[Subgroup Analysis of the Non-interventional REASON Study: PFS and OS According to Age, Smoking History, Gender, and Histology in NSCLC Patients Treated with Gefitinib or Chemotherapy]. / Subgruppenanalyse aus der nicht-interventionellen Beobachtungsstudie REASON: PFS und OS gemäß Alter, Raucherstatus, Geschlecht und histologischem Subtyp unter Verwendung von Gefitinib bzw. chemotherapeutischer Behandlung bei NSCLC-Patienten.

PURPOSE: Assessment of several clinical factors on progression-free (PFS) and overall survival (OS) in NSCLC patients (pts.) (stage IV) with mutated epidermal growth factor receptor (EGFRm+) treated with gefitinib (gef) or with chemotherapy (CT) under real-world conditions. METHODS: 285 EGFRm+ pts. of the non-interventional REASON study treated with gef (n = 206) or CT (n = 79) as first-line therapy or with gef (n = 213) or CT (n = 61) in any line throughout the course of therapy were analyzed according to age, gender, smoking history and histology. RESULTS: Compared with CT, patients treated with gef showed prolongation of PFS and OS in all subgroups. PFS was significantly increased in women and non-smokers. OS was significantly increased in women, non-smokers, (ex)-smokers, patients with adenocarcinoma and elderly patients when treated with gef compared to CT. Female gender turned out to be an independent positive predictive factor for OS in patients treated with gef (HRmale: 1.74, p = 0.0009). CONCLUSION: A clinical benefit of gef was shown for all analyzed clinical subgroups of EGFRm+ pts. This was confirmed for the female gender in a multivariate analysis.

[Exon-dependent Subgroup-analysis of the Non-interventional REASON-Study: PFS and OS in EGFR-mutated NSCLC Patients Treated with Gefitinib or Chemotherapy]. / Exonabhängige Subgruppenanalyse der nicht-interventionellen REASON-Studie: PFS und OS beim EGFR-mutierten NSCLC mit Behandlung von Gefitinib versus Chemotherapie.

PURPOSE: To analyze the influence of the localization of mutations in the epidermal growth factor receptor (EGFR) gene on progression-free (PFS) and overall survival (OS) in patients (pts) with locally advanced or metastatic non-small cell lung cancer (NSCLC) treated with gefitinib (gef) or chemotherapy (CT) under real world conditions within the REASON study. METHODS: Subgroups of pts with mutations in exon 19 (n = 141), 18/20 (n = 43), and 21 (n = 104) were analyzed for PFS and OS according to gef or CT treatment and compared using the log-rank test. RESULTS: Pts with mutations in exon 19 and 18/20 treated with gef as first line therapy showed increased PFS and OS compared to CT. This increase was statistically significant in pts with exon 19 mutation (11.3 vs. 6.5 months), but was not found in pts with exon 21 mutation (9.1 vs. 9.3 months). Also, OS was significantly increased in patients with mutation in exon 19 treated with gef ever over all treatment lines compared to CT (21.8 vs. 10.6 months), whereas this was not found in pts with mutation in exon 21 (14.1 vs. 13.9 months). CONCLUSION: Localization and nature of EGFR mutations influences gefitinib treatment outcomes under routine conditions and should therefore be analyzed in detail.

VEGF-A, VEGFR-1, VEGFR-2 and Tie2 levels in plasma of premature infants: relationship to retinopathy of prematurity.

AIM: To study prospectively the plasma levels of vascular endothelial growth factor (VEGF-A), its soluble receptors sVEGFR-1, sVEGFR-2 and soluble Tie2 in premature infants. To identify their changes related to the onset of retinopathy of prematurity (ROP). METHODS: Blood samples of 63 preterm infants born at a postmenstrual age (PMA) of 23-32 weeks were obtained between 5 days and 15 weeks after birth. 42 infants had no ROP, two had stage 1, nine stage 2 and 10 stage 3. Of these, four infants were treated with retinal photocoagulation. VEGF-A, sVEGFR-1, sVEGFR-2, and sTie2 were measured in the plasma with a sandwich enzyme immunoassay using factor-specific monoclonal mouse antibodies. The time course of concentrations plotted by kernel smoothing in infants with and without ROP were compared and a paired subgroup with analysis of variance was analysed. RESULTS: ROP patients had raised plasma levels of sVEGFR-2 and sTie2 compared with premature infants without ROP. VEGF-A and sVEGFR-1 levels were similar in both groups. Analysis of a subgroup with pairs of measurements, one before 32 weeks and one after 36 weeks, showed a significant increase in sTie2 after 36 weeks of PMA independent of ROP (p = 0.03). CONCLUSION: This is the first study to measure plasma levels of angiogenic factors in ROP. Similar VEGF-A plasma levels in infants with and without ROP suggest that pathogenic retinal angiogenesis in ROP is mainly driven by local VEGF-A synthesis. Elevated plasma levels in active ROP were observed for sVEGFR-2 and sTie2. These increases have yet to be confirmed as predictive values for ROP.

