ALBERT

Description: Abstract 1561 In recent years, the microRNA (miRNA) pathway has emerged as a crucial regulation system in tumorogenesis. miRNA expression is deregulated in the tumor, and miRNAs can function both as oncogenes and tumor suppressor genes. miR-SNPs are a novel class of single nucleotide polymorphisms that can affect miRNA biogenesis and target sites and can alter the expression and functions of miRNAs. We have evaluated 9 miR-SNPs and investigated whether a distinct haplotype of miR-SNPs predicts clinical outcome in HL. One hundred and forty-one adult patients (median age, 32 yrs; range, 13–89; males 51.1%) diagnosed with HL in our institution between September 1995 and June 2005 were included in the study. Distribution according to histology: nodular sclerosis (58.9%), mixed cellularity (17.7%), lymphocyte rich (6.4%), lymphoid depletion (4.3%), and nodular lymphocyte predominant (7.1%). Epstein-Barr Virus was present in 38.1% of the samples. SNP analysis was performed by allelic discrimination on ABI Prism 7500 (TaqMan assays) in DNA obtained from formalin-fixed, paraffin-embedded lymph nodes. We examined 9 miR-SNPs: 4 in miRNA genes (MIR196A2 rs11614913; MIR149 rs2292832; MIR423 rs6505162; MIR146 rs2910164); 2 in miRNA binding sites (KRT81 rs3660; FAM179B rs1053667); and 3 in miRNA-processing machinery (XPO5 rs11077; RAN rs14035; TRBPrs784567). miR-SNP genotypes were correlated with probability of treatment failure, treatment-related toxicity, disease-free survival (DFS) and overall survival (OS). The median follow-up was 50 months (range, 1–143). Of 141 patients, 119 (84.4%) achieved complete response, 7 (5%) showed a partial response, and 14 (9.9%) were chemoresistant. We observed an increased probability of treatment failure in patients carrying the XPO5 AA or CC genotype (P=0.036). In 14 patients with neurological toxicity, an association was observed with the KRT81 genotype (P=0.047). In 7 patients with bleomicine-associated pulmonary toxicity, we observed an association with the XPO5 genotype (P=0.048). XPO5 and TRBP genotypes emerged as significant markers for DFS. Mean DFS for 57 patients (56%) with the XPO5 AC genotype was 111 months vs 82 months for patients with the AA or CC genotype (P=0.044). Mean DFS for 37 patients (31.6%) with the TRBP CC genotype was 124 months vs 90 months for patients with the TT or TC genotype (P=0.022). A trend towards an association between the MIR196A2 genotype and DFS was also observed (P=0.07). Only the XPO5 genotype was associated with OS. Mean OS for 66 patients (54%) with the XPO5 AC genotype was 134 months vs 111 months for patients with the AA or CC genotype (P=0.038). Given the evidence for the influence of TRBP and XPO5 as individual markers, we then investigated the combined effect of these miR-SNPs on DFS and OS. We found a significant correlation between the TRBP/XPO5 haplotype and DFS (P=0.005) and OS (P=0.005). Patients with the XPO5 AA/CC and TRBP TT/TC genotypes had the worst prognosis (DFS: 71 vs 114 months; OS: 95 vs 135 months). In the multivariate analyses, the TRBP/XPO5 haplotype (OR, 2.977; 95%CI, 1.1–7.4; P=0.01) emerged as an independent variable for DFS. Only the Hasenclever prognostic index (OR, 5.7; 95%CI, 2–18; P=0.004) emerged as an independent variable for OS, but we observed a trend towards significance for the TRBP/XPO5 haplotype as well (P=0.06). In conclusion, miR-SNPs are a novel class of SNPs that can add useful prognostic information on the clinical outcome of HL, specifically in the detection of chemoresistant patients and patients likely to relapse. The TRBP/XPO5 haplotype has surfaced as a promising prognostic factor that warrants further investigation to confirm its role as a biomarker in HL. Figure. Kaplan-Meier curves for DFS (A) and OS (B) according to the TRBP/XPO5 haplotype. Figure. Kaplan-Meier curves for DFS (A) and OS (B) according to the TRBP/XPO5 haplotype. Disclosures: No relevant conflicts of interest to declare.

