ALBERT

Description: 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.

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: 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 3369 Background: Patients with Ph-negative Myeloproliferative Neoplasms (MPN) such as Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) can be exposed during the course of these MPN to thrombotic and bleeding complications, with increased morbidity and mortality. Age, previous history of thrombosis, increased White Blood Cell (WBC) and Jak2 allele burden have been proposed as risk factors for Venous (VTE) and Arterial (ATE) thromboses while bleeding has been previously associated with abnormalities of the von Willebrand factor (VWF). Aims: To investigate any significant role of ADAMTS-13 and VWF activities in the thrombotic and bleeding complications observed in a small but well characterized cohort of MPN patients. Patients and Methods: 88 consecutive patients were diagnosed at the Hematology and Transfusion Medicine Division, L.SACCO University Hospital of Milan, according to WHO criteria. Patients signed an informed consent to participate in this clinical study with a protocol approved by local IRB and they showed MPN type (%), mean age (range), gender M/F and Jak2 positivity (%) as follows: PV[n=42 (48%), 68 (36–86), 18/24; 85.7%]; ET [n=34 (38%), 66 (30–93), 10/24, 61.7%]; PMF [n=12 (14%), 67 (37–88), 7/5, 58%]. Thrombotic and bleeding episodes were recorded and managed from the time of diagnosis and associated with the use of aspirin (ASA) and of other MPN therapies. Among additional lab parameters, plasmatic ADAMTS-13 and VWF activities were also measured at enrolment as endothelial/platelet marker. These activities were assayed with Technozym ADAMTS-13 activity (Technoclone GmbH, Austria), Innovance VWF-GPIb activity (Siemens AG, Germany) and HemosIL-VWF antigen (Instrumentation Laboratory, USA). Multimeric analyses were also tested using very sensitive intermediate SDS-agarose gel electrophoresis. Statistical analyses were performed by SPSS-17.2. Results: 59/88 (67%) patients did not show any thrombotic or bleeding complications during the 6-year follow-up. In these cases mean (range) values of VWF:GPIb and VWF:Ag were 104 (29–202) and 133 (52–288) U/dL while ADAMTS-13 was 102 (63–143). 20/88 (23%) cases showed at least one thrombotic event (13ATE/7VTE): AMI (6), STROKE (6), TIA (2), PE (1), DVT (7). Patients with thromboses showed relatively higher values VWF:GPIb and lower ADAMTS-13 and this was confirmed in multivariate analysis especially for ET [VWF:GPIb=135 (61–237) U/dL, p=0.004 and ADAMTS-13=89(62–134), p=0.009]. Major bleeding episodes mainly mucosal (5 gastrointestinal, 3 post-surgery, 1 severe menorrhagia) requiring blood transfusions or hysterectomy were observed in 9/88 (10%) patients. At the multivariate analysis, major bleedings were significantly associated with lower VWF:GPIb [68 (25–111) U/dL, p=0.022), lower VWF:Ag [93 (35–146) U/dL, p=0.016] and to the ASA intake (p=0.006). Most of these bleeders showed also a relative loss of the highest molecular weight multimers. Conclusions: Based on these observations, we confirm that thrombotic events in MPN may certainly have multiple risk factors: however, lower ADAMTS-13 and higher VWF activities might play a role as additional risk factors especially in ET. Conversely, lower levels of VWF with loss of the largest multimers are important risk factors for bleeding in MPN especially in patients treated with ASA. Disclosures: No relevant conflicts of interest to declare.

Description: Multiple Myeloma (MM) is the most common form of plasma cell dyscrasia, characterized by a marked heterogeneity of genetic lesions and clinical course. It may develop from a premalignant condition (monoclonal gammopathy ofundetermined significance, MGUS) or progress from intra-medullary to extra-medullaryforms (plasma cell leukemia, PCL). To provide insights into the molecular characterization of plasma cell dyscrasias and to investigate the contribution of specific genetic lesions to the biological and clinical heterogeneity of MM, we analyzed the gene expression profiles of plasma cells isolated from 7 MGUS, 39 MM and 6 PCL patients by means of DNA microarrays. MMs resulted highly heterogeneous at transcriptional level, whereas the differential expression of genes mainly involved in DNA metabolism and proliferation distinguished MGUS from PCLs and the majority of MM cases. The clustering of MM patients was mainly driven by the presence of the most recurrent translocations involving the immunoglobulin heavy-chain locus. Distinct signatures have been found to be associated with different lesions: the overexpression of CCND2 and genes involved in cell adhesion pathways was observed in cases with deregulated MAF and MAFB, whereas genes upregulated in cases with the t(4;14) showed apoptosis related functions. In addition, we identified a set of cancer germ-line antigens specifically expressed in a sub-group of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.

