Researchers have indicated that performance in endurance athletes is related to specific physiological parameters (Joyner & Coyle, 2008) as the maximal oxygen uptake (VO2max.) (Maldonado-Martin, ...Mujika & Padilla, 2004) and anthropometric characteristics appropriate (Legaz-Arrese & Eston, 2005). The aim of the study was to determine the changes induced in anthropometric parameters and VO2max in endurance runners after a period of training, and its possible relationship to performance. Before and after of three months of training, abdominal, subscapular, iliac crest, triceps, front thight and medial calf skindfolds were measured at twenty one middle and long distance runners highly trained (age 23±3 years, weight 65.5±7.3 kg, height 177±0.05 m, VO2 max. 68.1±4.6 ml/min/kg) all with at least 4 years of experience and a training volume 105.9±16.8 kms/week; then the subjects performed an incremental test on a treadmill to determine VO2max. There was a significant increase p <0.05 in abdominal and iliac crest skinfold, and decrease p <0.01 in the calf medium skindfold with training. In addition, the maximum speed reached by the subjects in the incremental test performance had a significant increase p <0.01, while the VO2max. had not significance. Improvements in the performance of highly trained in endurance runners appear to be associated with loss fat weight in specific muscle groups in training.
Abstract 4155
Beyond the conventional criteria of lymphoma classification (integrated clinical, morphological, immunophenotypic, and molecular features) additional markers are still required for a ...more precise differential diagnosis and a better understanding of lymphoma pathogenesis.
MicroRNAs (miRNA) are non-coding small RNAs that play an important role in gene expression regulation, contributing to cell differentiation and tumorigenesis. Specifically, miRNAs have been already described to play a relevant role in B cell differentiation, and in some cases proposed to constitute lymphoma-type specific markers and possible therapeutic targets.
We explore the potential diagnostic application of miRNA expression in a large series of 147 cases including all B-cell non-Hodgkin lymphomas (NHL) major types and appropriate controls. As an example of a practical application, data were also used to identify miRNAs differentially expressed when comparing Burkitt Lymphoma (BL) and Diffuse Large B-Cell Lymphoma (DLBCL) in paraffin-embedded samples.
Each lymphoma type (BL, CLL, DLBCL, FL, MCL, MZL/MALT, NMZL and SMZL) was compared to the whole series of NHL by Significant Analysis of Microarray (SAM) method. The analysis identified a set of 128 characteristic miRNAs (FDR<0.01 and Fold change >1.5 log2). All lymphoma types were characterized by specific miRNA signatures, reflecting cell of origin and/or discrete oncogene alterations. Of interest is also the comparison with reactive lymphoid tissues, since it revealed a specific B-cell lymphoma miRNA profile, which includes a cluster of downregulated miRNAs, such as let7 family, miR-1 and miR-200 family.
Burkitt Lymphoma was also directly compared to DLBCL, and 43 miRNA selected by SAM analysis were studied in a new series of 28 BL and 43 DLBCL samples using quantitative RT-PCRIn this second step, the differential expression of a set of 19 miRNAs was confirmed between BL and DLBCL. (FDR < 0.05 after t-test (limma)).
These findings expand the potential diagnostic markers in lymphoma diagnosis and provide useful information on lymphoma pathogenesis.
No relevant conflicts of interest to declare.
Abstract 3493
Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of very aggressive malignancies lacking efficient therapy. Unfortunately, there are neither animal models nor representative ...cell lines for most PTCL types, making functional and pharmacodynamic studies even more difficult. PI3K signaling is essential for cell proliferation and survival, is frequently altered in human cancer and seems to play a critical role in T-cell development and activation. The aim of this work is to determine the efficiency of PI3K inhibition in PTCL, looking for pharmacodynamic biomarkers, and to identify markers that could distinguish responders from non-responders.
Twenty two PTCL cases and seven reactive lymph nodes were studied using gene expression profiling. We performed an in silico analysis using the Connectivity Map program to identify drugs that could potentially reverse the PTCL gene expression signature. Among them, several PI3K/mTOR inhibitors were found.
Moreover, genomic studies using Gene Set Enrichment Analysis identified PIK3CD gene (encoding for the delta isoform of PI3K) to be the only one significantly correlated to the activation of CD40, NF-kB and TCR pathways. Quantitative RT-PCR confirmed the strong overexpression of PIK3CD in 6 PTCL-derived cell lines compared to normal T cells from healthy donors. Sequence analyses for the coding region of the PIK3CD gene identified a point mutation in one of these cell lines, described as activating in solid tumors.
