar lymph nodes.

Vitamin D deficiency and changes in serum vitamin D levels with treatment among tuberculosis patients in South Korea.

BACKGROUND AND OBJECTIVE: Vitamin D deficiency has been reported to be associated with the development of active tuberculosis (TB), but many discrepancies exist among studies. The aims of this study were to compare the frequency of vitamin D deficiency in a Korean population of TB patients and control subjects, and to monitor the changes in vitamin D levels during TB treatment. METHODS: Patients with newly diagnosed TB were prospectively enrolled. In addition, healthy volunteers or patients with diseases other than TB were enrolled as controls. Baseline serum 25-hydroxyvitamin D (25-OHD) levels were measured in both groups and compared. In the TB patients, measurements of serum 25-OHD were repeated 1 month after the initiation of treatment and again after completion of treatment. RESULTS: In total, 116 patients with TB and 86 control subjects were recruited. The median 25-OHD concentration was not different in TB patients at diagnosis (13.9 ng/mL; interquartile range (IQR) 8.80-21.8) compared with control subjects (13.2 ng/mL; IQR 9.6-19.3) (P = 0.97). The frequency of vitamin D deficiency (≤ 10 ng/mL) was also not different in TB patients (36.2%) compared with controls (27.3%) (P = 0.21). In TB patients, the median 25-OHD concentration decreased significantly during treatment, to 12.5 ng/mL at 1 month and 11.0 ng/mL on completion of treatment (P = 0.01). CONCLUSIONS: Vitamin D levels do not appear to be associated with the development of TB in the Korean population. The median 25-OHD concentration decreased after treatment for TB.

Outcome of patients with connective tissue disease requiring intensive care for respiratory failure.

Occasionally acute respiratory failure (ARF) develops in patients with connective tissue disease (CTD), but the etiologies of ARF in these patients are not fully elucidated. The objective of this study is to find out the causes of ARF leading to intensive care unit (ICU) admission in these patients and to assess their clinical outcome. The medical records of 1,870 consecutive patients admitted to the ICU in Seoul National University Hospital since January 2005-September 2008 were reviewed. A total of 66 patients with CTD were analyzed. The median age was 58 years, and 45 patients were women. The median length of ICU stay was 16 days with a median duration of mechanical ventilation support of 15 days. The distribution of underlying CTD was 17 patients with systemic lupus erythematosus; 15 with rheumatoid arthritis; 14 with systemic vasculitis; and nine with polymyositis-dermatomyositis. Pneumonia was the leading cause of ARF in 24 patients (36%). We could not identify the cause of ARF in 14. Other causes of ARF were acute pulmonary edema for nine patients, diffuse alveolar hemorrhage for eight and Pneumocystis pneumonia for four. Forty-one patients (62%) died during admission, and the mortality rate was the lowest in those with acute pulmonary edema. Use of norepinephrine was statistically higher in nonsurvivors. We could identify the cause of ARF leading to ICU admission in at least 80% of patients with CTD. However, these patients still showed a high mortality rate regardless of etiology. Their survival might be influenced by hemodynamic status.

Positive tuberculin skin test or interferon-gamma release assay in patients with radiographic lesion suggesting old healed tuberculosis.

Radiographic lesions suggesting old healed tuberculosis (TB) is considered a risk factor for the subsequent development of active TB. The aim of this study was to estimate the positive rates of tuberculin skin test (TST) and interferon-gamma release assay (IGRA) in persons with old healed TB. Participants with lesions suggesting old healed TB on chest images and controls without such lesions were prospectively enrolled between January 1, 2010, and January 31, 2011. TST and the QuantiFERON-TB Gold In-Tube test (QFT-GIT) were performed. In total, 193 participants with old healed TB and 126 controls were recruited. The rates of positive TST and QFT-GIT among patients with old healed TB were 54.6% and 77.7%, respectively. The rates of positive TST and QFT-GIT among patients without old healed TB were 38.9% and 61.9%. Sixteen percent of participants with old healed TB showed negative results by both TST and QFT-GIT. The positive rate of TST waned among participants with old healed TB who were older than 60 yr, whereas QFT-GIT positivity was unaffected by age. The positive rates of TST and IGRA among participants with radiographic lesions suggesting old healed TB was higher than without those lesions. In addition, IGRA may be more accurate than TST for the detection of latent TB infection, especially in populations of individuals older than 60 yr.