Description: Chromosomal translocations involving the immunoglobulin heavy chain (IGH@) locus and variuos partner loci are frequently associated with multiple myeloma (MM). We investigated the expression profiles of FGFR3/MMSET, CCND1, CCND3, MAF and MAFB genes, respectively involved in t(4;14)(p16.3;q32), t(11;14)(q13;q32), t(6;14)(p21;q32), t(14;16)(q32;q23) and t(14;20)(q32;q12), in purified plasma cell populations from 39 MMs and six plasma cell leukemias (PCL) using DNA microarray analysis, and compared the results with the presence of translocations as assessed by dual-color FISH or RT-PCR. The t(4;14) was found in six MMs, t(11;14) in 9 MMs and 1 PCL, t(6;14) in one MM, t(14;16) in 2 MMs and 1 PCL, and t(14;20) in one PCL. The translocations were associated with the spiked expression of target genes in all cases. Furthermore, gene expression profiling allowed the identification of putative translocations dysregulating CCND1 (1 MM and 1 PCL) and MAFB (1 MM and 1 PCL) without any apparent involvement of immunoglobulin loci. Notably, all of the translocations were mutually exclusive. Markedly increased levels of MMSET expression were found in one MM showing associated FGFR3 and MMSET signals on an unidentified chromosome. Our data suggest the importance of using combined molecular cytogenetic and gene expression approaches to detect genetic aberrations 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: The deregulation of CCND1, CCND2 and CCND3 genes represents a common event in multiple myeloma (MM), being at least one of them deregulated in almost all MM tumors. A recently proposed TC classification1 grouped MM patients into five classes on the basis of their cyclins D expression profiles and the presence of the main translocations involving the immunoglobulin heavy-chain (IGH) locus at 14q32. The aim of our study was to identify the putative transcriptional fingerprints associated with the deregulation of the different D-type cyclins and the presence of IGH translocations. The cyclin D expression levels obtained by high-density oligonucleotide microarray analysis of purified plasma cells from 50 MM cases were used to stratify the samples into the five TC classes, along with the molecular characteristics. The cyclin D expression data were validated by means of real-time quantitative PCR analysis; fluorescence in-situ hybridization was used to investigate the cyclin D loci arrangements, and to detect the main IGH translocations and the chromosome 13q deletion. A multi-class classification analysis was performed on the gene expression data and used to identify the transcriptional fingerprints of the 5 TC groups. 112 probe sets were selected as characterizing the TC1, TC2, TC4 and TC5 groups, whereas the TC3 samples showed heterogeneous phenotypes and no marker genes. In particular, TC1, TC4 and TC5 groups were characterized by the molecular signatures associated with the primary IGH translocations target genes. The TC2 group, showing significantly extra copies of the CCND1 locus (P=5.9×10−3) and neither IGH translocations nor the chromosome 13q deletion (P=1.7×10−3), was characterized by the overexpression of 30 genes, mainly involved in protein biosynthesis at translational level. Among the most specifically modulated transcripts within the group we identified a novel gene containing a BTB/POZ domain, typical of many zinc finger transcription factors and associated with transcriptional repression activity. A meta-analysis performed on two publicly available MM datasets, containing almost 250 cases, validated the identified gene expression signatures with a global classification rate (indicating the correct prediction of the TC class for the independent set) of 86% and 90%, respectively. Our data contribute to the understanding of the molecular and biological features of distinct MM subtypes; the identification of a distinctive gene expression pattern in TC2 patients may improve risk stratification and indicate novel therapeutic targets.