A panel of 6 PTCL cell lines belonging to different PTCL subgroups was treated with 3 PI3K inhibitors (LY294002, ETP-45658, GDC-0941). Moreover, genetic inhibition was also carried out using small interference RNA to specifically abolish the expression of alpha and delta isoforms of PI3K (PIK3CA and PIK3CD genes, respectively).
In vitro studies showed very similar results with the three pharmacological PI3K inhibitors we used: they induced G1 cell cycle arrest in all cell lines, and apoptosis in some of them, in a time/dose-dependent manner. We also observed a decrease in the levels of pAKT(S473) in all cell lines, while pGSK3B(S9) and p-p70S6K(T389) were reduced after treatment only in sensitive cell lines.
Our results indicate that genetic inhibition of PI3K delta isoform could induce apoptosis in those PTCL cell lines that were sensitive to PI3K inhibitors, but not in the resistant cell lines; while genetic inhibition of PI3K alpha isoform did not display such effects.
Taken together these results could highlight the relevance of PI3K delta isoform in at least a subset of PTCL, indicating that PI3K inhibition, especially delta isoform, could be an effective therapeutic approach for PTCL and identifying potential markers for patients' stratification and pharmacodynamic assessment.
No relevant conflicts of interest to declare.
Abstract 3494
The search of an efficient therapy for Peripheral T-cell lymphomas (PTCL) patients is still a challenge, in part due to the very little knowledge about the PTCL pathogenesis, and the ...absence of appropriate models. This heterogeneous group of very aggressive malignancies can not be cured with conventional therapies; therefore, new therapeutic strategies are needed to improve the poor outcome in these patients. PIM family is composed of 3 kinases (PIM1, PIM2 and PIM3) which play an essential role in cell proliferation and survival. They are mainly activated through JAK/STAT signaling pathway, and are frequently altered in human malignancies by amplification, mutation and overexpression. The aim of this study is to determine the efficiency and the mechanism of action of PIM inhibition in PTCL.
Gene expression profiling of twenty two PTCL cases and seven reactive lymph nodes was performed. We observed a strong overexpression of the three PIM family genes in PTCL cases, especially PIM2. In addition, Gene Set Enrichment Analysis identified an overexpression of STAT3 and IL-2 pathways in PTCL cases, probably responsible for the strong expression of PIMs we found. Furthermore, PIM genes expression was confirmed by quantitative RT-PCR in 6 PTCL-derived cell lines compared to normal T cells from healthy donors, highlighting again the relevance of PIM2.
Genetic inhibition was carried out using small interference RNA to specifically abolish the expression of each PIM1, PIM2 and PIM3 in a panel of 6 PTCL cell lines belonging to different PTCL subgroups. Additionally, pharmacological inhibition with one PIM inhibitor (ETP-39010) was performed.
Surprisingly, genetic inhibition of each of the PIM gene alone did not show any cellular effect, neither cell cycle arrest nor apoptosis. But interestingly, we found that specific inhibition of each of the PIM genes caused an increased expression of the other PIM family members, probably leading to a compensatory mechanism among these kinases balancing the lack of one of them, avoiding pro-apoptotic effects and allowing cell survival.
Accordingly, a simultaneous inhibition of PIM1, PIM2 and PIM3 using the pharmacological pan-PIM inhibitor produced a decrease in cell viability and a strong induction of apoptosis in all cell lines, without cell cycle arrest. Several PIM inhibitor biomarkers have been identified at the mRNA level, involving the DNA damage response signaling.
In conclusion, our results indicate that PIM kinases inhibition could be an effective therapeutic approach for PTCL.
No relevant conflicts of interest to declare.
