Fasciclin 2 engages EGFR in an auto-stimulatory loop to promote imaginal disc cell proliferation in Drosophila.

How cell to cell interactions control local tissue growth to attain a species-specific organ size is a central question in developmental biology. The Drosophila Neural Cell Adhesion Molecule, Fasciclin 2, is expressed during the development of neural and epithelial organs. Fasciclin 2 is a homophilic-interaction protein that shows moderate levels of expression in the proliferating epithelia and high levels in the differentiating non-proliferative cells of imaginal discs. Genetic interactions and mosaic analyses reveal a cell autonomous requirement of Fasciclin 2 to promote cell proliferation in imaginal discs. This function is mediated by the EGFR, and indirectly involves the JNK and Hippo signaling pathways. We further show that Fasciclin 2 physically interacts with EGFR and that, in turn, EGFR activity promotes the cell autonomous expression of Fasciclin 2 during imaginal disc growth. We propose that this auto-stimulatory loop between EGFR and Fasciclin 2 is at the core of a cell to cell interaction mechanism that controls the amount of intercalary growth in imaginal discs.

The utility of multiparametric flow cytometry in the detection of primary effusion lymphoma (PEL).

Primary effusion lymphoma (PEL) is a rare B cell lymphoproliferative disorder that arises predominantly in body cavities causing malignant effusions. The incidence of PEL is very low, accounting for approximately 4% of all HIV-associated non-Hodgkin lymphomas. PEL has also been described in elderly patients and after solid-organ transplantation. It is associated in all cases with human herpes virus 8 (HHV8). We describe a case of PEL in a 88-year-old HIV-negative woman who presented with progressive dyspnea and moderate right-sided pleural effusion without significant lymphadenopathies or other effusions. The cytological study of the pleural fluid revealed a dense proliferation of large plasmablastic cells. A six-color multiparametric flow cytometry immunophenotyping study was carried out, and revealed 45% of large in size and high cellular complexity cells positive for CD45 (dim), CD38, CD138, CD30 and HLA-DR; and negative for CD19, CD20, cytoplasmatic CD79a, surface and cytoplasmic light chains Kappa and Lambda, CD3, CD4, CD5, CD7, CD8, CD28, CD56, CD81, and CD117. In situ hybridization for EBV-encoded smalI RNA was negative and immunohistochemistry for Kaposi sarcoma herpesvirus (HHV8) confirmed the diagnosis of PEL. Our results confirm that flow cytometry bring useful data in the diagnosis of large-cell lymphomas involving body cavities. © 2018 International Clinical Cytometry Society.

Interobserver variance in myelodysplastic syndromes with less than 5 % bone marrow blasts: unilineage vs. multilineage dysplasia and reproducibility of the threshold of 2 % blasts.

Previous studies have shown the reproducibility of the 2008 World Health Organization (WHO) classification in myelodysplastic syndromes (MDS), especially when multilineage dysplasia or excess of blasts are present. However, there are few data regarding the reproducibility of MDS with unilineage dysplasia. The revised International Prognostic Scoring System R-IPSS described two new morphological categories, distinguishing bone marrow (BM) blast cell count between 0-2 % and >2- < 5 %. This distinction is critical for establishing prognosis, but the reproducibility of this threshold is still not demonstrated. The objectives of our study were to explore the reliability of the 2008 WHO classification, regarding unilineage vs. multilineage dysplasia, by reviewing 110 cases previously diagnosed with MDS, and to study whether the threshold of ≤2 % BM blasts is reproducible among different observers. We used the same methodology as in our previous paper [Font et al. (2013) Ann Hematol 92:19-24], by encouraging investigators to include patients with <5 % BM blasts. Samples were collected from 11 hospitals and were evaluated by 11 morphologists. Each observer evaluated 20 samples, and each sample was analyzed independently by two morphologists. Discordance was observed in 36/108 suitable cases (33 %, kappa test 0.503). Diagnosis of MDS with unilineage dysplasia (refractory cytopenia with unilineage dysplasia (RCUD), refractory anemia with ring sideroblasts (RARS) or unclassifiable MDS) was assessed in 33 patients, by either of the two observers. We combined this series with the cases with RCUD or RARS included in our 2013 paper, thus obtaining 50 cases with unilineage dysplasia by at least one of the observers. The whole series showed very low agreement regarding RCUD (5/23, 21 %) and RARS (5/28, 18 %). Regarding BM blast count, the threshold of ≤2 % was not reproducible (discordance rate 32/108 cases, kappa test 0.277). Our study shows that among MDS WHO 2008 categories, interobserver discordance seems to be high in cases with unilineage dysplasia. We also illustrate that the threshold of ≤2 % BM blasts as settled by the R-IPSS may be not easy to reproduce by morphologists in real practice.