Description: MicroRNAs (miRNAs) are negative regulators of gene expression that play an important role in hematopoiesis and tumorigenesis. We analyzed miRNA expression in classic Hodgkin lymphoma (cHL) and the influence of Epstein-Barr virus (EBV) infection on the miRNA expression profiles. The expression of 157 miRNAs in lymph nodes from 49 cHL patients and 10 reactive lymph nodes (RLNs) was analyzed by real-time polymerase chain reaction (PCR). Hierarchic clustering revealed 3 well-defined groups: nodular sclerosis cHL, mixed cellularity cHL, and RLNs. A distinctive signature of 25 miRNAs differentiated cHL from RLNs, and 36 miRNAs were differentially expressed in the nodular sclerosis and mixed cellularity subtypes. These results were validated in a set of 30 cHLs and 5 RLNs, and in 3 cHL cell lines. miR-96, miR-128a, and miR-128b were selectively down-regulated in cHL with EBV. Our findings suggest that miRNAs play an important role in the biology of cHL and may be useful in developing therapies targeting miRNAs.

Description: Hodgkin lymphoma (HL) is a neoplasm characterized by the presence of relatively few tumoral cells, Hodgkin and Reed-Sternberg cells in an important non-neoplastic environment. The mature microRNAs (miRNAs) are small RNA molecules (20–25 nt), that act inhibiting the mRNA translation to protein by binding to 3′ UTR region of mRNA. In a previous work we detect a 25 miRNA signature of HL. Some of them inhibit the expression of key genes related to B lymphomagenesis. The aim of the study was to determine if the differential expression of miRNAs in tumoral tissue versus reactive lymph nodes, is due to genetic alterations in the Hodgkin/Reed Sternberg cells or alterations in the non-tumoral microenvironment. Fluorescein (FITC) 5′ labelled locked-nuclei-acid-incorporated (LNA) miRNA ribo probes for miR-21, 134, 138, 155 (miRCURY™ LNA detection, Exiqon) were used in 20 cases of HL formalin-fixed paraffin embedded tissue sections on silane coated slides (Vision BioSystem). Chromogenic in situ hybridization was done in an automated platform Bond Max (Vision Biosystems) with minor modifications. Pre-treatment of the slides was performed with Protease 1 for 10 min at 37°C. A total amount of 300 microliter of 25nM probe was hybridized in 1x sodium chloride-sodium citrate hybridization buffer (SSC) (Innogenetics) up to 50° C for 2 hours. We used a pre-diluted mouse anti-FITC antibody (Vision BioSystems) for 20–60 minutes followed by a goat anti-mouse linked to thousands of horse radish peroxidase (HRP) sites (Refine Detection System, Vision BioSystems). DAB was used as a chromogen reacting for 10 minutes and hematoxilyn was used as a counterstain. By functional and target analysis, we selected three miRNAs, miR-21 (PTEN), miR-134 (J-Chain) and miR-138 (PU.1), from the cHL signature that seemed to have a role in tumorigenesis process and analyzed them by CISH. In all cases a cytoplasmic signal was demonstrated in Hodgkin and Reed-Sternberg cells. Moreover, a nuclear signal was identified in reactive tumor infiltrating lymphocytes for miR-21 and miR-138. This nuclear signal may be due to crossreactivity with primary miRNA in these cells. We analyzed miR-155 as positive control of in situ hibridization and we demonstrated a cytoplasmic signal in Hodgkin and Reed Sternberg cells, as well as in scattered reactive lymphocytes and activated histiocytes as previously reported. In conclusion, miR-21, miR-134 and miR-138 play a role in HL lymphomagenesis since their expression is preferentially localized in Hodgkin/Reed Sternberg cells. The study of the other miRNAs of our signature can help explain the changes that appear in the atypical cells as well in the reactive cellular microenvironment in HL.

