Combined expression of the non-receptor protein tyrosine kinases FAK and Src in primary colorectal cancer is associated with tumor recurrence and metastasis formation.

PURPOSE: The protein tyrosine kinase focal adhesion kinase (FAK) and Src in association with phosphorylation of the adapter protein paxillin are essential in tumor metastasis formation. Elevated levels of FAK, Src and paxillin may increase the metastatic potential of colorectal tumor cells. The aim of the current study was to examine the expression of FAK, Src, and paxillin using immunohistochemistry in the context of disease progression and to evaluate its clinical significance as a prognostic factor. EXPERIMENTAL DESIGN: The relationship between FAK, Src and paxillin levels and colorectal cancer progression was evaluated by immunohistochemistry in 104 colorectal cancer specimens with clinical follow up. In addition, FAK, Src and paxillin expression levels were quantified in 68 colorectal tumors and corresponding liver metastases. RESULTS: FAK and paxillin expression individually did not significantly impact time to recurrence (p=0.09, and p=0.89 respectively). Src expression was associated with tumor recurrence p=0.03. However, tumors that expressed both high FAK and Src levels had a significant shorter time to recurrence (p=0.004, hazard ratio: 2.98, 95% CI 1.14-6.31). FAK, Src and paxillin showed equivalent levels in corresponding liver metastases compared to the primary tumors (p=0.67, p=0.28 and p=0.34 respectively). CONCLUSIONS: These findings show that high levels of FAK and Src combined were predictive for recurrence of colorectal cancer. In addition, expression of FAK, Src and paxillin in colorectal cancer were maintained in corresponding distant metastases.

Improved lung function and symptom control with formoterol on demand in asthma.

Many asthma patients remain symptomatic despite maintenance therapy with inhaled corticosteroids (ICS) and salbutamol as rescue medication. In the present study the relative efficacy and preference for as-needed formoterol compared with salbutamol was examined. In total, 211 patients with a mean age of 45 yrs (mean forced expiratory volume in one second (FEV1) 77% predicted normal), using ICS, were randomised to 3 weeks' double-blind treatment with as-needed formoterol 4.5 microg Turbuhaler and with as-needed salbutamol 100 mug Turbuhaler in a cross-over fashion. Overall, lung function and symptom control were better with as-needed formoterol than with as-needed salbutamol. During as-needed formoterol treatment daytime and night-time symptom scores were lower, peak expiratory flow and FEV1 were higher and patients experienced fewer disturbed nights (34%) compared with as-needed salbutamol. Patients preferred the formoterol treatment to salbutamol. Of the 162 patients expressing a preference, formoterol was preferred by 68% (95% confidence interval: 60-75). Subjective assessment of effectiveness also favoured formoterol, which was perceived as slightly faster acting than salbutamol. In conclusion, as-needed formoterol improved symptoms and lung function compared with salbutamol and was perceived as more effective and at least as fast acting for symptom relief.

[The treatment of asthma: indications for a change in approach]. / Behandeling van astma: aanwijzingen voor een verandering in aanpak.

In both children and adults with persistent asthma, treatment with an inhaled corticosteroid (ICS) is recommended. Moreover, inhaled bronchodilating agents have a clear role to play. The minimum effective dose of an ICS in the individual patient can be determined either by starting with a low dosage of ICS and increasing the dosage gradually on the basis of the symptoms (the 'step-up' approach), or by starting with a high dosage and, if the results are good, decreasing it to the pointwhere adequate control is maintained (the 'step-down' approach). In a study of the step-up approach with the ICS fluticasone, with or without salmeterol as a long-acting beta2-agonist (LABA) in adult patients with asthma, the approach with salmeterol produced the best results, namely, good asthma control in 71% of the patients and total control in 41%. In a study involving both children and adults with asthma, good results were obtained from treatment with a relatively low maintenance dose of ICS (budesonide) combined with a LABA (formoterol), whereby patients were permitted to use additional inhalations of the combination ICS and LABA. How the different therapeutic concepts result in long-term control, what the side effects are in the long term, and whether, in addition to the clinical symptoms, laboratory findings are also important as a therapeutic criterion are all unknown.

