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The β2-Adrenergic receptor (β2-AR) is a G protein-coupled receptor (GPCR), involved in the development of many cancers, among which HNSCC. In this contest, β2-AR signaling interacts with different pathways, such as PI3K and MAPK, commonly activated by TK receptors. For this reason, TK blockade is one of the most adopted therapeutic strategies in HNSCC patients. In our study we investigated the effects of the β2-AR blocking in HNSCC cell lines, using the selective inhibitor ICI118,551 (ICI), in combination with the MAPK inhibitor U0126. We found that ICI leads to the blocking of p38 and NF-kB oncogenic pathways, strongly affecting also the ERK and PI3K pathways. Cotreatment with U0126 displays a synergic effect on cell viability and pathway alteration. Interestingly, we found that the β2-AR blockade affects Nrf2-Keap1 stability and its nuclear translocation leading to a drastic ROS increase and oxidative stress. Our results are confirmed by a TCGA dataset analysis, showing that NFE2L2 gene is commonly overexpressed in HNSC, and correlated with a lower survival rate. In our system, the PI3K pathway inhibition culminated in the blocking of pro-survival autophagy, a mechanism normally adopted by cancer cells to became less responsive to the therapies. The mTOR expression, commonly upregulated in HNSC, was reduced in patients with disease-recurrence. It is well known that mTOR has a strong autophagy inhibition effect, therefore its downregulation promoted pro-survival autophagy, with a related increase recurrence rate. Our findings highlight for the first time the key role of β2-AR and related pathway in HNSCC cell proliferation and drug resistance, proposing it as a valuable therapeutic molecular target. The β2-Adrenergic receptor (β2-AR) is a G protein-coupled receptor (GPCR), involved in the development of many cancers, among which HNSCC. In this contest, β2-AR signaling interacts with different pathways, such as PI3K and MAPK, commonly activated by TK receptors. For this reason, TK blockade is one of the most adopted therapeutic strategies in HNSCC patients. In our study we investigated the effects of the β2-AR blocking in HNSCC cell lines, using the selective inhibitor ICI118,551 (ICI), in combination with the MAPK inhibitor U0126. We found that ICI leads to the blocking of p38 and NF-kB oncogenic pathways, strongly affecting also the ERK and PI3K pathways. Cotreatment with U0126 displays a synergic effect on cell viability and pathway alteration. Interestingly, we found that the β2-AR blockade affects Nrf2-Keap1 stability and its nuclear translocation leading to a drastic ROS increase and oxidative stress. Our results are confirmed by a TCGA dataset analysis, showing that NFE2L2 gene is commonly overexpressed in HNSC, and correlated with a lower survival rate. In our system, the PI3K pathway inhibition culminated in the blocking of pro-survival autophagy, a mechanism normally adopted by cancer cells to became less responsive to the therapies. The mTOR expression, commonly upregulated in HNSC, was reduced in patients with disease-recurrence. It is well known that mTOR has a strong autophagy inhibition effect, therefore its downregulation promoted pro-survival autophagy, with a related increase recurrence rate. Our findings highlight for the first time the key role of β2-AR and related pathway in HNSCC cell proliferation and drug resistance, proposing it as a valuable therapeutic molecular target.

S-Adenosyl-l-methionine (AdoMet) is a naturally and widely occurring sulfonium compound that plays a primary role in cell metabolism and acts as the principal methyl donor in many methylation reactions. AdoMet also exhibits antiproliferative and proapoptotic activities in different cancer cells. However, the molecular mechanisms underlying the effects exerted by AdoMet have only been partially studied. Inthe current study, we evaluated the antiproliferative effect of AdoMet on Cal-33 oral and JHU-SCC-011 laryngeal squamous cancer cells to define the underlying mechanisms. We demonstrated that AdoMet induced apoptosis in Cal-33 and JHU-SCC-011 cells, involving a caspase-dependent mechanism paralleled by an increased Bax/Bcl-2 ratio. Moreover, we showed, for the first time, that AdoMet induced ER-stress in Cal-33 cells and activated the unfolded protein response, which can be responsible for apoptosis induction through the activation of CHOP and JNK. In addition, AdoMet-induced ER-stress was followed by autophagy with a consistent increase in the levels of the autophagic marker LC3B-II, which was indeed potentiated by the autophago-lysosome inhibitor chloroquine. As both escape from apoptosis and decreased activation of JNK are mechanisms of resistance to cisplatin (cDPP), an agent usually used in cancer therapy, we have evaluated the effects of AdoMet in combination with cDPP on Cal-33 cells. Our data showed that the combined treatment resulted in a strong synergism in inhibiting cell proliferation and in enhancing apoptosis via intrinsic mechanism. These results demonstrate that AdoMet has ER-stress-mediated antiproliferative activity and synergizes with cDDP on cell growth inhibition, thus providing the basis for its use in new anticancer strategies.

Background To get insight into the molecular mechanisms underlying the anti-tumor activity of S-adenosyl-l-methionine (AdoMet), we analyzed AdoMet-induced modulation of microRNAs (miRNAs) expression profile in MCF-7 breast cell line and its correlation with cancer-related biological pathways. Methods MiRNA expression profiling was performed using a TaqMan MiRNA Array, following 500 µM AdoMet-treatment. The results were confirmed by Quantitative real-time PCR analysis. MCF-7 were transfected with miR-34a, miR-34c and miR-486-5p, mimics and inhibitors in presence or not of 500 µM AdoMet for 72 h. Apoptosis and autophagy were analyzed by flow cytometry and the modulation of the main antiproliferative signaling pathways were evaluated by Western blotting. The potential mRNA targets for each miRNA were identified by the TargetScan miRNA target prediction software. Results Twenty-eight microRNAs resulted differentially expressed in AdoMet-treated MCF-7 cells compared to control cells. Among them, miRNA-34a and miRNA-34c were up-regulated while miRNA-486-5p was down-regulated. Moreover, we confirmed the ability of AdoMet to regulate these miRNAs in MDA-MB 231 breast cancer cell line. We demonstrate that, in MCF7 cells, the combination of either miR-34a or miR-34c mimic with AdoMet greatly potentiated the pro-apoptotic effect of AdoMet, by a caspase-dependent mechanism and activates p53 acetylation by inhibiting SIRT1 and HDAC1 expression. We also showed that miR-486-5p inhibitor induces autophagy and enhances AdoMet-induced autophagic process by increasing PTEN expression and by inhibiting AKT signaling. Conclusions Our findings provide the first evidence that AdoMet can regulate miRNA expression in MCF-7 increasing our knowledge on the molecular basis of the antitumor effect of the sulfonium compound and suggest the use of AdoMet as an attractive miRNA-mediated chemopreventive and therapeutic strategy in breast cancer.