Description: Myelodysplastic syndromes (MDS) are clonal hematopoietic stem-cell disorders that include a broad spectrum of entities characterized by ineffective hematopoiesis and risk of transformation into acute myeloid leukemia (AML). MicroRNAs (miRNA) act as negative expression regulators of important genes as those participating in cellular proliferation, apoptosis and carcinogenesis. In this study we analyzed the expression patterns of 26 miRNA hematopoietic-related genes in patients with MDS. Total RNA was extracted from 20 MDS patients (BM and PB) and 5 normal controls. Median age of patients was 70 (range, 55–82) years. Seven patients had refractory anemia with excess of blasts (RAEB-I, n=5; RAEB-II, n=2), 7 refractory cytopenia with multilineage dysplasia (RCMD), 3 RCMD with ringed sideroblasts (RCMD-RS), 1 chronic myelomonocytic leukemia (CMML), 1 refractory anemia with ring sideroblasts (RARS) and 1 MDS associated with isolated del(5q). 15 patients presented low risk IPSS (LR) (low or intermediate-1) and 5 patients had high risk IPSS (HR) (intermediate-2 and high risk). Twenty-six mature miRNAs were assessed by Stem-loop RT-PCR and Real time PCR in ABI PRISM 7500. MiRNA expression data was normalized to overall median and relative quantification was calculated with the 2−ΔΔCt method. The data were presented as log10 of relative quantity of target miRNA. Median of normal controls was used as calibrator for all samples. Data were analyzed by class comparison methods using BRB Array Tools version 3.4.0 and TIGR multiexperiment viewer. Hierarchical clustering analysis of BM categorized two clusters corresponding to BM-MDS and BM-controls. Then, miR-142-5p (p

Description: MicroRNAs (miRNA) are small RNAs that act inhibiting the translation of mRNA to protein by complementary binding to his 3′UTR region. In a previous study we analyze the profile of miRNAs in classical Hodgkin lymphoma (HL) and found a 25 miRNAs signature that allows us to discriminate between HL and reactive lymph nodes. And some of them act inhibiting the translation of key genes in B lymphomagenesis. The aim of the study was to analyze if the expression of this miRNAs in the diagnostic samples predict clinical outcome in HL. Eighty nine adult patients (median age, 29 yrs; range, 13–89; males 47%) diagnosed with HL at a single institution between September 1995 and June 2005 have been studied. Seven patients (7.8%) were HIV+. Distribution according to histological subtypes was: nodular sclerosis (79%) and mixed cellularity (21%). Epstein-Barr Virus (EBV) was present in 28% of the samples. First-line treatment consisted of CMOPP/ABV or ABVD. Total RNA was extracted from formalin-fixed paraffin embedded lymph nodes using RecoverAll Total Nucleic Acid Isolation (Ambion). Mature miRNA expression was analyzed by stem loop RT-PCR and Real Time PCR (TaqMan MicroRNA Assay Protocol) in a 7500 Sequence Detection system(AB). Statistical analysis was performed with BRB Array Tools and SPSS 14.0. The characteristics considered were: age (45), ECOG, Hasenclever prognostic index (5), HIV status, Ann Arbor (I–II vs. III–IV), WHO histological classification, bulky disease, EBV (LMP1+ vs LMP1−), ESR (EORTC criteria), and the expression of above-mentioned miRNAs. Clinical outcomes analyzed were relapse rate, disease-free survival (DFS) and overall survival (OS). Out of 89 patients, 75 (84.3%) achieved CR, 7 (7.8%) partial response, 7 (7.8%) were chemoresistant. After a median follow-up of 43 months (1–128), OS was 83% and DFS 74%. Of the 25 miRNAs, miR-135a appears underexpressed in HL samples versus reactive lymph nodes. Moreover, we found two groups of patients relative to miR-135a expression: patients with low expression (Ct35.4). In the Kaplan Meier analysis the patients without expression of miR-135a had a higher probability of relapse than the patients with miR-135a expression (Log Rank p=0.045). In the multivariate analysis the miRNA was the only prognostic factor for relapse (RR=6.533 p=0.021). In conclusion, miR-135a has as putative target HIF1A, a transcription factor related with hypoxia state that have been related in other tumors with tumoral growth and relapse when it’s overexpressed. Maybe through this target miR-135a play a role in relapse as prognostic value, but a higher number of patients and a functional analysis will be required to validate this new prognostic factor for HL.

