ALBERT

Description: p18INK4c is a cyclin-dependent kinase (CDK) inhibitor that interferes with the Rb-kinase activity of CDK6/CDK4. Disruption of p18INK4c in mice impairs B-cell terminal differentiation and confers increased susceptibility to tumor development; however, alterations of p18INK4c in human tumors have rarely been described. We used a tissue-microarray approach to analyze p18INK4c expression in 316 Hodgkin lymphomas (HLs). Nearly half of the HL cases showed absence of p18INK4c protein expression by Reed-Sternberg (RS) cells, in contrast with the regular expression of p18INK4c in normal germinal center cells. To investigate the cause of p18INK4c repression in RS cells, the methylation status of the p18INK4c promoter was analyzed by methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing. Hypermethylation of the p18INK4c promoter was detected in 2 of 4 HL-derived cell lines, but in none of 7 non-Hodgkin lymphoma (NHL)–derived cell lines. We also detected p18INK4c hypermethylation, associated with absence of protein expression, in 5 of 26 HL tumors. The correlation of p18INK4c immunostaining with the follow-up of the patients showed shorter overall survival in negative cases, independent of the International Prognostic Score. These findings suggest that p18INK4c may function as a tumor suppressor gene in HL, and its inactivation may contribute to the cell cycle deregulation and defective terminal differentiation characteristic of the RS cells.

Description: The stem cell factor c-kit signaling pathway (SCF/c-kit) has been previously implicated in normal hematopoiesis, melanogenesis, and gametogenesis through the formation and migration of c-kit+ cells. These biologic functions are also determinants in epithelial–mesenchymal transitions during embryonic development governed by the Snail family of transcription factors. Here we show that the activation of c-kit by SCF specifically induces the expression of Slug, a Snail family member. Slug mutant mice have a cell-intrinsic defect with pigment deficiency, gonadal defect, and impairment of hematopoiesis. Kit+ cells derived from Slug mutant mice exhibit migratory defects similar to those of c-kit+ cells derived from SCF and c-kit mutant mice. Endogenous Slug is expressed in migratory c-kit+ cells purified from control mice but is not present in c-kit+cells derived from SCF mutant mice or in bone marrow cells from W/Wv mice, though Slug is present in spleen c-kit+ cells of W/Wv (mutants expressing c-kit with reduced surface expression and activity). SCF-induced migration was affected in primary c-kit+ cells purified from Slug−/− mice, providing evidence for a role of Slug in the acquisition of c-kit+ cells with ability to migrate. Slug may thus be considered a molecular target that contributes to the biologic specificity to the SCF/c-kit signaling pathway, opening up new avenues for stem cell mobilization.

Description: Rho GTPases control many facets of cell polarity and migration; namely, the reorganization of the cellular cytoskeleton to extracellular stimuli. Rho GTPases are activated by GTP exchange factors (GEFs), which induce guanosine diphosphate (GDP) release and the stabilization of the nucleotide-free state. Thus, the role of GEFs in the regulation of the cellular response to extracellular cues during cell migration is a critical step of this process. In this report, we have analyzed the activation and subcellular localization of the hematopoietic GEF Vav in human peripheral blood lymphocytes stimulated with the chemokine stromal cell–derived factor-1 (SDF-1α). We show a robust activation of Vav and its redistribution to motility-associated subcellular structures, and we provide biochemical evidence of the recruitment of Vav to the membrane of SDF-1α–activated human lymphocytes, where it transiently interacts with the SDF-1α receptor CXCR4. Overexpression of a dominant negative form of Vav abolished lymphocyte polarization, actin polymerization, and migration. SDF-1α–mediated cell polarization and migration also were impaired by overexpression of an active, oncogenic Vav, although the mechanism appears to be different. Together, our data postulate a pivotal role for Vav in the transmission of the migratory signal through the chemokine receptor CXCR4.