Efficacy and safety of budesonide/formoterol single inhaler therapy versus a higher dose of budesonide in moderate to severe asthma.

OBJECTIVES: This study evaluated the efficacy and safety of a novel asthma management strategy--budesonide/formoterol for both maintenance and symptom relief (Symbicort Single Inhaler Therapy)--compared with a higher maintenance dose of budesonide in patients with moderate to severe asthma. METHODS: This was a 12-month, randomised, double-blind, parallel-group study. Symptomatic patients with asthma (n = 1890; mean age 43 years [range 11 years-80 years], mean baseline forced expiratory volume in 1 s [FEV(1)] 70% of predicted, mean inhaled corticosteroid [ICS] dose 746 microg/day) received either budesonide (160 microg, 2 inhalations twice daily) plus terbutaline 0.4 mg as needed or a daily maintenance dose of budesonide/formoterol (160/4.5 microg, 2 inhalations once daily) with additional inhalations of budesonide/formoterol 160/4.5 microg as needed. Time to first severe exacerbation (hospitalisation/emergency room [ER] treatment or systemic steroids due to asthma worsening or a fall in morning peak expiratory flow [PEF] to < or = 70% of baseline on 2 consecutive days) was the primary outcome variable. RESULTS: A total of 1890 patients were randomised, of whom 1563 (83%) had severe asthma. The time to first severe exacerbation was prolonged by budesonide/formoterol single inhaler therapy (p < 0.001) compared with a higher dose of budesonide. The risk of having a severe exacerbation was 39% lower with budesonide/formoterol single inhaler therapy compared with budesonide (p < 0.001). The number needed to treat to prevent one severe exacerbation per year with budesonide/formoterol compared with budesonide was 5. The budesonide/formoterol group had 45% fewer severe exacerbations requiring medical intervention per patient compared with the budesonide group (p < 0.001). Budesonide/formoterol patients had fewer hospitalisations/ER treatments (15 vs 25 events, respectively [descriptive statistics]) and fewer treatment days with systemic steroids (1776 days vs 3177 days, respectively [descriptive statistics]) compared with budesonide patients. Budesonide/formoterol single inhaler therapy patients used less as-needed medication compared with budesonide patients (0.90 vs 1.42 inhalations/day; p < 0.001). The mean daily ICS dose was lower in the budesonide/formoterol group than in the budesonide group (466 microg/day vs 640 microg/day). Over the 12-month study period, the budesonide/formoterol group achieved asthma control sufficient to not require any additional as-needed medication on 60% of days. Overall, budesonide/formoterol single inhaler therapy gave 31 more asthma control days (a night and day with no asthma symptoms and no as-needed medication use) per patient-year and 12 additional undisturbed nights per patient-year compared with a higher dose of budesonide. Both treatments were well tolerated. CONCLUSION: Budesonide/formoterol single inhaler therapy has the potential to provide a complete asthma management approach with one inhaler, demonstrating a high level of efficacy in patients with moderate to severe asthma.

Adjustable maintenance dosing with budesonide/formoterol compared with fixed-dose salmeterol/fluticasone in moderate to severe asthma.

