ALBERT

Description: 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

Description: Abstract 2423 Chronic lymphocytic leukemia (CLL) is characterized by an extremely variable clinical course. Mutational status of the immunoglobulin heavy-chain variable (IGHV) region defines two disease subsets with different prognosis. A fraction of CLL cases carries highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3). We performed sequence analysis to characterize IGHV regions in a panel of 1133 CLL patients investigated by a multicenter Italian study group. A total of 1148 rearrangements were identified; the analysis of stereotyped subsets was performed based on previously reported criteria (Messmer et al, J Exp Med 2004; Stamatopuolos et al, Blood 2007). Specifically, we compared all our sequences with those found in three different publicly available data sets (Stamatopoulos et al, Blood 2007; Murray et al, Blood 2008 and Rossi et al, 2009 Clin Cancer Res). In addition, a pairwise alignment within all sequences was performed in order to discover novel potential subsets (HCDR3 identity 〉 60%). Based on the 2% cut-off used to discriminate between Mutated (M) and Unmutated (UM) cases, 777 sequences (67.59%) were classified as M, while 371 sequences (32.3%) as UM. The most represented IGHV genes within mutated cases were IGHV4-34 (104/118) and IGHV3-23 (85/96), whereas IGHV1-69 (97/112) was the most frequently used in the UM group. Interestingly, the IGHV3-21 gene, reported to be frequently expressed in CLL patients from Northern Europe, was present in only a small fraction of cases (24; 2.07%), confirming a previous finding reported by Ghia et al (Blood 2005) in a smaller panel. In our series, stereotyped HCDR3 sequences were found in 407/1148 (35.45%) patients, 177 of whom were M and 230 were UM cases. Overall, we observed that stereotyped sequences were significantly associated with UM IGHV status (Fisher's exact test, P

Description: Abstract 797 We have previously shown that the pan-HDAC inhibitor ITF2357 has strong cytotoxic activity against cells from patients with myeloproliferative neoplasms (MPN) bearing JAK2 mutation at position 617. Indeed ITF2357 inhibited colony growth of JAK2V617F positive cells at doses 5–10 fold lower than those required to block JAK2 wild type cells. We have therefore investigated here the molecular mechanism of this effect. Three cell lines homozygotes (HEL, UKE1) or heterozygotes (SET2) for the JAK2V617F mutation were used along with cell lines bearing JAK2 wild type (K562 and KG1). We confirmed the higher sensitivity of mutated with respect to unmutated cell lines in colony formation assay (mean IC50 42 nM versus 179 nM) and alamar blue assay (mean IC50 84 nM vs 325 nM, respectively). In proliferation assays measuring number of live and dead cells at different time points, we observed that 100 nM ITF2357 blocked the proliferation of both JAK2 mutated and unmutated cell lines to a similar extent, with mean inhibition of 31–69% at 72 hours, but induced apoptosis more efficiently in JAK2 mutated (mean 34%) versus unmutated cells (mean 2%). By cell cycle analysis we could show a block in G1 phase of cell cycle in JAK2V617F cells treated with 100 nM drug. In order to unravel the mechanism of specific inhibition of JAK2 mutated cells by ITF2357, we first investigated expression of HDAC isoforms in the different cell lines. We could detect HDAC1, HDAC2 and HDAC3 proteins in Western blots but these were not differentially expressed in a panel of 3 JAK2 mutated and 3 wild type cell lines. We then set out to analyse the molecular mechanism of action of ITF2357 by global gene expression analysis. Using the Rank Product method with a false positive prediction (pfp) of 0.05 and a 2 fold change cut off parameters, we observed 716 and 863 genes modulated at 6 hours by 250 nM ITF2357 in HEL and UKE-1 cell lines, respectively; 293 of these, (179 up- and 114 down-regulated), were common between both cell lines and 10 were subsequently validated by Q-RT-PCR. Among differentially expressed genes, a number are known to play an important role in the control of proliferation and /or apoptosis, most notably APAF1, BCL2L11, CCNG2, NFKB2, MXD1 and TP53INP1, while additional 6 genes (C-MYB, A-MYB, TAL1, NFE2, MLF1, NOTCH2) are involved in the control of hematopoietic differentiation. Of particular interest is NFE2, which was down modulated 2.7 fold by ITF2357 at 6 hours at the RNA level and by about 2 fold at 24 hours at the protein level. NFE2 has been reported to be hyperexpressed in JAK2V617 MPN patients. We also showed that ITF2357 downmodulated NFE2 expression 2 fold also in CD34+ cells purified from these patients. Given the accepted role of NFE2 in the control of erythroid progenitor cell proliferation and differentiation, and its enhanced expression in MPN patients, our data suggest that NFE2 down-regulation by ITF2357 may at least partially explain the drug effect on growth of MPN progenitor cells. The regulation of NFE2 expression and that of other hematopoietic transcription factors and regulatory proteins in response to ITF2357 is under investigation in our laboratory and data will be presented. Disclosures: Fossati: Italfarmaco SpA: Employment. Rambaldi:Italfarmaco SpA: Research Funding. Golay:Italfarmaco SpA: Research Funding.