Treatment response and long term follow-up results of nonspecific interstitial pneumonia.

The purpose of this study was to investigate the long-term clinical course of non-specific interstitial pneumonia (NSIP) and to determine which factors are associated with a response to steroid therapy and relapse. Thirty-five patients with pathologically proven NSIP were included. Clinical, radiological, and laboratory data were reviewed retrospectively. The male-to-female ratio was 7:28 (median age, 52 yr). Thirty (86%) patients responded to steroid therapy, and the median follow-up was 55.2 months (range, 15.9-102.0 months). Five patients (14%) showed sustained disease progression and three died despite treatment. In the five with sustained disease progression, NSIP was associated with various systemic conditions, and the seropositivity of fluorescent antinuclear antibody was significantly associated with a poor response to steroids (P = 0.028). The rate of relapse was 25%, but all relapsed patients improved after re-treatment. The initial dose of steroids was significantly low in the relapse group (P = 0.020). In conclusion, progression is associated with various systemic conditions in patients who show progression. A low dose of initial steroids is significantly associated with relapse.

Effects of consumer-centered u-health service for the knowledge, skill, and attitude of the patients with chronic obstructive pulmonary disease.

Through the use of ubiquitous health, or u-Health, services, medical information can be adapted and made accessible via computer and Internet to provide quality healthcare to anybody, any place, and any time. We developed and implemented u-Health services for patients with chronic obstructive pulmonary disease and studied their experiences with respect to their knowledge of chronic obstructive pulmonary disease and skill and attitude toward the u-Health devices. The u-Health services were composed of telemonitoring and teleconsultation supplemented with home visits. To determine its effectiveness, the u-Health service system was implemented for 2 years with 144 chronic obstructive pulmonary disease patients in a clinical experiment. The subjects were divided into three experimental groups, each provided with different services, compared before and after intervention, and among groups. The analysis of data gathered through the system suggested that u-Health services can support patients with chronic obstructive pulmonary disease, improve patients' knowledge about chronic obstructive pulmonary disease self-management, build u-Health device usage skills, and foster a positive attitude toward u-Health devices. The u-Health services for the chronic obstructive pulmonary disease patients were both feasible and effective from the patients' perspective.

IL-7 promotes CXCR3 ligand-dependent T cell antitumor reactivity in lung cancer.

We are evaluating the immune enhancing activities of cytokines for their optimum utility in augmenting cellular immune responses against lung cancer. In this study, we evaluated the mechanism of antitumor responses following IL-7 administration to mice bearing established Lewis lung cancer. IL-7 decreased tumor burden with concomitant increases in the frequency of CD4 and CD8 T lymphocyte subsets, T cell activation markers CXCR3, CD69, and CD127(low), effector memory T cells, and T cell cytolytic activity against parental tumor cells. Accompanying the antitumor responses were increases in IFN-gamma, CXCL9, CXCL10, and IL-12. Individual neutralization of CD4, CD8 T lymphocytes, or the CXCR3 ligands CXCL9 and CXCL10 reversed the antitumor benefit of IL-7, indicating their importance for optimal responses in vivo. Furthermore, IL-7 decreased the tumor-induced apoptosis of T cells with subsequent decrease of the proapoptotic marker Bim. We assessed the impact of IL-7 treatment on regulatory T cells that negatively impact antitumor immune responses. IL-7 decreased regulatory T Foxp3 as well as cell suppressive activity with a reciprocal increase in SMAD7. These results indicate that IL-7 induces CXCR3 ligand-dependent T cell antitumor reactivity in lung cancer.

Risk factors of postoperative pneumonia after lung cancer surgery.