The naturally-occurring sulfonium compound S-adenosyl-L-methionine (AdoMet) is an ubiquitous sulfur-nucleoside that represents the main methyl donor in numerous methylation reactions. In recent years, it has been shown that AdoMet possesses antiproliferative properties in various cancer cells, but the molecular mechanisms at the basis of the effect induced by AdoMet have been only in part investigated. In the present study, we found that AdoMet strongly inhibited the proliferation of breast cancer cells MCF-7 by inducing both autophagy and apoptosis. AdoMet consistently enhanced the levels of the autophagy markers beclin-1 and LC3B-II, and caused a significant increase of pro-apoptotic Bax/Bcl-2 ratio paralleled by poly (ADP ribose) polymerase (PARP) and caspase 9, and 6 cleavage. Notably, AdoMet, already at low doses, raised the percentage of cells in G2 /M phase of cell cycle by down-regulating the expression of cell cycle-regulatory proteins cyclin B and cyclin E with a remarkable increase of p53, p27 and p21. We also evaluated the combination of AdoMet and the autophagy inhibitor chloroquine (CLC) showing that autophagy block is synergistic in inducing both growth inhibition and apoptosis. These effects were paralleled by a strong inhibition of the activity of AKT and of the downstream effector mTOR and by an increased cleavage of caspase-6 and PARP. These data suggest, for the first time, that autophagy can act as an escape mechanism from the apoptotic activity of AdoMet, and that AdoMet could be used in combination with CLC or its analogs in the treatment of breast cancer. This article is protected by copyright. All rights reserved.

NOTCH1 mutations have recently emerged as new genetic lesions significantly correlated with survival in chronic lymphocytic leukemia (CLL). NOTCH1 c.7541_7542delCT is by far the most frequently observed NOTCH1 mutation in the disease. To estimate the prevalence and clonal evolution of NOTCH1 c.7541_7542delCT mutation, and prospectively investigate its clinical significance in early stage CLL and clinical monoclonal B cell lymphocytosis (cMBL), we analyzed by next generation sequencing (NGS) 384 cases at diagnosis enrolled in the GISL O-CLL1 multicenter trial. The patient cohort included 100 cMBL and 284 Binet stage A CLL cases, 48 of whom were also longitudinally investigated at progression or during follow-up (32 and 16, respectively) in absence of treatment. Deep sequencing of the NOTCH1 mutation hotspot was performed by Roche 454 pyrosequencing on the Genome Sequencer Junior instrument. NOTCH1 mutation was validated by an extremely sensitive PCR-based approach and Sanger sequencing. The association between NOTCH1c.7541_7542delCT and clinical, molecular and biological variables, as well as its impact on progression free survival (PFS), were tested. Deep sequencing analysis of NOTCH1 mutation hotspot in our cohort (median depth of coverage 1510x, ranging from 605 to 2842) revealed a mutant allele frequency ranging from 0.02% to 75% of total reads in 145 cases. The occurrence of the mutation was subsequently assessed by an extremely sensitive ARMS (amplification refractory mutation system)-PCR, which allowed to confirm the presence of delCT in the 49 cases with frequency of mutated sequencing reads greater than 0.7%, specifically in 11% of cMBL (11/100) and 13.4% of CLL patients (38/284). Furthermore, mutated samples were subjected to DNA Sanger sequencing: in line with the expected sensitivity of the method, the mutation was identified only in samples with higher mutation loads according to NGS (mutant allele frequency ≥ 7%, n=25). Our data revealed that often NOTCH1 mutational activation affected a neoplastic sub-clone, especially in cMBL patients. NOTCH1 mutated patients utilized unmutated IGHV genes more frequently, and had higher expression of CD38 and ZAP-70 (P=3.2e-11, P=2.6e-08, P=3.4e-05, respectively). Trisomy 12 was more frequent in this patient group (P=5.4e-04), whereas 13q14 deletion was less represented than in the NOTCH1 wild-type patients (P=2.8e-03). NOTCH1 mutation was associated with the occurrence of stereotyped HCDR3 (P=5.6e-03); in addition, compared with other major BCR subsets, CLL subset #10 was significantly enriched in NOTCH1 mutations (P=0.032). The prevalence of the analyzed dinucleotide deletion was not significantly different between cMBL and CLL patients, even if only Rai 0 cases (28/197 cases, 14.2% mutation frequency) were considered. The percentages of variant sequencing reads in NOTCH1-mutated cases were slightly higher in CLL (median 19.6%) than in cMBL (median 4.2%), a finding confirmed by a regression analysis that highlighted the association of the CLL presentation with higher percentages of NOTCH1 delCT reads (P=0.033). NOTCH1-mutated cases, both at sub-clonal and clonal levels, displayed a significant reduction in median PFS (P=0.0018), although NOTCH1 mutation prognostic value, in multivariate analysis, was not independent if 11q and/or 17p deletion, IGHV mutational status, and cMBL or CLL status were considered. Finally, sequential analyses in a representative fraction of cases of our dataset indicated that (i) NOTCH1 mutation did not occur during the course of the disease and that (ii) the mutational load in positive cases was stable over time. These findings highlight the importance of using high sensitive methods for an accurate detection of NOTCH1 mutation in cMBL/early stage CLL. This is required for a better prognostic stratification and also to obtain useful information for potential therapeutic approaches, since sub-clonal mutations in untreated CLL can possibly anticipate the dominant genetic composition of the relapsing tumor. Disclosures: No relevant conflicts of interest to declare.