BACKGROUND: Current asthma guidelines recommend that patients are educated to adjust their medication according to their asthma severity using physician-guided self-management plans. However, many patients take a fixed dose of their controller medication and adjust their reliever medication according to asthma symptoms. OBJECTIVES: This study examined whether asthma control improved if patients adjusted the maintenance dose of budesonide/formoterol (Symbicort Turbuhaler* 160/4.5 microg) according to asthma severity compared with traditional fixed dosing (FD) regimens. METHODS: Symptomatic patients with asthma (n = 658, mean symptom score 1.5, mean inhaled corticosteroids 735 microg/day, mean forced expiratory volume in 1 second [FEV(1)] 84% predicted) were randomised after 2 weeks' run-in to either: budesonide/formoterol adjustable maintenance dosing (AMD), budesonide/formoterol FD or salmeterol/fluticasone (Seretide Diskus dagger 50/250 microg) FD. In a 4-week double-blind period, both budesonide/formoterol AMD and FD groups received two inhalations twice daily (bid) and salmeterol/fluticasone FD patients received one inhalation bid. In the following 6-month open extension, both FD groups continued with the same treatment. Patients in the AMD group with well-controlled asthma stepped down to one inhalation bid; others continued with two inhalations bid. All AMD patients could increase to four inhalations bid for 7-14 days if symptoms worsened. All patients used terbutaline or salbutamol for symptom relief throughout. The primary variable was the odds of achieving a well-controlled asthma week (WCAW). RESULTS: The odds ratio for achieving a WCAW did not differ between the FD regimens; however, during the open period, budesonide/formoterol AMD increased the odds of achieving a WCAW vs. budesonide/formoterol FD (odds ratio 1.335; 95% CI: 1.001, 1.783; p = 0.049) despite a 15% reduction in average study drug use. Budesonide/formoterol AMD patients had a lower exacerbation rate over the study: 40% lower vs. salmeterol/fluticasone FD (p = 0.018); 32% lower vs. budesonide/formoterol FD (NS). During the double-blind period, there were no clinically relevant differences between the budesonide/formoterol FD and salmeterol/fluticasone FD groups. Budesonide/formoterol AMD patients used less reliever medication in the open extension: 0.58 vs. 0.92 occasions/day for budesonide/formoterol FD (p = 0.001) and 0.80 occasions/day for salmeterol/fluticasone FD (p = 0.011). CONCLUSIONS: Adjustable maintenance dosing with budesonide/formoterol provides more effective asthma control by reducing exacerbations and reliever medication usage compared with fixed-dose salmeterol/fluticasone.

The role of the epidermal growth factor receptor in sustaining neutrophil inflammation in severe asthma.

BACKGROUND: The extent of epithelial injury in asthma is reflected by expression of the epidermal growth factor receptor (EGFR), which is increased in proportion to disease severity and is corticosteroid refractory. Although the EGFR is involved in epithelial growth and differentiation, it is unknown whether it also contributes to the inflammatory response in asthma. OBJECTIVES: Because severe asthma is characterized by neutrophilic inflammation, we investigated the relationship between EGFR activation and production of IL-8 and macrophage inhibitory protein-1 alpha (MIP-1alpha) using in vitro culture models and examined the association between epithelial expression of IL-8 and EGFR in bronchial biopsies from asthmatic subjects. METHODS: H292 or primary bronchial epithelial cells were exposed to EGF or H2O2 to achieve ligand-dependent and ligand-independent EGFR activation; IL-8 mRNA was measured by real-time PCR and IL-8 and MIP-1alpha protein measured by enzyme-linked immunosorbent assay (ELISA). Epithelial IL-8 and EGFR expression in bronchial biopsies from asthmatic subjects was examined by immunohistochemistry and quantified by image analysis. RESULTS: Using H292 cells, EGF and H2O2 increased IL-8 gene expression and release and this was completely suppressed by the EGFR-selective tyrosine kinase inhibitor, AG1478, but only partially by dexamethasone. MIP-1alpha release was not stimulated by EGF, whereas H2O2 caused a 1.8-fold increase and this was insensitive to AG1478. EGF also significantly stimulated IL-8 release from asthmatic or normal primary epithelial cell cultures established from bronchial brushings. In bronchial biopsies, epithelial IL-8, MIP-1alpha, EGFR and submucosal neutrophils were all significantly increased in severe compared to mild disease and there was a strong correlation between EGFR and IL-8 expression (r = 0.70, P < 0.001). CONCLUSIONS: These results suggest that in severe asthma, epithelial damage has the potential to contribute to neutrophilic inflammation through enhanced production of IL-8 via EGFR- dependent mechanisms.

Formoterol in patients with chronic obstructive pulmonary disease: a randomized, controlled, 3-month trial.

