ALBERT

Description: The t(11;14)(q13;q32) chromosomal translocation, which is the hallmark of mantle cell lymphoma (MCL), is found in approximately 30% of multiple myeloma (MM) tumors with a 14q32 translocation. Although the overexpression of cyclin D1 has been found to be correlated with MM cell lines carrying the t(11;14), rearrangements of theBCL-1/cyclin D1 regions frequently involved in MCL rarely occur in MM cell lines or primary tumors. To test whether specific 11q13 breakpoint clusters may occur in MM, we investigated a representative panel of primary tumors by means of Southern blot analysis using probes derived from MM-associated 11q13 breakpoints. To this end, we first cloned the breakpoints and respective germ-line regions from a primary tumor and the U266 cell line, as well as the germ-line region from the KMS-12 cell line. DNA from 50 primary tumors was tested using a large panel of probes, but a rearrangement was detected in only one case using the KMS-12 breakpoint probe. Our results confirm previous findings that the 11q13 breakpoints in MM are scattered throughout the 11q13 region encompassing the cyclinD1 gene, thus suggesting the absence of 11q13 breakpoint clusters in MM.

Description: 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.

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: Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at chromosome 14q32 represent a common mechanism of oncogene activation in lymphoid malignancies. In multiple myeloma (MM), the most consistent chromosomal abnormality is the 14q+ marker, which originates in one third of cases through a t(11; 14)(q13; q32) chromosomal translocation; in the remaining cases, the identity of the partner chromosomes has not been well established. We used a Southern blot approach based on the linkage analysis of the joining (J) and the constant (C) μ, α, and γ regions to detect cases bearing IGH switch-mediated chromosomal translocations. We evaluated DNA of 88 nonkaryotyped patients with MM (78 cases) or plasma cell leukemia (PCL) (10 cases) and found the presence of “illegitimate” rearranged IGH fragments (no comigration between the J and C regions) in 21 cases. To confirm this analysis, we cloned the illegitimate rearranged fragments from three samples, and the molecular and fluorescent in situ hybridization (FISH) analyses indicated the presence of chromosomal translocations juxtaposing a switch IGH region to sequences from chromosomes 11q13 (one PCL case) or 4p16.3 (two MM cases). Interestingly, the breakpoints on 4p16.3 occurred about 14 kb apart in a genomic region located approximately 50 kb centromeric to the fibroblast growth-factor receptor 3 (FGFR3) gene. Moreover, Southern blot analysis using 4p16.3 genomic probes detected a rearrangement in an additional MM tumor. FISH analysis of the MM-derived KMS-11 cell line, reported to be associated with a t(4; 14)(p16.3; q32), showed that the FGFR3 gene was translocated on 14q32. High levels of FGFR3 mRNA expression were observed in the cloned MM tumors and KMS-11 cell line, but not in the cases that were apparently negative for this lesion. Furthermore, a point mutation at codon 373 in the transmembrane domain of the FGFR3 gene resulting in an amino acid substitution (Tyr → Cys) was detected in the KMS-11 cell line. These findings indicate that the t(4; 14)(p16.3; q32) represents a novel, recurrent chromosomal translocation in MM, and suggest that the FGFR3 gene may be the target of this abnormality and thus contribute to tumorigenesis in MM.

Description: Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at chromosome 14q32 represent a common mechanism of oncogene activation in lymphoid malignancies. In multiple myeloma (MM), variable chromosome partners have been identified by conventional cytogenetics, including the 11q13, 8q24, 18q21, and 6p21 loci. We and others have recently reported a novel, karyotypically undetectable chromosomal translocation t(4;14)(p16.3;q32) in MM-derived cell lines, as well as in primary tumors. The 4p16.3 breakpoints are relatively scattered and located less than 100 kb centromeric of the fibroblast growth factor receptor 3 (FGFR3) gene or within the recently identified WHSC1 gene, both of which are apparently deregulated by the translocation. To assess the frequency of the t(4;14)(p16.3;q32) translocation in MM, we performed a double-color fluorescent in situ hybridization (FISH) analysis of interphase nuclei with differently labeled probes specific for the IGH locus (a pool of plasmid clones specific for the IGH constant regions) or 4p16.3 (yeast artificial chromosome (YAC) 764-H1 spanning the region involved in breakpoints). Thirty MM patients, the MM-derived cell lines KMS-11 and OPM2, and six normal controls were examined. The identification of a t(4;14) translocation, evaluated as the presence of a der(14) chromosome, was based on the colocalization of signals specific for the two probes; a cutoff value of 15% (mean + 3 standard deviation [SD]) derived from the interphase FISH of the normal controls (range, 5% to 11%; mean ± SD, 8.16 ± 2.2) was used for the quantification analysis. In interphase FISH, five patients (one in clinical stage I, two in stage II, one in stage III, and a plasma cell leukemia) were found to be positive (≈15%). FISH metaphases with split or colocalized signals were detected in only two of the translocated cases and confirmed the pattern found in the interphase nuclei. Furthermore, in three of the five cases with the translocation, FISH analysis with the IGH joining probe (JH) showed the presence of the reciprocal product of the translocation [der(4) chromosome]. Overall, our study indicates that the t(4;14)(p16.3;q32) chromosomal translocation is a recurrent event in MM tumors and may contribute towards the detection of this lesion and our understanding of its pathogenetic and clinical implications in MM.

Description: 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.

Description: To date, little evidence of miRNA expression/deregulation in multiple myeloma has been reported. To characterize miRNA in the context of the major multiple myeloma molecular types, we generated miRNA expression profiles of highly purified malignant plasma cells from 40 primary tumors. Furthermore, transcriptional profiles, available for all patients, were used to investigate the occurrence of miRNA/predicted target mRNA pair anticorrelations, and the miRNA and genome-wide DNA data were integrated in a subset of patients to evaluate the influence of allelic imbalances on miRNA expression. Differential miRNA expression patterns were identified, which were mainly associated with the major IGH translocations; particularly, t(4;14) patients showed specific overexpression of let-7e, miR-125a-5p, and miR-99b belonging to a cluster at 19q13.33. The occurrence of other lesions (ie, 1q gain, 13q and 17p deletions, and hyperdiploidy) was slightly characterized by specific miRNA signatures. Furthermore, the occurrence of several allelic imbalances or loss of heterozygosity was found significantly associated with the altered expression of miRNAs located in the involved regions, such as let-7b at 22q13.31 or miR-140-3p at 16q22. Finally, the integrative analysis based on computational target prediction and miRNA/mRNA profiling defined a network of putative functional miRNA-target regulatory relations supported by expression data.