The purpose of this study was to investigate risk factors of postoperative pneumonia (POP) after lung cancer surgery. The 417 lung cancer patients who underwent surgical resection in a tertiary referral hospital were included. Clinical, radiological and laboratory data were reviewed retrospectively. Male and female ratio was 267:150 (median age, 65 yr). The incidence of POP was 6.2% (26 of 417) and in-hospital mortality was 27% among those patients. By univariate analysis, age ≥ 70 yr (P < 0.001), male sex (P = 0.002), ever-smoker (P < 0.001), anesthesia time ≥ 4.2 hr (P = 0.043), intraoperative red blood cells (RBC) transfusion (P = 0.004), presence of postoperative complications other than pneumonia (P = 0.020), forced expiratory volume in 1 second/forced vital capacity (FEV(1)/FVC) < 70% (P = 0.002), diffusing capacity of the lung for carbon monoxide < 80% predicted (P = 0.015) and preoperative levels of serum C-reactive protein ≥ 0.15 mg/dL (P = 0.001) were related with risk of POP. Multivariate analysis showed that age ≥ 70 yr (OR = 3.563, P = 0.014), intraoperative RBC transfusion (OR = 4.669, P = 0.033), the presence of postoperative complications other than pneumonia (OR = 3.032, P = 0.046), and FEV(1)/FVC < 70% (OR = 3.898, P = 0.011) were independent risk factors of POP. In conclusion, patients with advanced age, intraoperative RBC transfusion, postoperative complications other than pneumonia and a decreased FEV(1)/FVC ratio have a higher risk for pneumonia after lung cancer surgery.

Bioavailability and bioequivalence of two oral formulations of alendronate sodium 70 mg: an open-label, randomized, two-period crossover comparison in healthy Korean adult male volunteers.

BACKGROUND: Alendronate sodium is a Bisphosphonate drug used to treat and prevent osteoporosis and several other bone diseases. A new formulation has been developed and is currently awaiting regulatory approval, pending findings on bioequivalence. OBJECTIVES: The aims of the present study were to compare the bioavailability and pharmacokinetic (PK) properties, and to determine the bioequivalence, of a test and reference formulation of alendronate sodium 70 mg in a healthy Korean adult male population. METHODS: This open-label, randomized, 2-sequence, 2-period crossover study was carried out at Hanyang University Medical Center (Seoul, Republic of Korea). Healthy Korean adult male volunteers were randomly assigned to receive a single 70-mg dose of the test or reference formulation of alendronate sodium, administered with 240 mL of water, followed by a 7-day washout period and administration of the alternate formulation. The study drugs were administered after a 12-hour overnight fast. Serial blood samples were collected and adverse events were monitored by a clinical investigator via observation, personal interview, and vital signs (blood pressure, heart rate, and body temperature) over a 7-hour period (at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, and 7 hours) after drug administration. Plasma alendronate sodium concentrations were determined using a validated high-performance liquid chromatographic-postcolumn fluorescence derivatization method, with visible detection in the range of 2 to 100 ng/mL and lower limit of quantification set at 2 ng/mL. PK properties, including AUC(0-t), AUC(0-infinity), C(max), T(max), t(1/2), and the elimination constant (k(e)), were determined using non-compartmental analysis. The formulations were considered bioequivalent if the 90% CI ratios for C(max) and AUC were within the predetermined interval of 80% to 125%, the regulatory definition set by the US Food and Drug Administration (FDA). RESULTS: Twenty-three healthy male volunteers (mean [SD] age, 23.5 [2.0] years [range, 19-28 years]; height, 175.9 [5.4] cm [range, 162.0-185.0 cm]; and weight, 71.2 [9.5] kg [range, 61-96 kg]) were included in the study. No period or sequence effects were detected. The 90% CIs for the corresponding ratios of AUC(0-t), AUC(0-infinity) and C(max) were 84.97 to 114.47, 86.09 to 115.59, and 82.37 to 110.71, respectively. Additionally, the mean (range) of T(max) was 1.09 hours (0.5-2.0 hours), and the mean (SD) of t(1/2) and k(e) were 2.04 (0.97) hours and 0.34 (0.71) hour, respectively. The values for the test and reference formulations were within the FDA bioequivalence definition interval of 80% to 125%. No adverse events were reported in this study. CONCLUSIONS: Single doses of these formulations of alendronate sodium 70 mg met the criteria for bio-equivalence. No statistically significant differences in AUC(0-t), AUC(0-infinity), and C(max) were found in this healthy Korean adult male population.