Abstract 4040 Multiple myeloma (MM) is a clonal proliferation of malignant plasma cells (PCs) characterized by a marked genomic instability. Primary plasma-cell leukemia (pPCL) is an aggressive, rare variant of plasma cell dyscrasia characterized by extra-medullary proliferation of PCs, high genomic instability and very poor prognosis. To date, global genomics studies in pPCL are still limited. Highly purified PCs were obtained from 17 previously untreated pPCL patients, recruited in an open-label, exploratory, single-arm, two-stage study from the GIMEMA myeloma network aimed at the evaluation of safety and antitumor activity of the immunomodulatory agent lenalidomide in combination with low dose dexamethasone in previously untreated pPCL. All the samples were characterized for the main chromosomal aberrations by Fluorescence in-situ hybridization (FISH). Specifically, 13q and 17p deletions have been identified in 12/17 (70.6%) and 8/17 (47.1%) cases, respectively; the presence of t(11;14) was found in 4/17 patients (23.5%), t(4;14) in only 1/17 cases (5.9%) and MAF translocations in 8/17 (47.1%). To further investigate the genomic complexity of pPCL, we analyzed a subset of 13 samples by means of an integrative approach using different high-throughput microarray technologies: Human Mapping 250K Nsp SNP-array (Affymetrix) for the detection of copy number alterations (CNA), Gene 1.0 ST array (Affymetrix) for transcriptional profiling and miRNA Microarray v2 (Agilent) for the global miRNA expression. SNP-array data were concordant with FISH results for the detection of the main chromosomal aberrations, i.e. 13q (10/13=77%) and 17p (7/13=58.3%) deletions. The most recurrent CNA specifically identified by SNP-array was represented by 1q gain (8/13=61.5%); in addition. losses involving chromosomes 1p (5/13=38.5%), 8p (4/13=30.8%), 14q (5/13=38.5%), 16q (5/13=38.5%), gains affected 7q (4/13=30.8%) and 19p (4/13=30.8%) and one amplification at 17q21 in 6/13 pPCL (46.2%) were detected. One case displayed a near tetraploid karyotype and, interestingly, another one showed a hyperdiploid pattern. Mapping information was integrated with the gene expression and miRNA profiles of the tumor samples. A non-parametric analysis (Kendall's tau correlation at a p-value Disclosures: No relevant conflicts of interest to declare.

Abstract 2824 Poster Board II-800 Multiple myeloma (MM) is a malignant proliferation of bone marrow (BM) plasma cells (PCs), characterized by a profound genomic instability involving both numerical and structural chromosomal aberrations of potential prognostic relevance. The discovery of microRNA (miRNA) genes, encoding for a class of small non-coding RNAs involved in the regulation of cell cycle, survival and differentiation programs, has added a further level of complexity to normal and cancer cell biology; it has been suggested that chromosomal abnormalities and other types of genetic or epigenetic alterations might contribute to miRNA deregulation in cancer. To date, only little evidence of miRNA expression/deregulation in multiple myeloma (MM) has been reported. To characterize miRNA expression profiling of MM plasma cells (PCs) and integrate miRNA expression data with other molecular features of MM patients, global miRNA expression profiles of PCs isolated from BM biopsies of 38 newly diagnosed MM, 2 plasma cell leukemia patients, and 3 healthy donors were generated using the Agilent Human miRNA microarray V2 (representing 723 human mature miRNAs from the Sanger miRBase v10.1). All of the patients had previously been characterized by FISH for the main IGH translocations and other genetic abnormalities; they were also profiled for global gene expression by means of Affymetrix U133A arrays; nineteen of the patients profiled for miRNA expression underwent genome-wide DNA analysis using Affymetrix GeneChip® Human Mapping 50K XbaI microarrays. An unsupervised analysis of the samples, using conventional hierarchical clustering algorithm and based on the most variably expressed miRNAs across the dataset, grouped the PCs from healthy donors separately from MM PCs; among the pathological samples, the most striking finding was that the seven patients with t(4;14) (TC4) were tightly clustered, as were four out of the five samples with translocated MAF genes (TC5). A partial grouping of the TC2 cases (mostly hyperdiploid) was also observed, whereas the TC1, showing t(11;14), and TC3 (mostly expressing Cyclin D2), samples were dispersed along the dendrogram. A multiclass supervised analysis of the miRNA expression between the members of the 5 TC groups highlighted specific miRNA signatures, in particular characterizing the TC4 and TC5 groups. A slight miRNA signature was observed in t(11;14) cases and TC2 group, whereas we could not identify any TC3-specific miRNA signature. None of the differentially expressed miRNAs in patients with specific IGH translocations maps to the rearranged chromosomal regions. The levels of some differentially expressed miRNAs were quantified by means of Q-RT-PCR in all of the 43 samples, using specific TaqMan® microRNA assays (Applied Biosystems); a linear correlation analysis indicated very good correspondence between microarrays and Q-RT-PCR data. Furthermore, the expression of specific miRNAs was also evaluated in 14 additional cases, four carrying t(11;14), seven t(4;14), and three t(14;16) or t(14;20): as well, in this broaden panel the selected miRNAs confirmed their TC-specific over-expression. The occurrence of other lesions, such as 1q gain, 13q and 17p deletions, and hyperdiploidy, was slightly characterized by specific miRNA signatures. Furthermore, the integrated analysis with the genomic profiles revealed the occurrence of several allelic imbalances significantly associated with the altered expression of miRNAs located in the involved regions, such as let-7b at 22q13.31 (loss) and miR-142-3p at 17q22 (gain), or miR-140-3p at 16q22 (loss-of-heterozygosity). Finally, in an attempt to define the consequences of deregulated miRNA expression, we performed an integrative analysis based on computational target prediction, miRNA and mRNA profiling. Specifically, we searched for putative functional targeting relationships in MM cells supported by expression data, i.e. anti-correlations between miRNA and target mRNA expression profiles, and thus defined a global miRNAs/mRNAs regulatory network. Our data provide the first evidence of miRNA deregulation in the context of the molecular subtypes of MM, and represent the first attempt to define the complex of miRNAs/mRNAs regulatory relationships, aimed at deepening our understanding of the involvement of specific miRNAs and target genes in the pathology of the disease. Disclosures: No relevant conflicts of interest to declare.