Abstract 4157
In spite of the progress in the therapy of advanced stage classic Hodgkin lymphoma (cHL), still a significant proportion of patients fail to respond or relapse after complete remission. ...MicroRNAs (miRNA) are essential regulators of cell differentiation, emerging as robust predictor and prognostic markers. Specific miRNA signatures from the Hodgkin and Reed Sternberg cells (HRS) cells and their microenvironment have been proposed, but the potential prognostic role of them remains unclear. Here, we investigated whether miRNA signatures can allow to a better understanding of cHL pathogenesis and being applied in patient outcome prognostication.We have used mirRNA gene expression data from 32 samples of advanced cHL patients and 5 cHL derived cell lines (L540, L1236, L428, HDLM2, and KHMZ) to identify specific miRNA profiles from the tumoral cells and their non-tumoral microenvironment. A miRNA signature was identified in the cHL cell lines, probably reflecting functional properties of the HRS, whereas the others informed from properties of the reactive infiltrate. Selected miRNAs were further validated by performing laser capture microdissection using frozen samples in order to look for specific miRNAS preferentially expressed for either HRS cells and their surrounding microenvironment.
In addition, logistic regression and Cox analysis allowed us to identify a set of 102 miRNAs differentially expressed (p<0.05) among patients with favourable and unfavourable outcome that included members of the miR-17-92 cluster such us mir-17, mir-92a and mir-92b* and other miRNAs known to be involved in cancerogenesis. A restricted group of 32 miRNAS with better prognostic capacity was further selected to set up a quantitative RT-PCR assay and explore the potential diagnostic application of their expression in an independent series of 96 formalin-fixed paraffin-embedded (FFPE) cHL samples.
Bioinformatics target prediction (miRBase and TargetScan) and the web based computational tool DIANA-mirPath, were used in the identification of molecular pathways and target genes potentially regulated by the expression of these mirRNAs. Combined analysis of miRNAs, paired with bioinformatic target prediction, revealed a series of genes and pathways targeted by a small number of miRNAs, including essential pathways for lymphoma survival like MAPK signaling pathway, cell cycle, Jak-STAT signaling pathway and others.
In conclusion, specific miRNA signatures for the HRS cells and their microenvironment can be identified in cHL samples, and improve our understanding in cHL pathogenesis. Selected miRNAs can be used in paraffin-embedded tissue for prognosis of advanced cHL cases, and can be incorporated in an RT-PCR assay with potential clinical application in advanced cHL.
No relevant conflicts of interest to declare.
Abstract 4917
T-cell lymphomas (TCL) are a heterogeneous group of aggressive malignancies lacking specific and efficient therapy. Unfortunately, there are neither animal models nor representative ...cell lines for most TCL types, making functional and pharmacogenomics studies even more difficult. PI3K and PIM are kinases involved in cell proliferation, frequently altered in human cancer that seems to play a critical role in T-cell development and activation. Genomic studies have identified PIK3CD subunit to be significantly associated with in activation of CD40, NF-kB and TCR-pathways. The aim of this project is to determine the efficiency of PI3K inhibitors (PI3Ki) and PIM inhibitors (PIMi) in TCL, looking for biomarkers of their mechanism of action and to identify markers that could identify responders from non-responders.
Twenty PTCL and seven reactive lymph nodes were studied using gene expression microarrays. We performed an in silico analysis using the Connectivity Map program to identify drugs that could potentially reverse PTCL gene expression signature. Among them, several PI3K/mTOR inhibitors were found. A panel of 6 TCL cell lines belonging to different TCL subgroups were treated with 3 PI3Ki (LY294002, ETP-45658, GDC-0941) and one PIMi (ETP-39010). Functional studies were also done to establish the role of each of the targeted genes.
In vitro studies showed that PI3Ki induced G1 cell cycle arrest in all cell lines, and apoptosis in a portion of them, in a time/dose-dependent manner. We also observed a decrease in the levels of pAKT(S473), pGSK3B(S9) and p-p70S6K(T389) after treatment.
In addition, both the analysis of the PTCL gene expression signature as well as western blot studies on TCL cell lines has shown overexpression of PIM family genes, A decrease in cell viability, and a strong induction of apoptosis in all cell lines was seen after PIM inhibition, without cell cycle arrest. Several diagnostic and pharmacodynamic biomarkers of PIMi have been identified at the mRNA and protein level in both cell lines In conclusion, our results indicate that PI3Ki and PIMi are effective therapeutic approaches for TCLs, identifying potential markers for patient's stratification and pharmacodynamic assessment.
No relevant conflicts of interest to declare.
Abstract 969
Peripheral T-cell lymphoma (PTCL) has been the subject of a relatively limited number of studies to elucidate the molecular pathogenesis. As a result, molecular classification of PTCL is ...still to be developed, targeted drugs are in very early development and clinical outcome is dismal. Recently, new technologies in genomic analysis have offered the opportunity to improve the knowledge regarding microRNA and gene expression signatures in T-cell lymphoma as well as the potential of microRNAs as prognostic markers in this disease.