Description: 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

Description: Abstract 1780 Background: Biological features related to the development of autoimmune hemolytic anemia (AHIA) in patients with chronic lymphocytic leukemia (CLL) are crucial insights in the understanding of the pathogenesis of autoimmune phenomena in the course of the disease. Design and Methods: We retrospectively analyzed 585 CLL patients with available immunoglobulin heavy-chain variable (IGHV) gene status and B-cell receptor (BCR) configuration (HCDR3). Of them, 73 developed AIHA. The clinical characteristics at CLL diagnosis and follow-up were available in all patients, while cytogenetic analysis at the time of diagnosis was available in 409 patients. Results: Occurrence of AIHA was significantly associated with an IGHV unmutated (UM) status (p

Description: 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

Description: 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.

Description: Abstract 107 The considerable genetic predisposition to deep vein thrombosis (DVT) is only partially accounted for by known genetic risk variants. Genome-wide association studies investigating hundreds of thousands of common single nucleotide variants (SNVs) identified a number of associations, but missing heritability remains. Rare (i.e. minor allele frequency below 1%) and low-frequency (i.e. minor allele frequency below 5%) SNVs of the coding area may be responsible for at least part of this missing heritability. In order to investigate this, we sequenced 700,000 base pairs of genomic DNA including the protein coding exons and intron-exon boundaries of 186 hemostatic/pro-inflammatory genes. Indexed genomic DNA libraries were co-captured on NimbleGen HD2 2.1M-probe chips and capture products were sequenced on SOLiD 4 platforms. More than 70 billion base-pairs of raw sequence data were produced to sequence the target area with a median redundancy of 45X in 94 thrombophilia-negative patients with DVT and 98 controls. Most of the 4366 SNVs identified were rare, novel and nonsynonymous indicating pathogenetic potential. We tested the association of coding SNVs in the ADAMTS13 and VWF genes, encoding two interconnected proteins with fundamental roles in hemostasis. Sequencing of the two genes yielded 109 variants, 108 SNVs and a c.8241_8442del frameshift deletion in exon 51 of VWF. Being a carrier of rare coding (prevalence in DVT: 17% [n=16]; prevalence in controls 4% [n=4]; odds ratio [OR]: 4.8; 95% confidence interval [CI]: 1.6–15.0), rare nonsynonymous (prevalence in DVT: 11% [n=10]; prevalence in controls 3% [n=3]; OR: 3.8; 95% CI: 1.0–14.2) or low-frequency coding (prevalence in DVT: 36% [n=34]; prevalence in controls 23% [n=23]; OR: 1.9; 95% CI: 1.0–3.5) SNVs of ADAMTS13 was associated with DVT. Carrying rare or low-frequency SNVs of VWF was not associated with DVT. The 11 different rare missense variants of ADAMTS13 found in DVT patients had never been described in association with congenital thrombotic thrombocytopenic purpura. Patients carrying at least one ADAMTS13 mutation of the categories associated with DVT had lower plasmatic levels of ADAMTS13 activity compared to patients without mutations. The change in ADAMTS13 activity was −7% (95% CI: −24 to 10%) for patients with rare coding ADAMTS13 mutations, −12% (95% CI: −30 to 6%) for patients with rare nonsynonymous mutations and −29% (95% CI: −52 to −6%) for patients with missense mutations predicted to be damaging for protein function by SIFT. Our results uncover for the first time a link between ADAMTS13, an important regulator of hemostasis implicated in microvascular thrombosis, and DVT. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2847 Chronic lymphocytic leukemia (CLL) is frequently complicated by autoimmune disorders, primarily autoimmune hemolytic anemia and immune thrombocytopenia (ITP). In order to investigate biological features related to ITP development, we retrospectively analyzed 463 CLL patients characterized for immunoglobulin heavy-chain variable (IGHV) gene mutational status, cytogenetic features and B-cell receptor (BCR) configuration (HCDR3) at the time of diagnosis. Thirty-six patients (7.7%) had developed ITP, according to our previously reported criteria (Visco et al, Blood 2008): i) an otherwise unexplained rapid (〈 2 weeks) and severe fall (at least half of the initial level and below 100*109/L) of the platelet count; ii) normal or augmented number of megakaryocytes in the bone marrow; iii) no or limited (not palpable) splenomegaly and iv) no cytotoxic treatment in the last month. Stereotyped BCR configuration was defined by means of pair-wise alignment with known stereotyped sequences available from different public databases (Stamatopoulos et al, Blood 2007; Bomben et al, Br J Hematol 2009; Murray et al, Blood 2008), specifically excluding pairs of sequences whose length differed more than 3 amino acids and sharing more than 60% identity on alignments showing less than 3 gaps, as described by others and by our group (Stamatopoulos et al, Blood 2007; Maura et al, Plos One, in press). Our results indicated that the occurrence of ITP was strongly associated with unmutated (UM) IGHV (p