Multiple myeloma (MM) is a fatal malignancy of clonal bone marrow plasma cells characterized by a high genomic instability increasing with disease progression. We describe here a genomic amplification at 17p11.2–p12, an unstable chromosomal region characterized by a large number of low copy repeats which have been proven to mediate deletion and duplication in several genomic disorders and amplifications in solid tumors. An approximately 5 Mb 17p11.2–p12 amplified region was detected in KMS-26 myeloma cell line by SNP microarray analysis. Further FISH mapping showed two unidentified amplified chromosomes as well as a complex pattern of rearranged chromosomes 17. The analysis of transcriptional profiles in a proprietary data base of myeloma cell lines, identified 12 significantly overexpressed genes in KMS–26 amplified region, including TNFRSF13B/TACI, COPS3, and NCOR1. The evaluation of their expression levels in a data base including 11 monoclonal gammopathy of uncertain significance, 121 MM and 9 plasma cell leukaemia, showed a significant overexpression of at least one gene in 13 patients. FISH analyses of these patients identified one MM carrying a 3.8 Mb amplified region and two MMs with gains specifically involving the TACI locus. Interestingly, the complete inactivation of TP53 at 17p13.1 was found in KMS–26 whereas a monoallelic loss was identifiable in two of the three patients carrying gain/amplification. Our data suggest that, as in the case of solid tumors, amplification/gain of the 17p11.2–p12 in MM could be mediated by the presence of repeats located in this region and may provide insights for defining novel candidate myeloma-associated genes.

Karyotypic instability is strongly associated with multiple myeloma (MM). According to the chromosome number pattern, two major groups are recognized: hyperdiploid (H) tumors, associated with recurrent trisomies involving non-random chromosomes (3, 5, 7, 9, 11, 15, 19 and 21); and non hyperdiploid (NH) tumors associated with hypodiploid, pseudodiploid or near-tetraploid karyotypes. MM patients are approximately equally distributed between the two categories; notably, the most recurrent IGH translocations and chromosome 13 deletion appear to be prevalently associated with NH-MM, whereas recent evidences have suggested that H-MM correlates with a favorable prognosis. To molecularly characterize these two genetic categories, we performed a gene expression profiling analysis on 66 newly-diagnosed MM, characterized by FISH analyses for IGH translocations, 13q14 deletions and additional copies of chromosomes 1, 11 and 19. The ploidy status was investigated by combining two recently proposed FISH approaches (Wuilleme S. et al., 2004; Chng W.J. et al., 2005). The gene expression profiles of highly purified MM plasma cells have been generated by means of high-density oligonucleotide arrays (Affymetrix GeneChip U133A) and subsequently analyzed using unsupervised and supervised approaches (two-dimensional hierarchical clustering and SAM, respectively). The differential expression of 229 genes distinguished the 28 H-MM from the 38 NH-MM cases. The 208 upregulated genes in H-MM mapped mainly on the chromosomes involved in hyperdiploidy, while a significant percentage (29%) of the 21 genes upregulated in NH-MM were localized on 16q. The identified transcripts have been further validated on a publicly available gene expression dataset of an independent cohort of 64 MM patients (Carrasco et al., 2005). Notably, the global classification rate for the 64 cases resulted of 81%, confirming the validity of the identified transcriptional fingerprint. A functional analysis revealed a significant fraction of genes involved in protein biosynthesis (38%), transcriptional machinery and oxidative phosphorylation. Furthermore, an integrative genomic approach using a model-free statistical method (LAP, locally adaptive statistical procedure) supported these findings, allowing the identification in H-MM of globally upregulated regions on the chromosomes 3, 5, 9, 15 and 19, along with the downregulation of a region on 16q arm. Remarkably, two sub-groups are clearly distinguishable within H-MM group, one associated with chromosome 11 gain and the other showing 1q gain and chromosome 13 deletion. A supervised analysis of the H-11 vs H-13/1 patients identified 57 differentially expressed genes. Eleven of the 18 genes up-regulated in the H-11 group mapped to chromosome 11, whereas 21 of the 39 genes up-regulated in the H-13/1 group mapped to the 1q region. Notably, CCND2 resulted the most significantly upregulated gene in H-13/1 group. Our data reinforce the importance of combining cytogenetics and gene expression approaches for a better definition of the genetic alterations in MM and provide a molecular and genomic framework for dissection of disease pathogenesis and clinical management.

