Composition of receptor tyrosine kinase-mediated lipid micro-domains controlled by adaptor protein interaction.

Receptor tyrosine kinases (RTKs) are highly regulated, single pass transmembrane proteins, fundamental to cellular function and survival. Aberrancies in regulation lead to corruption of signal transduction and a range of pathological outcomes. Although control mechanisms associated with the receptors and their ligands are well understood, little is known with respect to the impact of lipid/lipid and lipid/protein interactions in the proximal plasma membrane environment. Given that the transmembrane regions of RTKs change in response to extracellular ligand binding, the lipid interactions have important consequences in influencing signal transduction. Fibroblast growth factor receptor 2 (FGFR2) is a highly regulated RTK, including under basal conditions. Binding of the adaptor protein, growth factor receptor-bound protein 2 (GRB2) to FGFR2 prevents full activation and recruitment of downstream signalling effector proteins in the absence of extracellular stimulation. Here we demonstrate that the FGFR2-GRB2 complex is sustained in a defined lipid environment. Dissociation of GRB2 from this complex due to ligand binding, or reduced GRB2 expression, facilitates the dispersion of FGFR2 into detergent-resistant membrane (DRM) micro-domains. This modification of the plasma membrane proximal to FGFR2 provides a further regulatory checkpoint which controls receptor degradation, recycling and recruitment of intracellular signalling proteins.

Drug Resistance in Glioma Cells Induced by a Mesenchymal-Amoeboid Migratory Switch.

Cancer cell invasion is a precondition for tumour metastasis and represents one of the most devastating characteristics of cancer. The development of drugs targeting cell migration, known as migrastatics, may improve the treatment of highly invasive tumours such as glioblastoma (GBM). In this study, investigations into the role of the cell adhesion protein Cellular communication network factor 1 (CCN1, also known as CYR61) in GBM cell migration uncovered a drug resistance mechanism adopted by cells when treated with the small molecule inhibitor CCG-1423. This inhibitor binds to importin α/ß inhibiting the nuclear translocation of the transcriptional co-activator MKL1, thus preventing downstream effects including migration. Despite this reported role as an inhibitor of cell migration, we found that CCG-1423 treatment did not inhibit GBM cell migration. However, we could observe cells now migrating by mesenchymal-amoeboid transition (MAT). Furthermore, we present evidence that CCN1 plays a critical role in the progression of GBM with increased expression in higher-grade tumours and matched blood samples. These findings support a potential role for CCN1 as a biomarker for the monitoring and potentially early prediction of GBM recurrence, therefore as such could help to improve treatment of and increase survival rates of this devastating disease.

A novel workflow for three-dimensional analysis of tumour cell migration.

The limitations of two-dimensional analysis in three-dimensional (3D) cellular imaging impair the accuracy of research findings in biological studies. Here, we report a novel 3D approach to acquisition, analysis and interpretation of tumour spheroid images. Our research interest in mesenchymal-amoeboid transition led to the development of a workflow incorporating the generation and analysis of 3D data with instant structured illumination microscopy and a new ImageJ plugin.

A developmental study of enteric neuron migration in the grasshopper using immunological probes.

Motility of enteric plexus neurons in the grasshopper Locusta migratoria depends critically on the NO/cGMP signaling cascade. This is reflected in a strong NO-dependent cGMP staining in migrating enteric midgut neurons. In contrast, first cGMP immunoreactivity (cGMP-IR) in the foregut enteric ganglia was detected clearly after the main migratory processes have taken place. Thus, expression of cGMP-IR in migrating neurons is a distinct phenomenon restricted to neurons forming midgut and foregut plexus, that does not occur during convergence of neurons forming the enteric ganglia. Analysis of time lapse video microscopy of migrating midgut neurons together with an immunofluorescence study of midgut cellular structures suggests a contribution of the midgut musculature to enteric neuron guidance. Additionally, during midgut plexus formation a fasciculating signal for enteric neuron neurites appears to be provided by the cell adhesion molecule Fasciclin I.

Regulation of enteric neuron migration by the gaseous messenger molecules CO and NO.