Description: Abstract 3919 Background: The constitutive activation of the JAK/STAT pathway plays an important role in the pathogenesis and proliferation of Hodgkin Lymphoma (HL). Although somatic activating point mutations in the JAK2 gene have been reported in myeloproliferative disorders (MPD), they are rarely described in HL, where JAK2 amplification is associated with mutations of regulator genes such as SOCS-1, constitutive activation of STAT proteins or miRNA deregulation. Recently, many JAK2 inhibitors, including Lestaurtinib (CEP701), have been reported to have clinical efficacy in MPD. CEP701 is a multitargeted tyrosine kinase inhibitor that potently inhibits FLT3 at nanomolar concentrations. Recent studies in MPD have further shown that CEP701 inhibitory activity is not limited to FLT3 and can suppress JAK2/STAT5 signaling through JAK2 inhibition. As a first step towards elucidating the potential role of CEP701 in HL therapy, we have analyzed its efficacy in vitro. Methods: Four HL cell lines, L-428, L-1236, HDMYZ and L-540, were assayed for proliferation, apoptosis and levels of proteins in the JAK2/STAT pathway (pJAK2, JAK2, pSTAT5, STAT5, Bcl-xL) after CEP701 treatment. 100,000 cells were plated in a 96-well plate in 100 ml culture medium with CEP701 or DMSO (vehicle control) at concentrations of 30–300 nM. After 1 or 24 hours of incubation with CEP701, the levels of the proteins and of FLT3 were analyzed by Western blot. Proliferation was analyzed with CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) and apoptosis by CaspaseGlo 3/7 after 48 hours of treatment. Results: The proliferation analysis showed an effective dose-dependent inhibition of cell growth in the 4 HL cell lines after treatment with increasing concentrations of CEP701. At 48h, in comparison to cells treated with DMSO alone (normalized to 100%), in cells treated with 100nM of CEP701, we observed a marked inhibition of 35% in L-428, 55% in L-1236, 15% in HDMYZ and 77% in L-540. Moreover, apoptosis increased by 38%, 31%, 21% and 25%, respectively. The protein analysis showed that after one hour, CEP701 inhibited phosphorylation of JAK2 (pJAK2) and its downstream target STAT5 (pSTAT5) in a dose-dependent manner, with no changes in the non-phosphorylated proteins. The downstream target Bcl-xL also decreased. Conclusions: Taken together, these data demonstrate that growth inhibition and apoptosis activation by CEP701 in HL cells correlates with the inhibition of the JAK2/STAT5-dependent signal transduction pathway. Here we present the first biological evidence that Lestaurtinib could be a promising new agent in the treatment of patients with HL. Supported by a FIS grant (PS09/00547). Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Long non-coding RNAs (lncRNAs) have recently emerged as important actors in the regulation of multiple cellular processes including cancer. Acute myeloid leukemia (AML) is a heterogeneous disease; most of the main cytogenetic AML subgroups harbor a specific gene expression profile. AML with translocation t(8;16)(p11;p13) (t(8;16) AML) is a subtype with specific clinical and biological characteristics including a distinctive gene (Camós et al, Cancer Research 2006) and microRNA (Díaz-Beyá et al, Leukemia 2013) expression profile. In this translocation, MYST3 on chromosome 8p11 fuses with CREBBP on chromosome 16p13.3. The MYST3-CREBBP fusion protein is able to interact with multiple transcription factors (TF) producing a disturbed transcriptional program. However, the lncRNA expression pattern of different cytogenetic AML subtypes, including t(8;16) AML, have not been described yet. Aims: To examine the expression profile of lncRNAs within different AML subtypes, and to characterize the expression pattern of lncRNAs in t(8;16) AML in comparison to other AML subtypes. Patients and Methods: 46 AML patients, 4 normal bone marrow (NBM) and 3 CD34+ NBM samples were included in the study. Samples included different AML subtypes: intermediate-risk cytogenetic AML (IR-AML, n=18), t(15;17) (APL, n=4), t(8;21) AML (n=4), inv(16) AML(n=2), t(6;9) AML (n=7), AML with monosomal karyotype (n=4), t(3;3) AML (n=1), t(9;11) AML (n=1) and t(8;16) AML (n=5). Within IR-AML patients with a different mutational profile: FLT3-ITD (n=7), NPM1 (n=5), CEBPA (n=7) and DNMT3A (n=6) were included. The lncRNA expression was studied using Affymetrix® Human Gene 2.1 ST platform which includes 9698 lncRNAs transcripts. The filtering and normalization of the array data was performed using oligo package from Bioconductor. Statistical analyses were performed with TiGR MultiExperiment Viewer, BRB tools and R. The Transcription factor Affinity Prediction Web Tool was used to determine the putative transcription factors binding to the differentially expressed lncRNAs promoters. Results: The hierarchical cluster analysis showed that all 4 NBM as well as all 3 CD34+ NBM clustered together according to their lncRNA expression. Interestingly, all 5 t(8;16) AML samples clustered together, as well as the 3 APL, the 7 t(6;9) AML and 5 out of 7 cases with CEBPA mutations. The specific lncRNA signature of APL was composed of 79 differentially expressed lncRNA and t(6;9) AML lncRNA signature comprised of 15 differentially expressed lncRNAs. When we focused on t(8;16) AML lncRNA profile, we identified an specific 113-lncRNA signature in the supervised analysis (Figure). Interestingly, when we analyzed which (TF) had motifs overrepresented in the promoters regions of the t(8;16) AML lncRNA signature, we identified GATA2 as the TF with significantly overrepresented motifs for GATA2 (p