Quantification of isradipine in human plasma using LC-MS/MS for pharmacokinetic and bioequivalence study.

A highly sensitive and rapid method for the analysis of isradipine in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed. The procedure involves a simple liquid-liquid extraction of isradipine and amlodipine (IS, internal standard) with methyl-t-butyl ether after alkaline treatment and separation by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 372.1-->m/z 312.2 and m/z 408.8-->m/z 237.9, for quantification of isradipine and IS, respectively. The standard calibration curves showed good linearity within the range of 10 to 5000 pg/mL (r(2)>or=0.9998). The lower limit of quantitation (LLOQ) was 10 pg/mL. The retention times of isradipine (0.81 min) and IS (0.65 min) suggested the potential for high throughput of the proposed method. In addition, no significant metabolic compounds were found to interfere with the analysis. This method offered good precision and accuracy and was successfully applied for the pharmacokinetic and bioequivalence studies of 5 mg of sustained-release isradipine in 24 healthy Korean volunteers.

Clinical significance of non-diagnostic pathology results from percutaneous transthoracic needle lung biopsy: experience of a tertiary hospital without an on-site cytopathologist.

BACKGROUND AND OBJECTIVE: More non-diagnostic pathology results may be reported following transthoracic needle lung biopsy (TTNB) when no on-site cytopathologist is available. This study was conducted to analyse the final outcomes in patients with non-diagnostic pathology results, and the factors related to the adequacy of specimens. METHODS: The medical records of consecutive patients who had undergone TTNB from January 2004 to January 2005 were retrospectively analysed. Non-diagnostic pathology results were classified into three groups: (i) atypical cells, (ii) non-specific inflammation and (iii) inadequate specimen. The final diagnosis and clinical outcome for each patient were analysed after additional diagnostic studies and clinical follow up. RESULTS: TTNB was performed on 291 patients. Specimens were adequate in 256 cases. The results were non-diagnostic for 103 patients, and the percentages of atypical cells, non-specific inflammation and inadequate specimen were 15.5% (16/103), 50.5% (52/103) and 40% (35/103), respectively. In 14 patients (87.5%) the diagnosis of atypical cells was confirmed, and in 10 (62.5%) these were due to malignancies. In two patients (3.8%) the lesions reported as non-specific inflammation were eventually confirmed as malignancies. After repeated attempts to confirm their diagnoses, 22 patients (62.9%) with initial reports of inadequate specimen were diagnosed with specific diseases. Specimen adequacy correlated with technical skill, size of the lesion, guidance method and biopsy method. CONCLUSIONS: Non-diagnostic pathology results from TTNB, in the absence of an on-site cytopathologist, are of value in assessing the clinical probability of malignancy and can be useful in the management of lung lesions. However, many other factors should be considered in patients with inadequate specimens.

Risk factors for bacterial pneumonia after cytotoxic chemotherapy in advanced lung cancer patients.

The purpose of this study was to demonstrate the prevalence of and risk factors for pneumonia after cytotoxic chemotherapy in advanced lung cancer patients. We retrospectively reviewed 193 out of 215 advanced lung cancer patients who were consecutively treated with cytotoxic chemotherapy at Seoul National University Hospital in 2005. The mean age was 61 years (range, 31-85 years). A total of 41 patients (21%) had Eastern cooperative oncology group (ECOG) performance status 2-3. Among 135 patients with pulmonary function test (PFT), 34% (n=46) of patients demonstrated an obstructive pattern of lung function. Pneumonia occurred in a total of 10 patients (5%). Occurrence rate was significantly higher in older patients (>70 years) than in younger ones (13% vs. 4%, p=0.05), higher in patients with ECOG 2-3 compared with those with ECOG 0-1 (15% vs. 3%, p<0.01), and higher in patients with an obstructive pattern of lung function compared with those without (11% vs. 1%, p=0.02). Pneumonia occurrence rate in patients with all three risk factors was 25%. Six out of 10 pneumonia patients required hospitalization, and one patient died from respiratory failure. We identified this high-risk group as a candidate for antibacterial prophylaxis after cytotoxic chemotherapy in advanced lung cancer patients.