The enteric nervous system (ENS) of insects is a useful model to study cell motility. Using small-molecule compounds to activate or inactivate biosynthetic enzymes, we demonstrate that the gaseous messenger molecules carbon monoxide (CO) and nitric oxide (NO) regulate neuron migration in the locust ENS. CO is produced by heme oxygenase (HO) enzymes and has the potential to signal via the sGC/cGMP pathway. While migrating on the midgut, the enteric neurons express immunoreactivity for HO. Here, we show that inhibition of HO by metalloporphyrins promotes enteric neuron migration in intact locust embryos. Thus, the blocking of enzyme activity results in a gain of function. The suppression of migratory behavior by activation of HO or application of a CO donor strongly implicates the release of CO as an inhibitory signal for neuron migration in vivo. Conversely, inhibition of nitric oxide synthase or application of the extracellular gaseous molecule scavenger hemoglobin reduces cell migration. The cellular distribution of NO and CO biosynthetic enzymes, together with the results of the chemical manipulations in whole embryo culture suggest CO as a modulator of transcellular NO signals during neuronal migration. Thus, we provide the first evidence that CO regulates embryonic nervous system development in a rather simple invertebrate model.

Presence of peripheral blasts in refractory anemia and refractory cytopenia with multilineage dysplasia predicts an unfavourable outcome.

The World Health Organization (WHO) assigns myelodysplastic syndrome (MDS) to RA/RCMD/RARS/RSCM/5q- syndrome, if medullary blasts are <5% and peripheral blast (PB) count < or =1%. In 1103 patients with these diagnoses, we analysed survival and risk of AML evolution depending on the presence of PB. Median survival in the group with 1% PB (n=74) was significantly lower as compared to those without PB (20 versus 47 months, p<0.00005). Cumulative risk of AML was significantly higher in patients showing PB (p<0.00005). Median survival of patients with PB was not different from that of RAEB I. We therefore propose to consider patients with PB, regardless of medullary blast, as RAEB I.

Time-dependent prognostic scoring system for predicting survival and leukemic evolution in myelodysplastic syndromes.

PURPOSE: The aims of this study were to identify the most significant prognostic factors in myelodysplastic syndromes (MDS) taking into account both their values at clinical onset and their changes in time and to develop a dynamic model for predicting survival and leukemic evolution that can be applied at any time during the course of the disease. PATIENTS AND METHODS: We studied a learning cohort of 426 MDS patients diagnosed at the Department of Hematology, San Matteo Hospital, Pavia, Italy, between 1992 and 2004, and a validation cohort of 739 patients diagnosed at the Heinrich-Heine-University Hospital, Düsseldorf, Germany, between 1982 and 2003. All patients were reclassified according to WHO criteria. Univariable and multivariable analyses were performed using Cox models with time-dependent covariates. RESULTS: The most important variables for the prognostic model were WHO subgroups, karyotype, and transfusion requirement. We defined a WHO classification-based prognostic scoring system (WPSS) that was able to classify patients into five risk groups showing different survivals (median survival from 12 to 103 months) and probabilities of leukemic evolution (P < .001). WPSS was shown to predict survival and leukemia progression at any time during follow-up (P < .001), and its prognostic value was confirmed in the validation cohort. CONCLUSION: WPSS is a dynamic prognostic scoring system that provides an accurate prediction of survival and risk of leukemic evolution in MDS patients at any time during the course of their disease. This time-dependent system seems particularly useful in lower risk patients and may be used for implementing risk-adapted treatment strategies.

Chronic myelomonocytic leukemia in the light of the WHO proposals.

The WHO classification moved CMML to myeloproliferative/myelodysplastic disorders, and defined CMML I and CMML II according to medullary and peripheral blast count. To confirm these proposals, we analyzed 266 patients with CMML I and 73 patients with CMML II. Median survival time was 20 months for CMML I, and 15 months for CMML II (p<0.005). The cumulative risk of AML evolution differed between patient groups (p=0,001). No conclusive differences in clinical, morphologic, hematologic or cytogenetic parameters were found. These data support the WHO proposals for the classifi-cation of CMML.

Intensive chemotherapy is not recommended for patients aged >60 years who have myelodysplastic syndromes or acute myeloid leukemia with high-risk karyotypes.