Description: In this study, we investigated whether distinct patterns of functional genomic polymorphisms in genes involved in drug metabolic pathways (GSTT1, GSTP1, GSTM1, SULT1C2, TOP2A, SXR-1), DNA repair (XPD, XPA, ERCC1, ERCC5, XRCC1, XRCC4, XRCC5), apoptosis and inflammatory cytokines (FAS, FASL, IL-10), and multidrug resistance (MDR1-2, MDR1-6) predict clinical outcome in patients with HL. One hundred and twenty-seven adult patients (median age, 33 yrs; range, 15–80; males 53%) diagnosed with HL at a single institution between September 1995 and June 2005 have been studied. Seventeen patients (13.4%) were HIV+. Distribution according to histological subtypes was: nodular sclerosis (56.7%), mixed cellularity (20.5%), lymphocytic predominance (5.6%), and lymphoid depletion (5.6%). Epstein-Barr Virus (EBV) was present in 35.4% of the samples. First-line treatment consisted of CMOPP/ABV (39.4%) or ABVD (52.8%). Allelic discrimination of single nucleotide polymorphisms (SNPs) (ABI Prism 7500; TaqMan) of DNA obtained from formalin fixed paraffin embedded lymph nodes was performed. The characteristics considered were: age (C polymorphism (RR=1.7; p=0.01) and the only adverse prognostic factor for relapse was IL-10 -1082 A〉G polymorphism (RR=2.1; p=0.02). A lower probability of DFS was associated with HIV+ status (RR=11.4; p=0.03), and a lower probability of OS to higher Hasenclever prognostic index (RR=2.3; p=0.02). Moreover, a close association between Asp5Glu genotype of the phase II drug-metabolizing enzyme SULT1C2 and pulmonary toxicity was found; thus, all eight patients with pulmonary toxicity due to bleomicine had the wild type SULT1C2 genotype (p=0.006). In conclusion, germline polymorphisms in FASL, IL-10 and SULT1C2, which can easily be analyzed in paraffin embedded samples, have prognostic value in patients with HL.

Description: Abstract 2934 Background: The analysis of polymorphisms in drug metabolism pathways and in DNA repair genes could help to identify patients with possible different treatment response and outcome. Single nucleotide polymorphisms (SNPs) are the most frequent type of genomic polymorphisms and have been described in association with prevalence, response to treatment, progression-free and overall survival in different tumors, including multiple myeloma (MM). The aim of the present study was to examine 22 SNPs related to DNA repair and drug metabolism, and correlate our findings with response, toxicity and survival in patients with MM after autologous stem-cell transplantation (ASCT). Patients and Methods: One hundred and eighty seven patients with MM (103M/84F, median age 55 years) intensified with melphalan-based ASCT have been studied in one institution. The median follow-up was 4 years (range 4 months to 18 years). None patient was lost to follow-up. Genomic DNA was isolated from bone marrow slides using a commercial assay (Qiagen). SNPs were analyzed by TaqMan assay in an ABI Prism 7500 Sequence Detection system (Applied Biosystems). The genes and SNPs evaluated in genomic DNA by allelic PCR were ERCC2 (rs13181, rs238406), ERCC5 (rs1047768, rs17655), XPA (rs1800975), XPC (rs2228001), XRCC1 (rs25487), XRCC5 (rs1051685, rs1051677), XRCC4 (rs963248), ERCC1 (rs3212948, rs735482) and BRCA1 (rs16941, rs799917) for DNA repair systems; NAT2 (rs1799930), CYP2C8 (rs11572080, rs2275622, rs10509681), TYMS (rs2790), SULT1 (rs1402467) and GST1 (rs1695) for phase I and II drug metabolisms, and ABCB1 (rs1045642) for drug transportation. These genes were selected based on their potential impact on prognosis in solid tumors in previous reports. Results: In the overall population, median PFS was 2.7 years (CI 95% 2.2 to 3.3 years), with a median OS of 6 years (CI 95% 4.5 to 8 years). OS was significantly shorter in patients with SNPs in ERCC5 (rs1047768; p=0.021), XPA (rs1800975; p=0.032) and GSTP1 (rs1695; p=0.015) (Figure). There was also a trend for CYP2C (rs2275622; p=0.054) and TYMS (p=0.107). The significance of the SNP in ERCC5 was retained in the group treated with conventional chemotherapy at induction (p=0.034), but not in those who received novel drugs (bortezomib, thalidomide and lenalidomide). Patients with SNP in ERCC1 achieved a lower CR rate (22.2% vs. 37.8%; p=0.033), with no prognostic significance. Polymorphism in GSTP1 was also associated with a shorter PFS (p=0.002), without differences in the complete remission (CR) rate. When only patients who received ASCT after first line treatment were considered, the effect over OS remained at significant level (p=0.039). Furthermore, the effect on PFS and OS was also significant in patients achieving CR after ASCT (p=0.03). NAT2 (rs1799930) and ERCC2 (rs238406) polymorphims were associated with clinically significant mucositis after conditioning, as well as TYMS (rs2790) with relevant gastrointestinal toxicity (p