Frequency and predictors of miliary tuberculosis in patients with miliary pulmonary nodules in South Korea: a retrospective cohort study.

BACKGROUND: Miliary pulmonary nodules are commonly caused by various infections and cancers. We sought to identify the relative frequencies of various aetiologies and the clinical and radiographic predictors of miliary tuberculosis (TB) in patients with miliary pulmonary nodules. METHODS: We performed a retrospective cohort study of patients who presented with micronodules occupying more than two-thirds of the lung volume, based on computed tomography (CT) of the chest, between November 2001 and April 2007, in a tertiary referral hospital in South Korea. RESULTS: We analyzed 76 patients with miliary pulmonary nodules. Their median age was 52 years and 38 (50%) were males; 18 patients (24%) had a previous or current malignancy and five (7%) had a history of TB. The most common diagnoses of miliary nodules were miliary TB (41 patients, 54%) and miliary metastasis of malignancies (20 patients, 26%). Multivariate analysis revealed that age 25% of total lung volume increased the probability of miliary TB. However, a history of malignancy decreased the probability of miliary TB. CONCLUSION: Miliary TB accounted for approximately half of all causes of miliary pulmonary nodules. Young age, an immune-compromised state, and several clinical and radiographic characteristics increased the probability of miliary TB.

Intrapulmonary administration of CCL21 gene-modified dendritic cells reduces tumor burden in spontaneous murine bronchoalveolar cell carcinoma.

The antitumor efficiency of dendritic cells transduced with an adenovirus vector expressing secondary lymphoid chemokine (CCL21) was evaluated in a murine model of spontaneous bronchoalveolar cell carcinoma. The transgenic mice (CC-10 TAg) express the SV40 large T antigen (TAg) under the Clara cell promoter, develop bilateral, multifocal, and pulmonary adenocarcinomas, and die at 4 months as a result of progressive pulmonary tumor burden. A single intratracheal administration of CCL21 gene-modified dendritic cells (DC-AdCCL21) led to a marked reduction in tumor burden with extensive mononuclear cell infiltration of the tumors. The reduction in tumor burden was accompanied by the enhanced elaboration of type 1 cytokines [IFN-gamma, interleukin (IL)-12, and granulocyte macrophage colony-stimulating factor] and antiangiogenic chemokines (CXCL9 and CXCL10) but a concomitant decrease in the immunosuppressive molecules (IL-10, transforming growth factor-beta, prostaglandin E(2)) in the tumor microenvironment. The DC-AdCCL21 therapy group revealed a significantly greater frequency of tumor-specific T cells releasing IFN-gamma compared with the controls. Continuous therapy with weekly intranasal delivery of DC-AdCCL21 significantly prolonged median survival by >7 weeks in CC-10 TAg mice. Both innate natural killer and specific T-cell antitumor responses significantly increased following DC-AdCCL21 therapy. Significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides a strong rationale for further evaluation of intrapulmonary-administered DC-AdCCL21 in regulation of tumor immunity and genetic immunotherapy for lung cancer.

Cyclooxygenase-2-dependent regulation of E-cadherin: prostaglandin E(2) induces transcriptional repressors ZEB1 and snail in non-small cell lung cancer.