BACKGROUND: It is unclear whether intensive chemotherapy is beneficial to patients with high-risk myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) if they are aged >/=60 years. METHODS: The authors studied 160 patients with a median age of 67 years who received intensive chemotherapy for MDS or AML with cytosine arabinoside and an anthracycline. RESULTS: At diagnosis, cytogenetic analysis was available in 146 patients. Karyotype was normal in 78 patients and abnormal in 68 patients. Of the abnormal karyotypes, 32 belonged to the high-risk category, ie, they involved either >/=3 chromosomes or chromosome 7. Complete remission (CR) was achieved by 94 patients (56%). CR rates were 70% among the patients who had a normal karyotype, 69% among the patients who had an abnormal (noncomplex) karyotype, but only 46% among the patients ho had a high-risk karyotype. The median survival was 9.5 months in the entire group, 18 months in patients with normal karyotype, 6 months in patients with abnormal, and 4 months in patients with a high-risk karyotype. A poor prognosis was attributable to low rates of CR and a high risk of early recurrence. CONCLUSIONS: According to the current data, elderly patients with AML or advanced MDS do not benefit from intensive chemotherapy if they show karyotype anomalies, especially those in the high-risk category.

Arsenic in the cerebrospinal fluid of a patient receiving arsenic trioxide for relapsed acute promyelocytic leukemia with CNS involvement.

We report on a 42-year-old patient whose relapse of acute promyelocytic leukaemia (APL) included meningeal infiltration. Since he had previously experienced ATRA syndrome, he received arsenic trioxide (ATO) plus intrathecal therapy with cytarabine, prednisone, and methotrexate. We measured the concentration of arsenic in his cerebrospinal fluid (CSF). Arsenic showed a peak CSF concentration of 0.008 mg/l (0.11 micromol/l) and a nadir of 0.002 mg/l (0.027 micromol/l), both representing about 14% of blood levels. ATO thus crosses the blood-CSF-barrier when administered intravenously, but the concentration in CSF is probably not sufficient for treatment of meningeal leukemia.

Embryonic differentiation of serotonin-containing neurons in the enteric nervous system of the locust (Locusta migratoria).

The enteric nervous system (ENS) of the locust consists of four ganglia (frontal and hypocerebral ganglion, and the paired ingluvial ganglia) located on the foregut, and nerve plexus innervating fore- and midgut. One of the major neurotransmitters of the ENS, serotonin, is known to play a vital role in gut motility and feeding. We followed the anatomy of the serotonergic system throughout embryonic development. Serotonergic neurons are generated in the anterior neurogenic zones of the foregut and migrate rostrally along the developing recurrent nerve to contribute to the frontal ganglion. They grow descending neurites, which arborize in all enteric ganglia and both nerve plexus. On the midgut, the neurites closely follow the leading migrating midgut neurons. The onset of serotonin synthesis occurs around halfway through development-the time of the beginning of midgut closure. Cells developing to serotonergic phenotype express the serotonin uptake transporter (SERT) significantly earlier, beginning at 40% of development. The neurons begin SERT expression during migration along the recurrent nerve, indicating that they are committed to a serotonergic phenotype before reaching their final destination. After completion of the layout of the enteric ganglia (at 60%) a maturational phase follows, during which serotonin-immunoreactive cell bodies increase in size and the fine arborizations in the nerve plexus develop varicosities, putative sites of serotonin release (at 80%). This study provides the initial step for future investigation of potential morphoregulatory functions of serotonin during ENS development.

Prospective validation of the WHO proposals for the classification of myelodysplastic syndromes.

BACKGROUND AND OBJECTIVES: The aim of this study was a prospective validation of the World Health Organization (WHO) proposals for the classification of myelodysplastic syndromes (MDS) with respect to their prognostic relevance. DESIGN AND METHODS: We classified 1095 patients with MDS diagnosed at our institution between November 1999 and December 2004 according to French-American-British (FAB) and WHO criteria by central morphologic review. The study was not population-based, but included all newly diagnosed patients from different regions in Germany. Patients were followed for survival and disease evolution to acute myeloid leukemia (AML) through December 31th, 2005. RESULTS: According to the WHO classification, there were 89 cases of refractory anemia (RA), 293 of refractory cytopenias with multilineage dysplasia (RCMD), 31 RA with ringed sideroblasts (RARS), 139 RCMD with ringed sideroblasts (RCMD-RS), 142 RA with excess blasts (RAEB) I and 149 RAEB II and 52 patients with 5q- syndrome. The median survival of patients with RA or RARS was not reached, the median survival of patients with RCMD was 31 months, that of patients with RCMD-RS was 28 months, that of 5q- patients was 40 months, of RAEB I 27 months and of RAEB II 12 months. The cumulative risk of AML evolution 2 years after diagnosis was 0% in RA and RARS, 8% in 5q-, 9% in RCMD, 12% in RCMD-RS, 13% in RAEB I and 40% in RAEB II. The number of high-risk karyotypes was lower in patients with RA/RARS than in those with RCMD/RCMD-RS and RAEB I/RAEB II. Karyotype findings were major prognostic variables. INTERPRETATION AND CONCLUSIONS: The WHO classification is feasible and provides valuable prognostic information, even in a short-term prospective study. Together with cytogenetic data and other prognostic parameters, the WHO classification is very useful for clinical decision making.