Description: Mesenchymal stem cells (MSC) are multilineage non hematopoietic progenitor cells that play a key role in supporting the lymphohematopoietic system. Their distribution in bone marrow and secondary lymphoid organs allows an intimate interaction with T and B-lymphocytes, which contribute to the normal lymph node development, but this interaction, can not be considered as a simple bi-directional cross-talk and other cell subsets, such as dendritic cells (DC), must be considered. We have analysed the effect of MSC on B-lymphocytes and the pathways involved in these effects. For these propose, we cultured B-cell with or without MSC and analysed different markers involved in the differentiation of B-cells. We found that MSC inhibited proliferation, arresting B lymphocytes in G0G1 phase of cell cycle (figure 1). However, the presence of MSC increased the viability ob B-lymphocytes (double number of viable B-cells: from 13012 to 22835 Annexin-V PE/7 ADD negative events within B-lymphocytes in absence versus presence of MSC). The exposure of B-cells to plasmocytoid DC (pDC) induced B-lymphocytes differentiation increasing both the percentage of CD38++/CD138++ cells as well as the mean fluorescence intensity of both markers (figure 2). Accordingly, different B-cell subpopulations could be identified which represented a continuum in B-cell maturation. While the levels of cytoplasmic immunoglobulin (cIg) were higher among CD38++ cells, the opposite occurred for the expression of surface Ig as well as CD19 and CCR7. Interestingly, the presence of MSC blocked B-cell differentiation. Regarding the pathways involved in these effects, the presence of MSC influenced on ERK 1/2 and p38 pathways, but these effects depended on the culture conditions. Thus, MSC induced phosphorilation of ERK 1/2 MAPK and inhibited phosphorilation of p38 in B-cells cultured with Ig plus CpG (low proliferative conditions) while the contrary occurred in B-cells cultured with TPA (highly proliferative conditions). Therefore we demonstrated that MSC increased viability and blocked cell cycle of B-lymphocytes. Furthermore the plasmocitoid dendritic cells favoured B-lymphocytes differentiation and this process in inhibited in presence with MSC. These effects are at least in part mediated through the ERK 1/2 and p38 pahtways. Figure Figure

Description: Despite the major advances in the treatment of classical Hodgkin Lymphoma (cHL) patients, around 30% to 40% of cases in advanced stages may relapse or die as result of the disease, and current markers to predict prognosis are rather unreliable. The identification of molecular events and biological processes associated with treatment failure are essential to develop new predictive tools. We used gene expression data from 29 samples of advanced cHL patients and HL-derived cell lines in order to identify transcriptional patterns from both tumoral cells and cell microenvironment. Student t-test was used to detect genes differentially overexpressed in cell lines and in tumor samples, thus creating two databases that report for genes expressed by the tumor HRS cells and genes expressed by the microenvironment. Using Gene Set Enrichment analysis (GSEA) we identified specific gene sets enriched in both databases in patients with favorable and unfavorable outcome, respectively. To validate these pathways we designed a novel Taqman low-density array (LDA) to examine the expression of the most relevant genes in 60 formalin-fixed, paraffin embedded (FFPE) tissue samples, and correlated the results with treatment outcome. Functional pathways related to unfavorable outcome significantly enriched in the HRS cells included the regulation of the G2/M checkpoint of the cell cycle, S phase and G1/S transition, chaperons, histone modification and other signaling pathways with an important representation of the MAPK pathway. On the other hand, genes reporting for specific T-cell populations (T-cytotoxic and T-regulatory cells) and macrophage activation were found to be overexpressed in the microenvironment. The final model presents a balanced representation of these genes, including also genes encoding factors implicated in drug resistance (RRM2, TYMS and TOP2A). RNA extracted from FFPE sections yielded analyzable data for 80% of samples. LDA analysis of the genes included in the model confirmed the feasibility of this approach, and the capacity for identifying cases with increased risk of failure.LDA provides an effective technique for analyzing gene expression in FFPE tissues, and it can be used for clinical prediction in diagnostic samples, using a selection of genes identified after GSEA analysis of the initial molecular signatures. This novel Taqman LDA will be used to develop a new molecular predictor of the outcome of patients with advanced cHL.

Description: Heterotypic interaction among tumor cells (TCs) and endothelial cells (ECs) may play a critical role during the vascular dissemination of neoplastic cells and during pathologic angiogenesis in tumors. To identify molecules involved in these processes, the distribution of vascular junctional proteins was first studied by immunofluorescence at sites of heterologous intercellular contact using TC-EC mosaic monolayers grown on 2-dimensional collagen. Several members of the tetraspanin superfamily, including CD9, CD81, and CD151, were found to localize at the TC-EC contact area. The localization of tetraspanins to the TC-EC heterologous contact area was also observed during the active transmigration of TCs across EC monolayers grown onto 3-dimensional collagen matrices. Dynamic studies by time-lapse immunofluorescence confocal microscopy showed an active redistribution of endothelial CD9 to points of melanoma insertion. Anti-CD9 monoclonal antibodies were found to specifically inhibit the transendothelial migration of melanoma cells; the inhibitory effect was likely caused by a strengthening of CD9-mediated heterotypic interactions of TCs to the EC monolayer. These data support a novel mechanism of tetraspanin-mediated regulation of TC transcellular migration independent of TC motility and growth during metastasis and a role for these molecules in the formation of TC-EC mosaic monolayers during tumor angiogenesis.