Neurotrophins support neuronal survival, development, and plasticity through processes requiring gene expression. We studied how vgf target gene transcription is mediated by a critical promoter region containing E-box, CCAAT and cAMP response element (CRE) sites. The p300 acetylase was present in two distinct protein complexes bound to this region. One complex, containing HEB (ubiquitous basic helix–loop–helix (bHLH)), bound the promoter in non-neuronal cells and was involved in repressing vgf expression. Neurotrophin-dependent transcription was mediated by the second complex, specific for neuronal cells, which included CRE binding protein and MASH1 (neuro-specific bHLH), bound the CCAAT motif, and was target of neurotrophin signalling. The interaction, mediated by p300, of different transcription factors may add specificity to the neurotrophin response.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by the monoclonal accumulation of B lymphocytes and a variable clinical course. Specific B-Cell Receptor (BCR) utilized by leukemic cells may influence disease progression and outcome. Highly homologous BCR, “stereotyped BCR”, are expressed in a recurrent fraction of patients with CLL and in some cases they were associated with distinct biological and clinical features. Stereotyped subset #4 have been reported to exhibit a favorable clinical course and to be the most frequent stereotyped BCR among the IGHV mutated (IGHV-M) cases. In this study we performed a comprehensive clinical, biological and molecular characterization of leukemic cells from 16 patients utilizing stereotyped subset #4 BCR (IGHV4-34) among a representative prospective cohort of 462 Binet stage A CLL patients enrolled in O-CLL1 protocol (clinicaltrial.gov identifier NCT00917540). In all cases, biological and molecular analyses were performed in peripheral CD19+ B-cells. All subset #4 patients were characterized by lower CD38 expression, unique IGHV-M configuration and absence of NOTCH1 and SF3B1 mutations. None subset #4 patients showed unfavorable cytogenetic deletions (i.e del11q23 and del17p13). Gene expression profiling (GEP) analysis was performed on 217 patients, including 9 subset #4 cases for whom RNA material was available. Supervised analysis comparing subset #4 vs all other patients (208) revealed 14 differentially expressed genes. Furthermore subset #4 patients were characterized by a significant downregulation of WDFY4, MEF2A and upregulation of PDGFA, FGFR1 and TFEC genes when compared with the remaining IGHV-M patients. miRNA profiles were analyzed in 229 patients including 10 subset #4 patients for whom RNA material was available. A specific miRNA expression pattern involving the upregulation of miR-497 and miR-29c was found in subset# 4 cases. Furthermore, we demonstrated that transfection of the miR-497 mimic in primary leukemic CLL cells induces, after 48 and/or 72 h, a downregulation of BCL2, known to be a validated target in different solid cancers. Our data provide a contribution to the biological definition of CLL patients with specific stereotyped IGHV4-34 BCR and identify for the first time distinct gene and miRNA expression profiles associated with this subset, providing further evidence of the putative leading role of HCDR3 conformation in CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 3955 Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs involved in the maturation of other RNA molecules and generally located in the introns of host genes. It is an emerging evidence that altered sno/scaRNAs expression may play a pathological role in cancer. Impaired sno/scaRNAs expression has recently been reported both in acute leukemia and smoldering myeloma that rapidly progressed to symptomatic disease. In addition, as regards multiple myeloma (MM), very recent data suggested an oncogenic role for SCARNA22 in those MM patients over-expressing SCARNA22/MMSET as a result of t(4;14) translocation. However, comprehensive information concerning the expression behavior of sno/scaRNAs in MM is still lacking. This study elucidates the patterns of sno/scaRNAs expression in MM by profiling purified malignant plasma cells from 55 MMs, 8 secondary plasma cell leukemias (sPCL) and 4 normal controls using Human Gene 1.0 ST arrays. Overall, a global sno/scaRNAs down-regulation was found in MMs and at more extent in sPCLs compared to normal plasma cells. Whereas SCARNA22 resulted the only sno/scaRNA characterizing the TC4 MM, TC2 group displayed a distinct sno/scaRNA signature overexpressing members of SNORD115 and SNORD116 families located in a region finely regulated by an imprinting center at 15q11 which, however, resulted overall hypomethylated in MMs independently of the SNORD115 and SNORD116 expression levels. In addition, impaired expression of sno/scaRNAs raised from the comparison between MM and sPCL, suggested a role in tumor progression. Furthermore, to uncover possible mechanisms at the basis of sno/scaRNAs deregulation, we investigated the correlation between sno/scaRNAs and the corresponding host-genes expression levels, outlining the coordinated expression of up to 50% of sno/scaRNAs/host-genes pairs. Finally, we investigated whether the sno/scaRNAs transcriptional pattern may be influenced by allelic imbalances involving their genomic location, as already demonstrated concerning mRNA expression, and revealed a dosage effect involving several chromosomal regions. Our data extend the current view of sno/scaRNAs deregulation in cancer and add novel information into the bio-molecular complexity of plasma cell dyscrasias. Furthermore, our findings may contribute to develop functional approaches to examine the activity of deregulated sno/scaRNAs in MM, as well as to further enlighten their possible role as targets of novel therapeutic agents. Disclosures: No relevant conflicts of interest to declare.

Abstract 3938 Primary plasma cell leukemia (PPCL) is a rare and very aggressive form of plasma cells dyscrasia, characterized by poorer outcome than multiple myeloma (MM). To provide insights into the biology of PPCL, we investigated 23 newly-diagnosed patients included in an open-label, multicenter, prospective, single arm, two-stage study aiming to explore efficacy and safety of lenalidomide and dexamethasone combination (LD) as first line therapy in previously untreated PPCL (median follow-up: 23 months; range 9–32). The primary endpoint of the study was the response rate, according to the criteria defined by International Myeloma Working Group, after 4-cycle therapy with LD over a 4-month schedule; among secondary endpoints were overall survival (OS) and eligibility to undergo autologous or allogeneic stem cells transplantation (SCT) after LD treatment. Herein, we took advantage of the FISH characterization and the microarray analysis of transcriptome, miRNome and copy number configurations of the PPCLs to investigate whether a correlation could exist between transcriptional features or allelic imbalances and clinical outcome. FISH was used to detect the main IGH translocations. The gene expression profiles of highly purified plasma cells from PPCLs cases were generated on GeneChip® Gene1.0 ST arrays. Expression values were normalized using robust multi-array average (RMA) procedure. MicroRNA profile were generated on Agilent Human miRNA Microarray V2. Expression values were extracted with Agilent Feature Extraction Software v10.1; quantile normalization was applied on raw data using R aroma.light package. GeneChip® Human Mapping 250K NspI arrays was used for genotyping. Copy number was estimated using circular binary segmentation and normalized on FISH data using R DNA.copy and FBN packages, respectively. To assess correlation between expression values and OS, R globaltest package was used to generate the linear regression model in which the distribution of the response variable is modeled as a function of the expression levels of each gene/miRNA. Our analysis indicated that all but three of the PPCLs had one among t(4;14) (13%), t(11;14) (39%) or MAF-associated translocation (35%). However, neither any of them nor any of the numerical alteration involving 1p, 6p, 8p, 13q, 14q, 16q, 17p (loss) and 1q (gain) as assessed by SNP-arrays were correlated with OS. As well, no correlation between response to treatment with LD and the prevalence of these cytogenetic alterations was evidenced. Of the 1145 most variable gene across the PPCL dataset and OS, 27 reached a highly significant correlation (P Finally, three genes (CYB5D2, EDEM3 and YIPF6) and four miRNAs (miR-497, miR-106b, miR-181a* and miR-181b) were identified having expression levels correlated with response to the first-line treatment with LD. Overall, this study represents the first integrated approach on a prospective study investigating genes and miRNAs expression and genotyping configuration in PPCL, indicating specific genes and miRNAs with relevance in the clinical outcome of the disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 3890 Chronic lymphocytic leukemia (CLL) is a genetically heterogeneous disease with a variable outcome. The identification of factors that could predict the clinical course of early-stage CLL represents a crucial objective. Although previous studies indicated that telomere length may be a useful independent prognostic factor in the risk stratification of CLL, limited information has been reported in asymptomatic early stage patients (Binet stage A). We investigate the association of telomere length with the major biological and cytogenetic markers known to predict clinical outcome in CLL. The global DNA methylation levels of Alu and LINE sequences, was also investigated. Correlation with disease progression, measured as the time elapsed from diagnosis to first treatment, was evaluated. We measured relative telomere length (RTL) by real-time PCR in a panel of highly purified (>90%) peripheral mononuclear CD19+ cells from 7 healthy donors and 77 untreated CLLs. All the cases were characterized by FISH for the most frequent chromosomal aberrations (Fabris et al. GCC, 2008). Molecular markers including mutation status of the heavy chain variable regions of immunoglobulin genes (IGVH), the expression of the 70-kd zeta-chain T-cell receptor-associated protein kinase (ZAP-70) and CD38 cell surface antigen protocols were previously reported (Cutrona et al., Haematologica, 2008). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu and LINE-1. We found a significantly lower RTL values in CLLs (median RTL=0.4 IQR 0.3–0.6) as compared with controls (median RTL=1.0 IQR 0.9–1.3) (P Disclosures: No relevant conflicts of interest to declare.