The aim of this study was to investigate formoterol, an inhaled long-acting beta2-agonist, in patients with chronic obstructive pulmonary disease (COPD). Six-hundred and ninety-two COPD patients, mean baseline forced expiratory volume in one second (FEV1) 54%, FEV1/forced vital capacity 75% of predicted, reversibility 6.4% pred, were treated with formoterol (4.5, 9 or 18 microg b.i.d.) or placebo via Turbuhaler for 12 weeks. Symptoms were recorded daily. Spirometry and the incremental shuttle walking test (SWT) were performed at clinic visits. Compared with placebo, 18 microg b.i.d. formoterol reduced the mean total symptom score by 13% and increased the percentage of nights without awakenings by 15%. Formoterol (9 and 18 microg b.i.d.) significantly reduced symptom scores for breathlessness (-7% and -9%, respectively) and chest tightness (-11% and -8%, respectively), reduced the need for rescue medication (-25% and -18%, respectively), and increased symptom-free days (71% and 86%, respectively). FEV1 improved significantly after all three doses of formoterol (versus placebo). No differences were found between groups in SWT walking distance. No unexpected adverse events were seen. In conclusion, 9 and 18 microg b.i.d. formoterol reduced symptoms and increased the number of symptom-free days in a dose-dependent manner in chronic obstructive pulmonary disease patients. Formoterol improved lung function at a dose of 4.5 microg b.i.d. and higher.

Unaltered perception of dyspnoea during treatment with long-acting beta2-agonists.

There is the possibility that during treatment with inhaled long-acting beta2-agonists that a loss of perception of dyspnoea might occur and that the forced expiratory volume in one second (FEV1) might fall precipitously during bronchial provocation. This study investigated these possibilities during methacholine provocation, continued until there was > or =30% fall in FEV1, mimicking a moderate asthma attack. Nineteen asthmatic patients were asked to score their dyspnoea as a Borg score during provocation with methacholine. One hour prior to this provocation, the patients used the last morning dose of 14 days treatment with either formoterol (twice daily 24 microg by Turbuhaler), salmeterol (twice daily 100 microg by Diskhaler) and placebo in a double-blind, randomized, double-dummy, cross-over design. The perception of dyspnoea, expressed as the Borg score divided by the change in FEV1 at > or =30% fall in FEV1, was similar on the three test days at 0.067, 0.076 and 0.074%(-1) after formoterol, salmeterol and placebo treatment, respectively (p=0.16). The slope of the methacholine dose response curve did not differ (p=0.52). In conclusion, no suggestion was found for an abnormal perception of dyspnoea or an exaggerated fall in forced expiratory volume in one second during provocation with methacholine under long-acting beta2-agonist treatment.

Compliance with inhaled glucocorticoids and concomitant use of long-acting beta2-agonists.

We investigated whether treatment with a long-acting beta2-agonist (LAbeta2) is associated with a decrease in patient compliance with regard to inhalation corticosteroids (ICS). Date on prescriptions collected by 15,760 patients suffering from airways disease were provided by 69 Dutch pharmacies. All prescriptions of ICS and LAbeta2 were analysed and divided in four groups by LAbeta2 use during 1997 and 1998. Date from 15,760 patients were available. In the 10,929 patients not treated with LAbeta2, compliance decreased slightly but not significantly. In 3281 patients receiving LAbeta2 compliance also decreased slightly but not significantly. In 404 patients, who used a LAbeta2 in 1997 and discontinued treatment in 1998, the compliance fell significantly (P<0.05). In 1147 patients who started to use a LAbeta2 in 1998, compliance with ICS significantly improved (P<0.05). These results suggest that the regular use of LAbeta2 improves compliance with ICS. Therefore, the concern that compliance with inhaled corticosteroid therapy will decrease under concomitant use of LAbeta2 appear to be unfounded.

Decreased bronchodilating effect of salbutamol in relieving methacholine induced moderate to severe bronchoconstriction during high dose treatment with long acting beta2 agonists.