Description: Disruption of the physiologic balance between cell proliferation and death is a universal feature of all cancers. In general terms, human B-cell lymphomas can be subdivided into 2 main groups, low- and high-growth fraction lymphomas, according to the mechanisms through which this imbalance is achieved. Most types of low-growth fraction lymphomas are initiated by molecular events resulting in the inhibition of apoptosis, such as translocations affecting BCL2, in follicular lymphoma, or BCL10 and API2/MLT1, in mucosa-associated lymphoid tissue (MALT) lymphomas. This results in cell accumulation as a consequence of prolonged cell survival. In contrast, high-growth fraction lymphomas are characterized by an enhanced proliferative activity, as a result of the deregulation of oncogenes with cell cycle regulatory functions, such asBCL6, in large B-cell lymphoma, or c-myc, in Burkitt lymphoma. Low- and high-growth fraction lymphomas are both able to accumulate other alterations in cell cycle regulation, most frequently involving tumor suppressor genes such asp16INK4a, p53, andp27KIP1. As a consequence, these tumors behave as highly aggressive lymphomas. The simultaneous inactivation of several of these regulators confers increased aggressivity and proliferative advantage to tumoral cells. In this review we discuss our current knowledge of the alterations in each of these pathways, with special emphasis on the deregulation of cell cycle progression, in an attempt to integrate the available information within a global model that describes the contribution of these molecular changes to the genesis and progression of B-cell lymphomas.

Description: Complications during induction chemotherapy (CT) for AML cause mortality in 5–20% of cases. It is relevant to define the risk of early death in this setting. In patients with a high probability of dying due to toxicity, the intensification of the supportive measures and/or alternative antineoplastic approaches could be appropriate. The aim of this study was to detect the features associated with lethal complications during induction CT for AML. We defined early deaths (ED) as those occurring before 42 days after the start of induction in the absence of evident leukemia. We analyzed all consecutive patients diagnosed with AML between June 1998 and February 2006 in 20 Spanish hospitals. These cases were treated according two multicenter trials CETLAM 99 (n=326) and CETLAM 2003 (n=248). Both schemes included idarubicin 12 mg/m2 days 1,3 and 5, etoposide 100 mg/m2 days 1–3 and cytarabine 500 mg/m2/12 hours days 1, 3, 5 and 7 as front-line treatment. In the CETLAM 2003, G-CSF (150 mg/m2 days 0–7) was added as priming therapy. The series included 574 patients, 248 (43%) female, with a median age of 48 years. Creatinine level was elevated (〉 1,2 mg/dL) in 11% of patients. The overall mortality rate during induction (ED) was 12% (n=69). 335 (58%) patients achieved a complete remission with a single course of CT, 108 (19%) a partial remission and 62 (11%) were refractory. The most common causes of death were infection (n= 28, 46%), bleeding (n=7, 11%), pulmonary failure not due to infection (n=6, 10%), and multiorgan failure (n=4, 7%). Univariate analysis showed that age older than 50 years old, male gender, M4 or M5 FAB subtype, leukocyte count higher than 100x109/L, blasts in the marrow 〉70% and creatinine level above 1,2 mg/dL were associated with more frequent ED. In multivariate analysis, elevated creatinine level [hazard ratio (HR) 2.6 (1.3–5.2); P=0.009], leukocytosis 〉100x109/L [HR 2.3 (1.1–4.6) P=0.021] and age 〉 50 years old [HR 2.1 (1.4–3.9) P=0.018] were independent risk factors. The remaining parameters, including among others the use of G-CSF, gender, blasts in the marrow, treatment protocol, cytogenetics at diagnosis and FLT-3 mutational status were not associated with higher incidence of ED. Taking into account the three significant variables identified as risk factors, we developed a scoring system to predict the probability of ED during induction. The probability of ED in the low risk (none risk factor, n=212), intermediate risk (1 risk factor, n=239) and high risk (2 or 3 risk factors, n=57) categories were 3%, 11% and 28% respectively (P