Abstract 2878 Primary plasma-cell leukemia (pPCL) is an aggressive, rare variant of plasma cell (PC) dyscrasia characterized by extra-medullary proliferation of PCs, high genomic instability and very poor prognosis. The present study was aimed at investigating global genomics in 17 pPCL recruited in an open-label, exploratory, single-arm, two-stage study from the GIMEMA myeloma network designed to evaluate the safety and antitumor activity of lenalidomide in combination with low dose dexamethasone as first-line therapy in pPCL. All the samples were characterized for the main chromosomal aberrations by Fluorescence In-Situ Hybridization (FISH). Specifically, 13q and 17p deletions have been identified in 13 (76.5%) and 6 (35.3%) cases, respectively; the presence of t(11;14) translocation was found in 7 patients (41.2%), t(4;14) in 2 (11.8%) and t(14;16) in 7 (41.2%). To better define the chromosomal alterations of this set of patients, we further investigated them by means of Human Mapping 250K Nsp SNP-array (Affymetrix). SNP-array data were fully concordant with FISH results as regards 13q and 17p deletions in the analyzed patients. Among the copy number alterations identified by mapping analysis the most frequently gained chromosomal region was represented by 1q (9 cases, 52.9%); 1p, 8p, 14q, and 16q arms were affected by loss of DNA material in more than 40% of cases. Moreover, four patients showed gain at 7q (23.5%), one case displayed a near tetraploid karyotype and another one had a hyperdiploid-like pattern. Most of the minimally altered regions identified on the different chromosomes encompassed genes that have been reported to be deregulated in PC dyscrasia, such as CDKN2C (mapped to 1p32.3), FAM46C (1p12), CKS1B (1q21.2), PARK2 (6q26), PPP2R2A (8p21.2), RB1 and MIR-15A/16-1 (13q14.2), TRAF3 (14q32.32), CYLD (16q12.1), WWOX (16q23.3-q24.1), and TP53 (17p13.1). The mutational analysis of the most frequently mutated exons (5–9) of TP53 gene revealed the presence of coding mutations in 4 patients (23.5%), three of which carried a monoallelic deletion including the gene locus. This supports the knowledge that the prevalence of TP53 mutations increases in more advanced disease and is strongly associated with hemizygosity. Genome-wide profiling data were then integrated with the transcriptional profiles generated on Gene 1.0 ST array (Affymetrix). Our analysis (Wilcoxon rank-sum test at a P Disclosures: No relevant conflicts of interest to declare.

Abstract 1814 Poster Board I-840 Background The growth and survival of multiple myeloma (MM) cells in the bone marrow microenvironment is regulated by functional complex interactions between the tumor cells and the surrounding bone marrow stromal cells mediated by adhesion molecules and the production of several cytokines of which interleukin-6 (IL-6) has been identified as the most important. Major advances in the investigation of MM biology were made possible by the availability of human myeloma cell lines (HMCLs). The IL-6-dependent CMA-03 cell line was established in our laboratory from a peritoneal effusion of a refractory relapsed MM patient. By gradually decreasing the IL-6 added to the culture, an IL-6-independent variant, CMA-03/06, could be obtained. Aims. To perform a biological and molecular characterization of this novel cell line, and to provide insights into the signaling pathways and target genes involved in the growth and survival of CMA-03/06. Methods. The growth, immunophenotypic, cytogenetic and fluorescence in situ hybridization (FISH) characterization of CMA-03/06 cell line was performed by means of standard procedures. IL-6 production into the culture media was determined using a high sensitivity IL-6 specific ELISA. Genome-wide profiling data were generated by means of Affymetrix GeneChip® Human Mapping 250K Nsp arrays; copy number (CN) alterations were calculated using the DNAcopy Bioconductor package, based on circular binary segmentation method. Global gene expression profiling (GEP) was performed by means of the GeneChip® Human Gene 1.0 ST Arrays (Affymetrix); the supervised analyses were done using the SAM software version 3.0. Results Unlike CMA-03, the addition of IL-6 to the culture medium of CMA-03/06 cells or co-culture with multipotent mesenchymal stromal cells did not induce an increase in CMA-03/06 proliferation. IL-6 was not detected in the supernatants from either CMA-03 or CMA-03/06 cell lines within 48 h, suggesting that the IL-6 independence of CMA03/06 cells is not a result of the development of an autocrine IL-6 loop. Nevertheless, IL-6 induced the activation of STAT3 and STAT1 in both cell lines, even if a slight constitutive STAT3 phosphorylation was found in CMA-03/06. The immunophenotypic analysis showed a significant difference in the expression of three antigens in the 2 cell lines: CD45 was considerably reduced in CMA-03/06 cells, whereas they were found positive for both chains of IL-6 receptor, CD126 and CD130, almost undetectable in CMA-03. Conventional cytogenetic and FISH analyses did not reveal differences between the 2 HMCLs. The genome-wide analysis allowed the identification of about 100 altered chromosomal regions common to both HMCLs, mostly DNA gains. Comparison of CMA-03/06 and CMA-03 cells evidenced a different CN in only 15 small chromosomal regions, 8 of which did not contain any transcript, whereas few genes were located on the other ones. GEP analysis of CMA-03/06 compared with CMA-03 identified 21 upregulated and 47 downregulated genes, many of which particularly relevant for MM biology, mainly involved in cellular signaling, cell cycle, cell adhesion, cell development, regulation of transcription, immunologic, inflammatory or defense activity, apoptosis. None of the genes differentially expressed in CMA-03/06 compared with CMA-03 except 1 were positioned on the chromosomal regions showing a different CN. Finally, CMA-03/06 cell line showed a lower susceptibility to camptothecin-induced apoptosis compared to CMA-03 cells. Conclusions Our data show the IL-6 independence of CMA-03/06 cell line in the absence of an autocrine IL-6 loop; the cells, however, maintain the IL-6 signaling pathway responsiveness. A consistent number of genes particularly relevant for MM biology were found deregulated in CMA-03/06 cell line compared with CMA-03. Furthermore, CMA-03/06 cell line shows an increased resistance to apoptosis. The novel CMA03/06 cell line may thus represent a suitable model for studies investigating molecular mechanisms involved in clonal evolution towards IL-6 and/or stroma-independent growth and survival of myeloma cells. Disclosures No relevant conflicts of interest to declare.