Exisulind induces apoptosis in advanced myelodysplastic syndrome (MDS) and acute myeloid leukaemia/MDS.

The influence of Exisulind on the viability and apoptosis of CD34(+) stem cells from patients with advanced myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML)/MDS was investigated. In eight out of 10 patient samples Exisulind reduced the fraction of viable cells by inducing apoptosis. We found evidence that Exisulind-mediated apoptosis depends on c-Jun NH(2)-terminal kinase (JNK) activation. Addition of a specific JNK-inhibitor to Exisulind-treated advanced MDS and AML/MDS cells partly abrogated apoptosis. We propose that Exisulind is tested in clinical phase I/II trials for the treatment of advanced MDS and AML/MDS.

Secondary myelodysplastic syndrome following long-term treatment with azathioprine in patients with multiple sclerosis.

Azathioprine (Aza) is a widely used immunosuppressive drug in multiple sclerosis (MS) treatment. Recently, the incidence of secondary myelodysplastic syndromes (sMDS) associated with a poor prognosis was found to be elevated in patients treated with Aza for non-malign disorders. Three hundred and seventeen MS patients were retrospectively analysed and complete blood counts were examined for those exposed to Aza. We identified one case of sMDS (cumulative dose 627 g) in a young patient and two further malignancies (cumulative doses 27 g and 54 g) in the Aza group (n = 81; 3.7%). In the non-Aza (n = 236) group, five malignancies (2.1%, P = 0.419) were identified. Including our patient, four cases of sMDS after long-term Aza therapy in MS have been reported so far. Cases suggest a time- and dose-dependent risk of sMDS in long-term therapy of MS with Aza. Long-term Aza therapy needs careful monitoring.

The histone deacetylase (HDAC) inhibitor valproic acid as monotherapy or in combination with all-trans retinoic acid in patients with acute myeloid leukemia.

BACKGROUND: Valproic acid (VPA) inhibits histone deacetylase activity and, synergizing with all-trans retinoic acid (ATRA), achieves differentiation induction of myeloid blast cells in vitro. METHODS: We used VPA in 58 patients with acute myeloid leukemia (AML) who were too old and/or medically unfit to receive intensive chemotherapy (32 AML secondary to myelodysplastic syndrome [MDS], 22 de novo AML, 4 AML secondary to myeloproliferative syndrome). VPA serum concentrations were 50-100 mug/mL. Thirty-one patients received VPA monotherapy. ATRA was added later in 13 patients who did not respond or who relapsed. Another 27 patients received VPA plus ATRA from the start. Median treatment duration was 93 days for VPA and 88 days for ATRA. RESULTS: The response rate was only 5% according to International Working Group (IWG) criteria for AML but was 16% when IWG response criteria for MDS were used, which capture hematologic improvement and stabilization of the disease. These endpoints, which are not necessarily correlated with diminishing blast counts, are relevant for the patients' quality of life. Among 23 patients with a peripheral blast count > 5%, 6 (26%) showed a diminishing blast count, and 5 of these had a complete peripheral blast clearance. CONCLUSIONS: Future trials should combine VPA with chemotherapy or demethylating agents.

Results of a phase 2 study of valproic acid alone or in combination with all-trans retinoic acid in 75 patients with myelodysplastic syndrome and relapsed or refractory acute myeloid leukemia.