Soluble markers for the assessment of biological activity with PTK787/ZK 222584 (PTK/ZK), a vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor in patients with advanced colorectal cancer from two phase I trials.

BACKGROUND: Plasma and serum biomarkers of angiogenesis and activated endothelial cells were evaluated to assess biological activity of PTK787/ZK 222584 (PTK/ZK), a novel oral angiogenesis inhibitor targeting all known vascular endothelial growth factor (VEGF) receptor tyrosine kinases. PATIENTS AND METHODS: Patients with colorectal cancer (CRC) (n=63) were enrolled into two phase I/II dose escalation trials of PTK/ZK in 28-day cycles until discontinuation. Patients with stable disease for > or =2 months were categorized as 'non-progressors'. Plasma markers of angiogenesis, VEGF-A and basic fibroblast growth factor (bFGF), and the serum markers of activated endothelial cells, sTIE-2 and sE-Selectin, were assessed at baseline, and pre-dose on days 1, 8, 15, 22 and 28 of every cycle, with additional assessments 10 h post-dose on days 1 and 15. The percentage change from baseline was subsequently correlated with AUC and C(max) of PTK/ZK on day 1, cycle 1 and clinical outcome. RESULTS: A dose-dependent increase in plasma VEGF-A and bFGF was observed in the first cycle of PTK/ZK treatment. The correlation of change in plasma VEGF-A with AUC and C(max) was characterized by an E(max) model, suggesting that a change of > or =150% from baseline VEGF-A correlated with non-progressive disease. Change from baseline plasma VEGF-A within the first cycle of treatment was significantly correlated with clinical outcome by logistic regression analysis (P=0.027). CONCLUSIONS: In patients with CRC treated with PTK/ZK, changes in plasma VEGF-A and bFGF demonstrate biological activity of PTK/ZK, may help to establish optimal dose and correlate with outcome.

Nerve growth factor induces process formation in meningeal cells: implications for scar formation in the injured CNS.

Nerve growth factor (NGF) induces the differentiation and supports the survival of subpopulations of neurons in the PNS and CNS. Here we report that meningeal cells in the pia mater express immunoreactivity and mRNA for both known NGF receptors, the low-affinity receptor p75 and the tyrosine kinase receptor trkA. NGF induces rapid tyrosine phosphorylation of trkA in meningeal cells in vitro. NGF does not stimulate proliferation of primary meningeal cells but induces process outgrowth. p75- and trkA-immunoreactive meningeal cells with long processes, resembling NGF-treated cells in vitro, are abundant in the scar tissue that forms at spinal cord lesions in rat and cat. These data suggest that NGF, which is expressed at increased levels in the brain and spinal cord after lesions, may be involved in scar formation in the injured CNS.

Neurotrophins and cerebellar development.

Neurotrophins are structurally related molecules which regulate the survival and differentiation of various populations of neurons during development. In the cerebellum, the neurotophins and their Trk receptors are expressed at a relatively high level, suggesting an important function for these factors during development. There is also a tight age-dependent and spatial regulation of the molecules in the various cerebellar neurons. Previous studies have shown that BDNF and NT-3 have distinct biological effects on survival and differentiation of cerebellar granule neurons and Purkinje cells. Aside from acting as survival and differentiation factors, the neurotrophins could also have more subtle effects on neuronal function. It is also becoming increasingly evident, not the least from studies in neurotrophin deficient and in cerebellar mutant mice, that the neurotrophins act in concert with other factors and molecules in controlling neuronal development. We will here review some of the recent developments in the neurotrophin field with regard to cerebellum and also discuss what is known about the signaling event following stimulation of cerebellar neurons with BDNF and NT-3. The characterization of specific maturation stages and of genes which are involved and regulated by neurotrophins in developing cerebellum will help us to understand the processes of neuronal survival and differentiation in general.

Cell density increases Bcl-2 and Bcl-x expression in addition to survival of cultured cerebellar granule neurons.

The proto-oncogene bcl-2 and its family members, bcl-x and bax are recognized as major regulators of cell death and survival. Although Bcl-2 and Bcl-x are expressed in brain, little is known how they are regulated in neurons. Here we have studied the expression of bcl-2, bcl-xL and bax mRNA in rat cerebellar granule neurons cultured under conditions which influence neuron survival. Insulin-like growth factor-1 and brain-derived neurotrophic factor supported the survival of these neurons, but affected neither the expression of bcl-2, bcl-xL nor bax mRNA. In contrast, bcl-2 and bcl-xL mRNAs were up-regulated in cerebellar granule neurons plated at high density exhibiting an increased neuronal survival. Western blots showed that cell density also increased Bcl-2 protein level. However, conditioned medium from dense cultures did not affect the level of bcl-2 mRNA nor survival of the neurons. This suggests that cell density promotes survival and regulates Bcl-2 expression in cerebellar granule neurons through a signaling pathway different from known neurotrophic factors.