B-cell chronic lymphocytic leukemia (B-CLL) is a genetically heterogeneous disease with a variable clinical course. Chromosomal changes have been identified by FISH in approximately 80% of patients, and the presence of specific lesions, such as trisomy 12 and 13q14, 11q23, 17p13.1 and 6q23 deletions represent prognostic markers for disease progression and survival. In order to characterize further the complexity of B-CLL genomic lesions, we performed high density, single nucleotide polymorphism (SNP) array analysis in highly purified neoplastic cells (>92%) from a panel of 100 untreated, newly diagnosed patients (57 males and 43 females; age, median 63 years, range 30–87) in Binet stage A. All patients were investigated by FISH for the presence of trisomy 12 (21 cases); 13q14 deletion (44 cases, 34 as the sole abnormality); 11q22.3, 17p13.1 and 6q23 (15, 7 and 2 patients, respectively). In addition, ZAP-70 and CD38 expression resulted positive in 42 and 46 patients, whereas IgVH genes were mutated in 45 patients. Genome-wide DNA profiling data were generated on GeneChip® Human Mapping 250K NspI arrays (Affymetrix); copy number alterations (CNA) were calculated using the DNA copy Bioconductor package, which looks for optimal breakpoints using circular binary segmentation (CBS) (Olshen et al, 2004). A total of 782 CNAs (ranging from 1 to 31 per sample, mean and median values 7.82 and 7, respectively) were detected; DNA losses (365/782=46.67% loss; 194/782=24.81% biallelic deletion) were found to be more frequent than gains (148/782=18.93% gain; 75/782=9.59% amplification). The most recurrent alterations detected by FISH were all confirmed by SNP array analysis, strengthening further the good reliability of such high-resolution technology. We identified 12 minimally altered regions (MARs) larger than 100 kb with a frequency higher than 5%. Among well known alterations, the largest was represented by chromosome 12 trisomy, followed by 6q, 17p and 11q23 deletions (32.87, 19.09 and 10.43 Mb, respectively) and 13q14 deletion (635 kb). Gain of 2p25.3 involves a common region of 4.39 Mb region in 7 patients, although it was extended to the whole short arm of chromosome 2 in 3 cases. Among those alterations previously described in B-CLL, we found losses at 14q32.33 (12 pts) and 22q11.2 (5 pts) involving the IGH and IGLλ loci, respectively. With regard to novel regions, we identified losses at 4q35.2 (5 pts) and 11q25 (6 pts). In addition we found a high frequency of losses/gains at 14q11.2 (42 pts) and 15q11.2 (33 pts), two genomic regions reported to be affected by DNA copy number variations in normal individuals. As regards correlations between CNAs and biological markers, we found that the number of CNAs is significantly higher in cases with unmutated IgVH (9.4; range 2–31) as compared with mutated IgVH (6; range 1–13) (p=0.002), while neither CD38 nor ZAP-70 expression showed significant correlation. In addition, a significant higher number of either CNAs (p=0.001), total MARs (p

The recent discovery of microRNAs (miRNAs), small noncoding RNAs involved in the regulation of cell cycle, survival, and differentiation programmes, has added a further level of complexity to normal and cancer cell biology. Loss or amplification of miRNA genes by broad cytogenetic abnormalities or minute molecular aberrations has been observed in a variety of human malignancies, with the consequent altered expression of these regulatory genes. Additionally, approximately one third of miRNAs are located within the intronic regions of coding transcription units, and recent evidence indicates that the expression of these miRNAs largely coincides with the transcription of the corresponding host genes. To date, no evidence of deregulated miRNA expression has been reported in multiple myeloma (MM). To provide insights into miRNA biology in MM, we performed an integrative analysis of genome-wide, gene expression and miRNA expression profilings in a panel of 16 human myeloma cell lines (HMCLs). Global miRNA and mRNA expression data were generated on Agilent miRNA microarrays (representing 470 human mature miRNAs) and GeneChip® HG-U133A arrays, respectively, and both quantile-normalized. Genome-wide profiling data were generated on GeneChip® Human Mapping 250K NspI arrays and copy number (CN) values were inferred using the circulary binary segmentation (DNAcopy R Bioconductor package). To measure the correlation between the expression levels of each miRNA and the corresponding CN value or host gene expression, conventional non-parametric analyses were performed (Kendall’s tau and Wilcoxon rank-sum tests). As regards miRNA gene CN, the most frequent alteration identified was represented by gain/amplification (for all miRNA genes investigated, an increased CN was present in at least 3 HMCLs, with an average frequency of 58%), followed by loss (5%) and biallelic deletion (0.3%). Our analysis revealed that 14 different miRNA transcripts (miR-15a, miR-19a, miR-21, miR-22, miR-30d, miR-99b, miR-130b, miR-132, miR-140, miR-185, miR-339, miR-491, miR-503, miR-768-3p) had concordant levels with the inferred CN value of the corresponding miRNA gene. Notably, the identified miRNAs mapped to different genomic regions, some of which are involved in recurrent CN alterations in MM, such as 8q24, 19q13.33, or chromosome arms 13q, 16q, 17p, 17q, 22q, and for some of the miRNAs a role in other types of cancer has already been suggested. As regards intragenic miRNAs, 187 miRNA/host gene pairs were obtained after localizing miRNAs within the absolute 5′ and 3′ regions of genes represented on the HG-U133A arrays; 25 of these showed a significant correlation between miRNA and mRNA levels. Among the most correlated miRNA/hostgene pairs we identified miR-152/COPZ2, miR-342-3p/EVL, miR-335/MEST, miR-25 and miR-106b/MCM7. For some of the identified pairs, miRNA expression levels were validated by means of Q-RT-PCR. In conclusion, we showed that miRNA expression in HMCLs could be affected by the presence of genomic lesions or may correlate with host-gene modulation, suggesting a possible role in the molecular pathogenesis of MM. Our integrative approach represents the basis for further investigations, also in primary tumors, aimed at functionally characterizing specific miRNAs in MM.