Valproic acid (VPA) inhibits histone deacetylase activity and induces differentiation of acute myeloid leukemia (AML) blasts in vitro. We observed clinical responses to VPA in patients with myelodysplastic syndrome (MDS) and AML. Here, we report follow-up data on 75 patients. Of these, 66 were started on VPA monotherapy, with later addition of all-trans retinoic acid (ATRA) in patients who did not respond or relapsed. Nine patients were treated with VPA + ATRA from the start. Median treatment duration was 4 months for VPA and 2 months for ATRA. Hematological improvement, according to international working group criteria for MDS, was observed in 18 patients (24%). Median response duration was 4 months. ATRA exerted no additional effect in patients receiving the combination from the start or benefited primary VPA nonresponders. However, of ten VPA responders who relapsed, four achieved a second response after addition of ATRA. Response rates were strongly dependent on disease type according to WHO classification. We found a response rate of 52% in MDS patients with a normal blast count (refractory sideroblastic anemia, refractory cytopenia with multilineage dysplasia, and refractory sideroblastic cytopenia with multilineage dysplasia). The response rate was 6% in refractory anemia with excess blasts (I + II), 16% in AML, and 0% in chronic myelomonocytic leukemia. Bone marrow blast count was the only variable that predicted responses. We conclude that VPA is clinically useful in low-risk MDS. For patients with high-risk MDS, VPA may be combined with chemotherapy or demethylating drugs. If patients relapse after an initial response to VPA, ATRA has the potential to induce a prolonged second response.

Difference in clinical features between Japanese and German patients with refractory anemia in myelodysplastic syndromes.

Several reports indicate that there might be differences in clinical features between Asian and Western myelodysplastic syndrome (MDS) cases. We analyzed refractory anemia (RA) in French-American-British (FAB) classification cases diagnosed in Japan and Germany to perform a more exact comparison between Asian and Western MDS types. In the first step, we analyzed agreement of morphologic diagnosis between Japanese and German hematologists. Blood and bone marrow slides of 129 patients diagnosed with FAB-RA, FAB-RA with ringed sideroblasts (RARS), or aplastic anemia were selected randomly and evaluated separately by each group. The agreements of diagnoses according to FAB and World Health Organization (WHO) classifications were 98.4% and 83.8%, respectively. Second, we compared clinical features between 131 Japanese and 597 German patients with FAB-RA. Japanese patients were significantly younger than German patients. Japanese patients had more severe cytopenias. However, prognosis of Japanese patients was significantly more favorable than that of German patients. Japanese patients had a significantly lower cumulative risk of acute leukemia evolution than did German patients. Frequency of WHO-RA in Japanese patients with FAB-RA was significantly higher than that in German patients. In conclusion, our results indicate that the clinical features of Japanese patients with FAB-RA differ from those of German patients.

Secondary myelodysplastic syndromes following treatment with azathioprine are associated with aberrations of chromosome 7.

We report 14 cases of secondary myelodysplastic syndromes (sMDS) following treatment with azathioprine for non-malignant disorders. Long-term treatment with azathioprine seems to be associated with an increased risk of MDS and subsequent leukemic transformation.

Ex vivo apoptosis, CD95 and CD28 expression in T cells of children with juvenile idiopathic arthritis.

We hypothesise that T-cell apoptosis and the percentage of T cells expressing molecules involved in apoptosis modulation (CD95, CD28) are altered at the inflammation site and in peripheral blood (PB) of children with juvenile idiopathic arthritis (JIA). Paired JIA samples of synovial fluid (SF) and PB ( n=7) and PB samples from age-matched normal children ( n=23) were analysed immediately ex vivo. Apoptosis was measured by detection of phophatidylserine (PS) externalisation on T cells. CD95 or CD28 was detected by FACS, and soluble CD95 and CD95 ligand levels were detected by enzyme-linked immunosorbent assay (ELISA). In SF, the mean percentage of apoptotic T cells was somewhat higher than in PB. However, the percentages of T cells expressing CD95 and soluble CD95 levels were markedly higher in SF (CD4 cells 96+/-2%, CD8 91+/-6%, soluble CD95 6,420+/-2,571 pg/ml) than in PB (CD4 32+/-10%, CD8 36+/-9%, soluble CD95 4,296+/-2,142 pg/ml). Peripheral blood T-cell apoptosis in JIA (CD4 20+/-8%, CD8 42+/-19%) was higher than in the control group (CD4 5+/-2%, CD8 9+/-6%). Interestingly, the percentage of PB CD4 cells expressing CD28 was lower in JIA than controls. In conclusion, systemic T-cell apoptosis was higher in JIA while a substantial number of SF T cells survived locally, despite the fact that almost all cells express CD95.