L1CAM binds ErbB receptors through Ig-like domains coupling cell adhesion and neuregulin signalling.

During nervous system development different cell-to-cell communication mechanisms operate in parallel guiding migrating neurons and growing axons to generate complex arrays of neural circuits. How such a system works in coordination is not well understood. Cross-regulatory interactions between different signalling pathways and redundancy between them can increase precision and fidelity of guidance systems. Immunoglobulin superfamily proteins of the NCAM and L1 families couple specific substrate recognition and cell adhesion with the activation of receptor tyrosine kinases. Thus it has been shown that L1CAM-mediated cell adhesion promotes the activation of the EGFR (erbB1) from Drosophila to humans. Here we explore the specificity of the molecular interaction between L1CAM and the erbB receptor family. We show that L1CAM binds physically erbB receptors in both heterologous systems and the mammalian developing brain. Different Ig-like domains located in the extracellular part of L1CAM can support this interaction. Interestingly, binding of L1CAM to erbB enhances its response to neuregulins. During development this may synergize with the activation of erbB receptors through L1CAM homophilic interactions, conferring diffusible neuregulins specificity for cells or axons that interact with the substrate through L1CAM.

Pathogenic human L1-CAM mutations reduce the adhesion-dependent activation of EGFR.

L1-cell adhesion molecule (L1-CAM) belongs to a functionally conserved group of neural cell adhesion molecules that are implicated in many aspects of nervous system development. In many neuronal cells the adhesive function of L1-type CAMs induces cellular signaling processes that involves the activation of neuronal tyrosine protein kinases and among other functions regulates axonal growth and guidance. Mutations in the human L1-CAM gene are responsible for a complex neurodevelopmental condition, generally referred to as L1 syndrome. Several pathogenic L1-CAM mutations have been identified in humans that cause L1 syndrome in affected individuals without affecting the level of L1-CAM-mediated homophilic cell adhesion when tested in vitro. In this study, an analysis of two different pathogenic human L1-CAM molecules indicates that although both induce normal L1-CAM-mediated cell aggregation, they are defective in stimulating human epidermal growth factor receptor tyrosine kinase activity in vitro and are unable to rescue L1 loss-of-function conditions in a Drosophila transgenic model in vivo. These results indicate that the L1 syndrome-associated phenotype might involve the disruption of L1-CAM's functions at different levels. Either by reducing or abolishing L1-CAM protein expression, by interfering with L1-CAM's cell surface expression, by reducing L1-CAM's adhesive ability or by impeding further downstream adhesion-dependent signaling processes.

Validity test study of JAK2 V617F and allele burden quantification in the diagnosis of myeloproliferative diseases.

Several sensitive methods for the detection of JAK2 V617F mutation have been published recently, most of them based on Real Time polymerase chain reaction (PCR). However, only some of them have performed studies of diagnostic validity. This study compares three methods based on Real Time PCR to detect JAK2 V617F mutation: two based on hybridization probes (HP) and peptide nucleic acid probe (PNA) and a third employing allele specific oligonucleotide primers for JAK2 V617F quantification. One hundred forty-nine healthy subjects, 61 essential thrombocythemia (ET), 32 polycythemia vera (PV), 38 secondary thrombocytoses, and 35 secondary erythrocytoses were included. Validity test study for JAK2 617 HP PCR in PV Sensitivity (Se) was 88% and in Specificity (Sp), 100%. In ET, Se was 57% and Sp, 100%. For JAK2 617 PNA PCR in PV, Se was 94% and Sp, 97.8%. In ET, Se was 70% and Sp, 95.7%. In JAK2 V671F allelo-specific-oligonucleotide (ASO) quantitative PCR (qPCR), cutoff point of 1% was established by receiving operating characteristic (ROC) curves. In PV, Se was 93.8% and Sp, 98.5%. In ET, Se was 80% and Sp, 95.9%. Two percent of the healthy subjects were positive by JAK2 617 PNA PCR and 2% by JAK2 617 ASO qPCR. JAK2 V617F mutation was detected in healthy subjects by cloning and sequencing. JAK2 617 HP is an adequate test in differential diagnosis for both erythrocytosis and thrombocytosis. When JAK2 V617F allele burden is low, JAK2 617 ASO qPCR should be performed. Simultaneous determination of JAK2 V617F and PRV-1 overexpression does not improve the diagnostic value of JAK2 V617F tests in MPD.

Activation of EGF receptor kinase by L1-mediated homophilic cell interactions.