Elevated tumor cyclooxygenase-2 (COX-2) expression is associated with tumor invasion, metastasis, and poor prognosis in non-small cell lung cancer (NSCLC). Here, we report that COX-2-dependent pathways contribute to the modulation of E-cadherin expression in NSCLC. First, whereas genetically modified COX-2-sense (COX-2-S) NSCLC cells expressed low E-cadherin and showed diminished capacity for cellular aggregation, genetic or pharmacologic inhibition of tumor COX-2 led to increased E-cadherin expression and resulted in augmented homotypic cellular aggregation among NSCLC cells in vitro. An inverse relationship between COX-2 and E-cadherin was shown in situ by double immunohistochemical staining of human lung adenocarcinoma tissue sections. Second, treatment of NSCLC cells with exogenous prostaglandin E(2) (PGE(2)) significantly decreased the expression of E-cadherin, whereas treatment of COX-2-S cells with celecoxib (1 mumol/L) led to increased E-cadherin expression. Third, the transcriptional suppressors of E-cadherin, ZEB1 and Snail, were up-regulated in COX-2-S cells or PGE(2)-treated NSCLC cells but decreased in COX-2-antisense cells. PGE(2) exposure led to enhanced ZEB1 and Snail binding at the chromatin level as determined by chromatin immunoprecipitation assays. Small interfering RNA-mediated knockdown of ZEB1 or Snail interrupted the capacity of PGE(2) to down-regulate E-cadherin. Fourth, an inverse relationship between E-cadherin and ZEB1 and a direct relationship between COX-2 and ZEB1 were shown by immunohistochemical staining of human lung adenocarcinoma tissue sections. These findings indicate that PGE(2), in autocrine or paracrine fashion, modulates transcriptional repressors of E-cadherin and thereby regulates COX-2-dependent E-cadherin expression in NSCLC. Thus, blocking PGE(2) production or activity may contribute to both prevention and treatment of NSCLC.

EGFR signaling is required for TGF-beta 1 mediated COX-2 induction in human bronchial epithelial cells.

Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins and thromboxanes from free arachidonic acid. Increasing evidence suggests that COX-2 plays a role in tumorigenesis. A variety of stimuli induce COX-2 and it is overexpressed in many tumors, including non-small cell lung cancer (NSCLC). We studied the regulation of COX-2 expression in immortalized human bronchial epithelial cells (HBECs) by transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF) because these two growth factors are present in both the pulmonary milieu of those at risk for lung cancer as well as in the tumor microenvironment. EGF significantly enhanced TGF-beta1-mediated induction of COX-2 and corresponding prostaglandin E2 (PGE2) production. TGF-beta1 and EGF induced COX-2 at the transcriptional and post-transcriptional levels. EGF receptor (EGFR) inhibition, neutralizing antibody against amphiregulin, or mitogen-activated protein kinase kinase (MEK) inhibition blocked TGF-beta1-mediated COX-2 induction. COX-2 induction by TGF-beta1 depended upon Smad3 signaling and required the activity of EGFR or its downstream mediators. Autocrine amphiregulin signaling maintains EGFR in a constitutively active state in HBECs, allowing for COX-2 induction by TGF-beta1. Thus, EGFR ligands, which are abundant in the pulmonary microenvironment of those at risk for lung cancer, potentiate and are required for COX-2 induction by TGF-beta1 in HBEC. These findings emphasize the central role of EGFR signaling in COX-2 induction by TGF-beta1 and suggest that inhibition of EGFR signaling should be investigated further for lung cancer prevention.

Tumor cyclooxygenase-2/prostaglandin E2-dependent promotion of FOXP3 expression and CD4+ CD25+ T regulatory cell activities in lung cancer.

Cyclooxygenase (COX)-2 and its product prostaglandin (PG) E2 underlie an immunosuppressive network that is important in the pathogenesis of non-small cell lung cancer. CD4+ CD25+ T regulatory (Treg) cells play an important role in maintenance of immunologic self-tolerance. CD4+ CD25+ Treg cell activities increase in lung cancer and appear to play a role in suppressing antitumor immune responses. Definition of the pathways controlling Treg cell activities will enhance our understanding of limitation of the host antitumor immune responses. Tumor-derived COX-2/PGE2 induced expression of the Treg cell-specific transcription factor, Foxp3, and increased Treg cell activity. Assessment of E-prostanoid (EP) receptor requirements revealed that PGE2-mediated induction of Treg cell Foxp3 gene expression was significantly reduced in the absence of the EP4 receptor and ablated in the absence of the EP2 receptor expression. In vivo, COX-2 inhibition reduced Treg cell frequency and activity, attenuated Foxp3 expression in tumor-infiltrating lymphocytes, and decreased tumor burden. Transfer of Treg cells or administration of PGE2 to mice receiving COX-2 inhibitors reversed these effects. We conclude that inhibition of COX-2/PGE2 suppresses Treg cell activity and enhances antitumor responses.