Multiple myeloma (MM) is a malignant proliferation of bone marrow plasma cells characterized by a wide spectrum of genetic and epigenetic changes. Global hypomethylation of repetitive genomic sequences such as long interspersed nuclear elements-1 (LINE-1) and Alu repetitive elements (approximately 500.000 and 1.4 million in the human genome) has been associated with chromosomal instability. Additionally, satellite alfa DNA (SAT-alpha DNA) hypomethylation has been reported to be associated to karyotypic instability in human cancer, possibly playing a role in centromere function. So far, the LINE-1/Alu and centromeric SAT-alpha DNA methylation patterns have not been investigated in the context of the different clinical and molecular MM subtypes. Global DNA methylation changes were investigated in a panel of 53 newly diagnosed, untreated MMs, 7 plasma cell leukemias (PCL) and 11 healthy subjects as controls. DNA was extracted from purified plasma cells, treated with bisulfite and analyzed by bisulfite-PCR and Pyrosequencing. Methylation of LINE-1 and Alu elements was shown to correlate with total 5mC content and thus used to estimate global DNA methylation. MMs showed a decrease of Alu (21.1%) and LINE-1 (70.0%) methylation average levels compared with controls (25.2% and 79.5% respectively). Lower median methylation levels were also found in centromeric SAT-alpha DNA of MMs (77.95%) compared to controls (89.5%). The median methylation level of PCLs was lower than MMs (16.7% versus 21.1% for Alu; 45.5% versus 70.0% for LINE-1; and 33.3% versus 77.9% for SATalpha DNA). Notably, a statistically significant association between SAT-alpha DNA and LINE-1 methylation (Spearman’s rank correlation, ρ = 0.94; P < 0.001) was found in MM. The comparison between methylation pattern and different molecular MM subgroups by means of non parametric tests, revealed that LINE-1 and SAT-alpha DNA methylation was significantly lower in the nonhyperdiploid versus hyperdiploid (HD) tumors (P = 0.01 and 0.02 respectively). Alu and SAT-alpha were significantly lower in the MMs with t(4;14) (P = 0.02 and 0.004 respectively). Finally, in the context of translocation/cyclin D (TC) classification, a statistically significant differences inside the five different groups were found for SAT-alpha DNA methylation (P = 0.008, Kruskal-Wallis test). These findings may provide insights into the molecular mechanisms of MM pathogenesis and suggest that our approach may contribute toward a more exhaustive stratification of the disease.

Multiple myeloma (MM) is characterized by a high genomic instability that involves both ploidy and structural rearrangements. Nearly half of MM tumors are non-hyperdiploid and are frequently associated with 13q deletion and chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32. The remaining tumors are hyperdiploid, showing low prevalence of both IGH translocations and chromosome 13 deletions. Our study was aimed at providing insights into the genomic heterogeneity associated with plasma cell neoplasms by defining the genome-wide pattern of genetic lesions in a representative and stratified panel of MM patients. To this end, genome-wide profiling data of 45 plasma cell dyscrasia patients (41 MM and 4 plasma cell leukemia) were generated on GeneChip® Human Mapping 50K Xba SNP arrays, and the local DNA copy number variations were calculated using the DNAcopy Bioconductor package. The patients were clustered using the non-negative matrix factorization (NMF) algorithm to identify, within the natural grouping of profiles, the strongest clusters on the basis of their genomic characteristics. We identified three consistent clusters, characterized byrecurrent gains of odd-chromosomes, suggestive of the hyperdiploid status (Group A),high frequency of chromosome 13 deletion and 1q gains (Group B), orhigh frequency of chromosomes 13, 14, 16 and 22 deletions and losses of 1p and 4p regions, together with some cases showing 1q gains (Group C). To determine whether peculiar transcription fingerprints characterized these groups, gene expression profiles of 40 out of 45 corresponding samples generated on GeneChip® HG-U133A arrays were analyzed using the Prediction Analysis of Microarray (PAM) software. The multi-class analysis identified 229 transcripts (corresponding to 195 genes), which specifically marked the three groups. In particular, Group A was characterized by the overexpression of genes involved in the translational machinery or thought to be involved in MM pathogenesis such as the HGF, the tumor necrosis factor ligand TRAIL, DKK1, and c-KIT. Upregulation of the CKS1B gene was present in Group B and C, most likely reflecting the high frequencies of 1q gains in tumors within group B and C and its consequent deregulation. Group C was marked by the specific downregulation of genes mainly mapped to 1p arm: AMPD1, CSDE1, AKR1A1 and the PRKACB kinase, suggesting a relationship with the recurrent 1p loss within the group. Our data further supported the notion that structural abnormalities in multiple myeloma are associated with gene expression imbalances, and provide novel analytical approaches for the identification of genetic lesions and molecular patterns of the disease.