Constitutive phosphorylation of TrkC receptors in cultured cerebellar granule neurons might be responsible for the inability of NT-3 to increase neuronal survival and to activate p21 Ras.

The neurotrophins brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are both expressed in developing cerebellum in addition to their tyrosine kinase receptors. TrkB and TrkC. In contrast to BDNF.NT-3 has only a negligible or a transient survival activity on cultured cerebellar granule neurons. The granule neurons however, express both TrkC and Trk B receptors which suggests a basic difference in signaling between BDNF and NT-3 in these neurons. Here we have studied whether this difference can be attributed to the presence of alternative TrkC receptor variants on the granule neurons and which signaling pathway is specifically activated by BDNF but not by NT-3 in these neurons. Using RT-PCR it was shown that the cerebellar granule neurons express the full length TrkC receptor, in addition to variant receptors containing small inserts in the receptor tyrosine kinase domain. There was no dramatic change in the relative amounts of different TrkC receptors during development. However, we found the TrkC receptor constitutively phosphorylated even in the absence of added ligand suggesting an interaction of TrkC with endogenously produced NT-3. In addition, NT-3 was able to phosphorylate the BDNF receptor, TrkB but only at higher concentration (50 ng/ml). There were also distinct differences in the activation of intracellular molecules by BDNF and NT-3. Thus, p21 Ras and PLC gamma were activated by BDNF but not by NT-3 whereas both BDNF and NT-3 increased calcium and c-fos mRNA in the granule neurons. These results show that differential activation of specific intracellular pathways such as that of p21 Ras determines the specific effects of BDNF and NT-3 on granule neuron survival. In addition, since calcium is increased by NT-3 in the cerebellar granule neurons, this neurotrophin might have some unknown important effects on these neurons.

Characterization of TrkB receptor-mediated signaling pathways in rat cerebellar granule neurons: involvement of protein kinase C in neuronal survival.

TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Here, we report that both truncated and full-length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT-4/5 increased the survival of cultured cerebellar granule neurons. BDNF and NT-4/5 also induced an autophosphorylation of TrkB receptors and subsequently resulted in a phosphorylation and binding of phospholipase C-gamma (PLC-gamma) and SH2-containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC-gamma, BDNF increased the phosphatidylinositol (PI) turnover and elevated intracellular calcium levels. To investigate the involvement of protein kinase C (PKC) in the survival of granular neurons, we show here activation of PKC after BDNF or TPA treatment and blocking of the observed survival-promoting effects of BDNF and TPA with calphostin C, a specific PKC inhibitor. In addition, BDNF activated c-ras in a concentration-dependent manner. These results suggest that two different pathways, the c-ras and the PLC-gamma pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF.

Differential regulation of p21ras activation in neurons by nerve growth factor and brain-derived neurotrophic factor.

Neurotrophins activate the Trk tyrosine kinase receptors, which subsequently initiate signaling pathways that have yet to be fully resolved, resulting in neuronal survival and differentiation. The ability of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) to activate GTP binding to p21ras was investigated using cultured embryonic chick neurons. In both sympathetic and sensory neurons, the addition of NGF markedly increased the formation of Ras-GTP. The magnitude of the effect was found to depend upon the developmental stage, peaking at embryonic day 11 in sympathetic neurons and at embryonic day 9 in sensory neurons, times when large numbers of neurons depend on NGF for survival. Surprisingly, following the addition of BDNF, no formation of Ras-GTP could be observed in neurons cultured with BDNF. When sensory neurons were cultured with NGF alone, both NGF and BDNF stimulated GTP binding to Ras. In rat cerebellar granule cells, while the acute exposure of these cells to BDNF resulted in the formation Ras-GTP, no response was observed following previous exposure of the cells to BDNF, as was observed with sensory neurons. However, this desensitization was not observed in a transformed cell line expressing TrkB. In neurons, the mechanism underlying the loss of the BDNF response appeared to involve a dramatic loss of binding to cell-surface receptors, as determined by cross-linking with radiolabeled BDNF. Receptor degradation could not account for the desensitization since cell lysates from neurons pretreated with BDNF revealed that the levels of TrkB were comparable to those in untreated cells. These results indicate that in neurons, the pathways activated by NGF and BDNF are differentially regulated and that prolonged exposure to BDNF results in the inability of TrkB to bind its ligand.