The study included a group of 22 patients with PTCL along with 7 reactive lymph nodes (LN) as controls. The global microRNA and gene expression profiles were examined using a commercially available Agilent platform. To identify microRNAs differentially expressed in PTCL versus LN samples and between different subgroups of PTCL, we used the significance analysis of microarrays (SAM) protocol and permutation tests (10,000 permutations). For analysis of pathways associated with PTCL pathogenesis, a gene set enrichment analysis (GSEA) was performed. Results were validated in an additional set of paraffin embedded samples.
A signature composed of 80 microRNAs was found to be differentially expressed in PTCL compared with LN, including the let-7 family, mir-10, mir-15, mir-16 and miR-101 (p<0.0001). Gene expression profiling (GEP) revealed twelve pathways significantly enriched in malignant tissue (FDR<0.1), including the ERK, EGF, CDK5, MET and cytokine induced signaling cascades. GEP data were analyzed trying to correlate the lymphoma cases with the signatures of different T-cell subpopulations including TH1, TH2, T-reg, TH17, TFH and cytotoxic T-cells. The analysis of lymphoma samples revealed a group of 5 cases with a null phenotype lacking any resemblance to normal T-cell subpopulations. These patients were CD4, CD8 double negative and had poorer prognosis than patients with tumors expressing T-cell differentiation markers. We compared microRNA and gene expression profiles of the cases with null-phenotype vs. differentiated-phenotype and found that the former group expressed oncogenic microRNAs, such as the miR-17-92 cluster (Oncomir-1) and miR-181 family. In addition, a set of 27 microRNAs was lost in the null-phenotype group (FDR<0.0001). These included miR-223, miR-100, let-7b, let-7c, miR-145, miR-195 and miR-497 which target genes of the insulin like growth factor 1 (IGF-1) pathway and oncogenic Ras family, signaling cascades that have been shown to function as potent proliferation stimuli. Consistently with the results as outlined above, GSEA analysis demonstrated RACCYCD (Ras and Rho), IGF1, Wnt and cell cycle regulation pathway enrichment in the null-phenotype group. In contrast, NK/T, T-cytotoxic, inflammatory cytokine, NF-κB and T-cell receptor (TCR) pathways were significantly upregulated in the differentiated group (FDR<0.1).
Molecular analysis of PTCL, facilitated by the comparison with normal T-cell subpopulations, revealed the existence of a null-phenotype PTCL, characterized by aggressive behavior and expressing a microRNA oncogenic signature. This research suggests possible and novel roles for microRNAs in the diagnosis and pathogenesis of T-cell lymphoma.
No relevant conflicts of interest to declare.
Abstract 3943
Poster Board III-879
The nuclear factor κB (NF-κB) family of transcription factors is required for the development of T and B lymphocytes and the regulation of the innate and adaptive ...immune response. Deregulated NF-κB activity has been associated with a number of malignancies and several types of lymphomas depend on NF-κB activity for cell proliferation and survival. A number of studies have been published in the last few years demonstrating the importance of the alternative NF-κB pathway in lymphomas. The NF-κB-Inducing Kinase (NIK or MAP3K14) is a serine/threonine kinase that is essential for the activation of the alternative NF-κB pathway. NIK induces the phosphorylation of the NF-κB member p100, which is followed by the processing of p100 to p52 and its subsequent nuclear translocation. Gene expression data from 106 lymphoma samples with different diagnosis (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), mucosa-associated lymphoid tissue (MALT), nodal marginal zone lymphoma (NMZL) and chronic lymphocytic leukemia (CLL)) were previously generated using Agilent oligonucleotide microarrays. Data analysis indicated variability in NIK expression among the different lymphoma samples, showing a higher expression in CLL and FL samples. NIK expression was closely associated with the expression of other members of the alternative NF-κB pathway, such as NFKB2, RELB and CD40. To investigate the correlation between NIK gene expression and functional pathways, we performed a gene set enrichment analysis (GSEA). We found that the expression of NIK was positively and significantly correlated with several biologically important pathways in both lymphomagenesis and normal leukocyte development and function, such as B- and T-cell receptor pathways, CD40 signaling pathway, and the classical and alternative NF-κB pathways. Twenty seven lymphoma-derived cell lines were examined by Western blot for expression of NIK and p100/p52. The pathway was found to be frequently activated in these cell lines since high levels of p52 were detected in the majority of MCL (5/9 cell lines), Hodgkin lymphoma (3/3), CLL (1/1), DLBCL (7/9) and T-cell lymphoma (5/5) cell lines. Two-thirds of these p52-positive cell lines also expressed NIK and three of them expressed a truncated form of p100. The expression of NIK and p52 was also associated with Epstein-Barr virus (EBV) infection, given that four out of five EBV-positive cell lines showed elevated levels of these proteins. p100/p52 expression was immunohistochemically analyzed using tissue microarrays including paraffin-embedded tissues from totally 356 DLBCL patients. Examination of these samples indicated that the alternative pathway is activated in a subset of these tumors, with nuclear p52 expressed in 17% of the cases. A significant positive correlation between EBV-infection and p52 expression was established and the majority of the p52-positive cases also expressed nuclear p50, suggesting that both the alternative and classical NF-κB pathways are frequently activated in the same tumors. Alternative and classical NF-κB activation was more frequently present in the non-GC DLBCL type, but also observed in a significant proportion of the GC-DLBCL cases. Furthermore, array based comparative genomic hybridization (aCGH) revealed mono-allelic loss of TRAF3, a negative regulator of NIK, in 5 out of 22 DLBCL cases. Taken together, our results show that the alternative NF-κB pathway is activated in a subset of human lymphoma samples and cell lines. This highlights the relevance of NIK as a potential therapeutic target in lymphoid malignancies.
No relevant conflicts of interest to declare.
Abstract 3660
Poster Board III-596
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. Current predictive systems, based on clinical and analytical parameters, fail to identify accurately this significant fraction of patients with short failure-free survival (FFS). Transcriptional analysis has identified genes and pathways associated with clinical failure, but the biological relevance and clinical applicability of these data await further development. Robust molecular techniques for the identification of biological processes associated with treatment response are necessary for developing new predictive tools.
We used a multistep approach to design a quantitative RT-PCR-based assay to be applied to routine formalin-fixed, paraffin-embedded samples (FFPEs), integrating genes known to be expressed either by the tumor cells and their reactive microenvironment, and related with clinical response to adriamycin-based chemotherapy. First, analysis of 29 patient samples allowed the identification of gene expression signatures related to treatment response and outcome and the design of an initial RT-PCR assay tested in 52 patient samples. This initial model included 60 genes from pathways related to cHL outcome that had been previously identified using Gene Set Enrichment Analysis (GSEA). Second, we selected the best candidate genes from the initial assay based on amplification efficiency, biological significance and treatment response correlation to set up a novel assay of 30 genes that was applied to a large series of 282 samples that were randomly split and assigned to either estimation (194) or validation series (88). The results of this assay were used to design an algorithm, based on the expression levels of the best predictive genes grouped in pathways, and a molecular risk score was calculated for each tumor sample.
Adequate RT-PCR profiles were obtained in 264 of 282 (93,6%) cases. Normalized expression levels (DCt) of individual genes vary considerably among samples. The strongest predictor genes were selected and included in a multivariate 10-gene model integrating four gene expression pathway signatures, termed CellCycle, Apoptosis, NF-KB and Monocyte, which are able to predict treatment response with an overall accuracy of 68.5% and 73.4% in the estimation and validation sets, respectively. Patients were stratified by their molecular risk score and predicted probabilities identified two distinct risk groups associated with clinical outcome in the estimation (5-year FFS probabilities 75.6% vs. 45.9%, log rank statistic p≈0.000) and validation sets (5-year FFS probabilities 71.4% vs. 43.5%, log rank statistic p<0.004). Moreover, this biological model is independent of and complementary to the conventional International Prognostic Score using multivariate Cox proportional hazards analysis.
We have developed a molecular risk algorithm that includes genes expressed by tumoral cells and their reactive microenvironment. This makes it possible to classify advanced cHL patients with different risk of treatment failure using a method that could be applied to routinely prepared tumor blocks. These results could pave the way for more individualized and risk-adapted treatment strategies of cHL patients, enabling subsets of patients to be identified who might benefit from alternative approaches
No relevant conflicts of interest to declare.
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