Neural cell adhesion molecules (CAMs) are important players during neurogenesis and neurite outgrowth as well as axonal fasciculation and pathfinding. Some of these developmental processes entail the activation of cellular signaling cascades. Pharmacological and genetic evidence indicates that the neurite outgrowth-promoting activity of L1-type CAMs is at least in part mediated by the stimulation of neuronal receptor tyrosine kinases (RTKs), especially FGF and EGF receptors. It has long been suspected that neural CAMs might physically interact with RTKs, but their activation by specific cell adhesion events has not been directly demonstrated. Here we report that gain-of-function conditions of the Drosophila L1-type CAM Neuroglian result in profound sensory axon pathfinding defects in the developing Drosophila wing. This phenotype can be suppressed by decreasing the normal gene dosage of the Drosophila EGF receptor gene. Furthermore, in Drosophila S2 cells, cell adhesion mediated by human L1-CAM results in the specific activation of human EGF tyrosine kinase at cell contact sites and EGF receptors engage in a physical interaction with L1-CAM molecules. Thus L1-type CAMs are able to promote the adhesion-dependent activation of EGF receptor signaling in vitro and in vivo.

[Cytogenetic risk categories in acute myeloid leukemia: a comparison between MRC (Medical Research Council) and SWOG (Southwest Oncology Group) models)]. / Grupos de riesgo citogenético en la leucemia mieloide aguda: comparación de los modelos adoptados por los grupos MRC (Medical Research Council, del Reino Unido) y SWOG (Southwest Oncology Group, de EE.UU.).

BACKGROUND AND OBJECTIVE: Classification of acute myeloid leukemia (AML) based on karyotype provides an important tool for therapy selection. There are two standardized criteria for the classification of patients into groups of cytogenetic risk. One of them was established by the UK Medical Research Council (MRC) and the other by the US Southwest Oncology Group (SWOG). They define three and four cytogenetic categories, respectively. The aim of this study was to define the frequency of chromosomal abnormalities and to compare the groups of cytogenetic risk in patients with AML who received intensive chemotherapy, as a guide for future investigations. PATIENTS AND METHOD: Chromosomal analysis was performed using standard techniques on bone marrow samples from 146 adult patients between January 1995 and December 2001. Kaplan-Meier and Cox's regression models were used for statistical analysis. RESULTS: Cytogenetic results were obtained in 142 patients. The incidence of a complex karyotype and del(5q) was higher in patients with secondary AML. Classification by cytogenetic risk was performed in 105 treated patients. The classification using both models was identical in 82 patients and different in 23. Results in univariate analysis were significant for EFS (p < 0.000 for MRC and p < 0.02 for SWOG). Nevertheless, only the MRC model was significant for OS (p < 0.001). In multivariate analysis, age and cytogenetics were the only variables having prognostic value. CONCLUSIONS: There was some relation between secondary AML, advanced age and adverse karyotype. Both classification models have a great prognostic value. In our experience, codification according to MRC criteria appears to be more effective to detect patients at high risk of relapse.

Tandem transplants with different high-dose regimens improve the complete remission rates in multiple myeloma. Results of a Grupo Español de Síndromes Linfoproliferativos/Trasplante Autólogo de Médula Osea phase II trial.

Between 1994 and 1999, 88 multiple myeloma (MM) patients were included in a phase II study to evaluate a tandem autologous stem cell transplantation (ASCT) programme. The first was conditioned with melphalan 200 mg/m2 (MEL200-ASCT1), and the second with cyclophosphamide, etoposide and BCNU (CBV-ASCT2). All patients were in response after MEL200-ASCT1. A control group of MM patients with response to a single ASCT was selected to compare outcomes. After MEL200-ASCT1, 26 patients (30%) achieved complete remission (CR). Of the remaining 48 evaluable patients, 16 (33%) achieved CR with CBV-ASCT2. The final CR rate was 48%. The 5-year survival (OS) was 55%[95% confidence interval (CI) 43-67%] while the event-free survival (EFS) was 28% (95% CI 15-39%). CR status after CBV-ASCT2 was the most important prognostic factor for OS and EFS (P = 0.00001), although no differences in outcomes were detected when the patients in CR after MEL200-ASCT1 were compared with those who obtained CR after CBV-ASCT2. Univariate and multivariate analyses showed improved OS and EFS for the tandem series as compared with the control series treated with a single MEL200-ASCT. However, in a stratified comparison by response, there were no prognostic differences between tandem patients and control patients treated with a single ASCT. In summary, our study suggests that the benefit of a second high-dose therapy course depends on its capacity to result in CR for MM patients who have not attained CR after ASCT1.