BACKGROUND: In vitro the long acting beta2 agonist salmeterol can, in contrast to formoterol, behave as a partial agonist and become a partial antagonist to other beta2 agonists. To study this in vivo, the bronchodilating effect of salbutamol was measured during methacholine induced moderate to severe bronchoconstriction in patients receiving maintenance treatment with high dose long acting beta2 agonists. METHODS: A randomised double blind crossover study was performed in 19 asthmatic patients with mean forced expiratory volume in one second (FEV1) of 88.4% predicted and median concentration of methacholine provoking a fall in FEV1 of 20% or more (PC(20)) of 0.62 mg/ml at entry. One hour after the last dose of 2 weeks of treatment with formoterol (24 microg twice daily by Turbuhaler), salmeterol (100 microg twice daily by Diskhaler), or placebo a methacholine provocation test was performed and continued until there was at least a 30% decrease in FEV1. Salbutamol (50 microg) was administered immediately thereafter, followed by ipratropium bromide (40 microg) after a further 30 minutes. Lung function was monitored for 1 hour after provocation. RESULTS: There was a significant bronchodilating and bronchoprotective effect after 2 weeks of active treatment. The dose of methacholine needed to provoke a fall in FEV1 of > or = 30% was higher after pretreatment with formoterol (2.48 mg) than with salmeterol (1.58 mg) or placebo (0.74 mg). The difference between formoterol and salmeterol was statistically significant: 0.7 doubling dose steps (95% CI 0.1 to 1.2, p=0.016). The immediate bronchodilating effect of subsequently administered salbutamol was significantly impaired after pretreatment with both drugs (p<0.0003 for both). Three minutes after inhaling salbutamol the increase in FEV1 relative to the pre-methacholine baseline was 15.8%, 7.3%, and 5.5% for placebo, formoterol and salmeterol, respectively (equivalent to increases of 26%, 14%, and 12%, respectively, from the lowest FEV1 after methacholine). At 30 minutes significant differences remained, but 1 hour after completing the methacholine challenge FEV1 had returned to baseline values in all three treatment groups. CONCLUSION: Formoterol has a greater intrinsic activity than salmeterol as a bronchoprotective agent, indicating that salmeterol is a partial agonist compared with formoterol in contracted human airways in vivo. Irrespective of this, prior long term treatment with both long acting beta2 agonists reduced the bronchodilating effect of an additional single dose of salbutamol equally, indicating that the development of tolerance or high receptor occupancy overshadowed any possible partial antagonistic activity of salmeterol. Patients on regular treatment with long acting beta2 agonists should be made aware that an additional single dose of a short acting beta2 agonist may become less effective.

A 49-year-old woman with well-differentiated fetal adenocarcinoma.

A 49-year-old woman underwent a pneumonectomy because of a mass in the middle lobe. Histological examination of the tumour showed a well-differentiated fetal adenocarcinoma (WDFA). This rare tumour appears to be associated with an excellent prognosis in the absence of metastases following surgical resection.

Bronchial angiogenesis in severe glucocorticoid-dependent asthma.

To examine the role of the bronchial microvasculature and adhesion molecule expression in severe asthma, the authors have performed an immunohistochemical study on bronchial biopsies comparing 15 glucocorticoid-dependent asthmatics, 15 mild asthmatics and eight control subjects. Serially cut glycol methacrylate-embedded sections were stained with monoclonal antibodies identifying the vessel marker EN-4, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, E- and P-selectin. Sections were also stained for lymphocyte function associated antigen (LFA)-1 and very late antigen (VLA)-4. By comparison with mild asthma and nonasthma, severe asthma was characterized by increased numbers of submucosal vessels (p=0.009) which was associated with increased numbers of vessels expressing ICAM-1 (p=0.005). A highly significant correlation was found between the total number of EN-4+ vessels and the vessels expressing ICAM-1 (r=0.85, p=0.01). In contrast, E-selectin expression was lower in severe as compared with mild asthma (p=0.01) but not different from normal. No differences were found between the three groups in the expression of VCAM-1 and P-selectin nor in numbers of LFA-1+ and VLA-4+ cells. The results of this study support the notion that mucosal neovascularization is an important feature of airways remodelling in severe asthma. This is associated with a relatively higher density of vessels expressing intercellular adhesion molecule-1, although the expression of this adhesion molecule per vessel was not raised.

Low-dose methotrexate treatment in severe glucocorticoid-dependent asthma: effect on mucosal inflammation and in vitro sensitivity to glucocorticoids of mitogen-induced T-cell proliferation.