Tumor cyclooxygenase 2-dependent suppression of dendritic cell function.

Dendritic cells (DCs) serve as professional antigen-presenting cells and are pivotal in the host immune response to tumor antigens. To define the pathways limiting DC function in the tumor microenvironment, we assessed the impact of tumor cyclooxygenase (COX)-2 expression on DC activities. Bone marrow-derived DCs were cultured in either tumor supernatant (TSN) or TSN from COX-2-inhibited tumors. After culture, DCs were pulsed with tumor-specific peptides, and their ability to generate antitumor immune responses was assessed following injection into established murine lung cancer. In vitro, DC phenotype, alloreactivity, antigen processing and presentation, and interleukin (IL)-10 and IL-12 secretion were evaluated. DCs cultured in TSN failed to generate antitumor immune responses and caused immunosuppressive effects that correlated with enhanced tumor growth. However, genetic or pharmacological inhibition of tumor COX-2 expression restored DC function and effective antitumor immune responses. Functional analyses indicated that TSN causes a decrement in DC capacity to (a) process and present antigens, (b) induce alloreactivity, and (c) secrete IL-12. Whereas TSN DCs showed a significant reduction in cell surface expression of CD11c, DEC-205, MHC class I antigen, MHC class II antigen, CD80, and CD86 as well as a reduction in the transporter-associated proteins, transporter associated with antigen processing 1 and 2, the changes in phenotype and function were not evident when DCs were cultured in supernatant from COX-2-inhibited tumors. We conclude that inhibition of tumor COX-2 expression or activity can prevent tumor-induced suppression of DC activities.

Intratumoral administration of dendritic cells overexpressing CCL21 generates systemic antitumor responses and confers tumor immunity.

To achieve in situ tumor antigen uptake and presentation, intratumoral administration of ex vivo-generated, gene-modified murine bone marrow-derived dendritic cells (DC) was used in a murine lung cancer model. To attract mature host DC and activated T cells at the tumor site, the DC were transduced with an adenoviral vector expressing secondary lymphoid tissue chemokine (CCL21/SLC). Sixty percent of the mice treated with 10(6) DC-AdCCL21 intratumorally (7-10 ng/ml/10(6) cells/24 h of CCL21) at weekly intervals for 3 weeks showed complete tumor eradication, whereas only 25% of mice had complete resolution of tumors when mice were treated with fibroblasts expressing CCL21. In contrast only 12% of the mice treated with unmodified or control vector modified DC (DC-AdCV) showed complete tumor eradication. DC-AdCCL21 administration led to increases in the CD4(+), CD8(+), and CD3(+)CXCR3(+) T cells, as well as DC expressing CD11c(+) DEC205(+). CD4(+)CD25(+) T-regulatory cells infiltrating the tumors were markedly reduced after DC-AdCCL21 therapy. The tumor site cellular infiltrates were accompanied by the enhanced elaboration of granulocyte macrophage colony-stimulating factor, IFN-gamma, MIG/CXCL9, IP-10/CXCL10, and interleukin 12, but decreases in the immunosuppressive mediators transforming growth factor beta and prostaglandin E(2). DC-AdCCL21-treated tumor-bearing mice showed enhanced frequency of tumor-specific T lymphocytes secreting IFN-gamma, and tumor protective immunity was induced after DC-AdCCL21 therapy. In vivo depletion of IP-10/CXCL10, MIG/CXCL9, or IFN-gamma significantly reduced the antitumor efficacy of DC-AdCCL21. These findings provide a strong rationale for the evaluation of DC-AdCCL21 in cancer immunotherapy.