Description: Introduction: Liver metabolism has two fundamental types of enzymes. Phase two enzymes include GlutathioneS-transferases (GST), with three main variants: GST-M1, GST-T1 and GST-P1. Individual differences in these enzymes could influence their detoxificating capacity. Aim: To study the influence of several genetic GST liver enzymes variants in the development of liver Sinusoidal Obstruction Syndrome (SOS) in patients with multiple myeloma (MM) that received BUMEL as conditioning regimen for Autologous Stem Cell Transplantation (ASCT). Patients: 91 patients with MM - included in Spanish protocol Myeloma 2000 that had received BUMEL as conditioning schedule for ASCT - have been studied; 12 of them had developed SOS. 62 healthy individuals as well as 12 patients with monoclonal gammapathy of uncertain significance (MGUS) were also studied. Methods: Three genotype variants of GST enzymes were studied: presence or absence of GST-M1, GST-T1 and GST-P1 polymorphism Ile105Val by real time PCR in light cycler v2 or ABI PRISM 7900HT thermocyclers. Associations between variables were studied by X2 and exact Fisher test. Logistic regression analysis was performed to calculate Odds ratios and relative risk of SOS development. Results: Significant differences (p〉0.0001) were found in the prevalence of homozygous genotype GST-P1 Ile105Val comparing the group of patients with MM and SOS (5 of 12, 41%) and the rest of MM patients without SOS (5 of 79, 6%). This genotype was present in 17% of healthy individuals and MGUS. The genotype absence of GST-M1 was observed in 33% (4/ 12) of MM patients with SOS, 46% (37/79) of MM patients without SOS (p=0.38), 56% of controls and 71% of MGUS. The genotype absence of GST-T1 was observed in 50% (6/ 12) of MM patients with SOS, 27% (22/79) without SOS (p=012), 19% (16/62) of healthy individuals and 21% of MGUS. 33% (4/12) of MM patients with SOS presented the absence of GST-T1 genotype and the presence of homozygous GST-P1 Ile105Val and none of the patients with MM without SOS (p=0,000) presented these genotypes. The only factor associated to development of SOS in regression study was the presence of genotype GST-P1 Ile105Val in homozygosis (OR 10.57 CI 2.45–45.6 p=0.002). Conclusions: The genotype absence of GST-T1 together with the homozygous GST-P1 Ile105Val polymorphism, especially the latter, should be considered as new risk factors in the development of SOS in MM undergoing ASCT conditioning regimen with BUMEL.

Description: Abstract 3219 Poster Board III-156 Introduction Dimethylsulfoxide (DMSO) containing cryoprotective solutions are routinely used for the storage of hematopoietic progenitors (HP). At room temperature, DMSO is toxic for the cells and may produce severe adverse reactions during their infusion, especially in the pediatric patients. These problems can be avoided by washing the cells prior to the infusion. Our objective was to test if an automatic washing method (Sepax S-100, Biosafe) allowed us to preserve the CD34+ cell numbers with an adequate viability and engraftment potential. Material and Methods Forty five peripheral blood HP apheresis that have been cryopreserved using autologous plasma plus 9% DMSO were studied. After rapid thawing in a water bath at 37° C, an automatic wash with the Sepax S-100 (2 washes cycle) was performed. Nucleated cell levels determined by an hematology analyzer, flow cytometry CD34+ cell counts and Trypan Blue cell viability test were performed on aliquots collected prior to and after the washing technique. The paired Student's t-test and the Pearson's correlation coefficient were used for the statistical analysis. Results The mean total nucleated cell (TNC) and CD34+ cell recovery was 75,47% ± 3, and 94,66% ± 4,62 respectively. In spite of the TNC significant loss (p500 cells /mL) and platelet engraftment (〉50.000 cells/mL) were 11 ± 0,2 and 20 ± 1,4 days respectively. Conclusions The Sepax S-100 automatic wash protocol of DMSO containing peripheral blood progenitor cells determines a good CD34+ cell recovery and preserves their viability and engraftment potential. This method avoids the DMSO infusion related adverse events and it constitutes a closed and easy to do procedure. Disclosures No relevant conflicts of interest to declare.