The authors have investigated whether the steroid-sparing effect of methotrexate (MTX) in severe orally glucocorticoid-insensitive asthmatics may be accounted for by the ability of this drug to increase the T-cell responsiveness sensitivity to dexamethasone in vitro. In addition the authors have investigated whether low-dose MTX treatment is associated with anti-inflammatory effects in peripheral blood and the bronchial mucosa. In eight patients with severe atopic asthma, using > or =15 mg x day(-1) prednisolone, the inhibitory effect of dexamethasone on mitogen stimulated peripheral blood mononuclear cells (PBMC) in vitro was tested before and after 8 weeks of uncontrolled treatment with MTX. Endobronchial biopsies were taken before and after MTX therapy in seven subsequent patients, and analysed using immunohistochemistry. In eight patients, serum was drawn for measuring levels of free interleukin (IL)-8. The in vitro sensitivity of PBMC to dexamethasone (at 1.6 x 10(-9) and 3.2 x 10(-10) mol x L(-1)) was significantly lower in the asthmatics before treatment when compared with the control subjects (p=0.03 and =0.001) but increased significantly after MTX treatment (p=0.04 and =0.02) to normal responsiveness. This was not associated with a decrease in peripheral blood T-cell numbers or activation. Except for a significant increase in the numbers of CD3+ (p=0.04), no significant numerical changes in activated T-cells, eosinophils, or mast cells were found (p>0.05). However, MTX treatment was associated with a significant fall in serum levels of free IL-8 (p=0.03). It is hypothesized that the steroid-sparing effect of methotrexate originates from increased sensitivity of lymphocytes to the inhibitory effects of glucocorticoids. The absence of an inhibitory effect on inflammatory cells in blood and mucosa suggests that this effect is achieved by modulating cell function rather than cell number.

Mucosal inflammation in severe glucocorticoid-dependent asthma.

To improve our understanding of the inflammatory mechanisms underlying severe disease, a biopsy study was performed comparing 15 clinically unstable glucocorticoid-dependent asthmatics, 10 mild asthmatics, and 10 control subjects. Compared with mild asthma, severe asthma was characterized by reduced mucosal eosinophilia. Whilst no significant differences were found in the numbers of mast cells, neutrophils, CD3+ and CD4+ T-cells between the three groups, up to a 4-fold increase In the numbers of activated T-lymphocytes bearing the interleukin (IL)-2 receptor (IL-2R) was found in the mucosa in severe asthma compared to mild asthma (p = 0.03) and control subjects (p = 0.003). Compared to control subjects, the mucosa of severe asthmatics contained significantly (p = 0.02) higher numbers of IL-5+ cells, with no differences between mild and severe disease. In contrast, staining for the anti-IL-4 monoclonal antibody 3H4 revealed that biopsies from mild asthmatics contained more IL4+ cells than biopsies from severe asthmatics and control subjects (p = 0.0008). In the severe asthmatics, a close correlation (r(s) = 0.76, p = 0.005) was found between the numbers of IL-2R-bearing cells and the variability in peak expiratory flow. In conclusion, persistent T-cell activation is a prominent feature of severe asthma. These results also indicate that interleukin-5, and not interleukin-4, is upregulated in severe disease.

Comparison of formoterol, salbutamol and salmeterol in methacholine-induced severe bronchoconstriction.

The onset of the bronchodilating effect of formoterol (12 microg by Turbuhaler) was compared with that of salbutamol (50 microg by Turbuhaler), salmeterol (50 microg by Diskhaler) and placebo in methacholine-induced severe bronchoconstriction. Seventeen subjects with mild-to-moderate asthma completed this randomized, double blind, cross-over, double-dummy study. On four study days, baseline forced expiratory volume in one second (FEV1) was recorded and the subjects were challenged with methacholine until FEV1 fell by at least 30%. Immediately thereafter, the study drugs were inhaled and lung function was assessed for 60 min. The geometric mean time for FEV1 to return to 85% of baseline was 7.2 min with formoterol, 6.5 min with salbutamol, 14.1 min with salmeterol and 34.7 min with placebo (p=0.0001, overall ANOVA). The difference between formoterol and salmeterol was statistically significant (p=0.01); there was no difference between formoterol and salbutamol (p=0.69). In conclusion, formoterol reversed methacholine-induced severe bronchoconstriction as rapidly as salbutamol and more rapidly than salmeterol. Classifying beta2-agonists as "fast"- and "slow"- acting may be supplemental to "short"- and "long"-acting.