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Purpose: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3′UTR in CCND2 overexpression in multiple myeloma.Experimental Design: Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3′UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3′ rapid amplification of cDNA ends (RACE)-PCR.Results: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3′RACE experiments revealed that changes in CCND2 3′UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3′UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction.Conclusions: Overall, these results highlight the impact of CCND2 3′UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma. Clin Cancer Res; 22(1); 207–17. ©2015 AACR.

Supplementary Tables Supplementary Table 1. Cytogenetic characteristics of MM samples Supplementary Table 2. Sequences of primers used in the study Supplementary Table 3. CCND2 mRNA FISH probes sequences Supplementary table 4. microRNAs predicted to target CCND1 or/and CCND2 Supplementary table 5. microRNAs expression in MM cell lines. Supplementary Figures Supplementary Figure 1. CCND2 mRNA level 24h after transfection of MM1S, RPMI and H929 cell lines with indicated miRNAs Supplementary Figure 2. Expression of miR-15a and miR-196b in H929 and MM1S cell lines after transfection Supplementary Figure 3. (A) Alignment of 3'RACE PCR product sequence from MM1S cell line with CCND2 mRNA (NM_001759.3). (B) Sequence of the 3'RACE PCR product withhighlighted APA signal. Supplementary Figure 4. CCND2 mRNA expression visualized with FISH probes Supplementary Figure 5. Luciferase activity in HEK923 cells cotransfected with various miRNAs, plasmid encoding GFP and plasmid containing luciferase and 3'UTR of CCND2 short isoform Supplementary Figure 6. Regulation of cyclin D2 level by CCND1/CCND3.

HLA studies have been used to determine the admixture of different populations within the Iberian Peninsula including neighbouring regions with shared origins, such as Portugal and Castile and Leon. These studies certainly can be used to study human migration that could establish populations currently settled according to genetic distant analysis based on the HLA diversity and language variety., This work was supported by the “Gerencia Regional de Salud de Castilla y Leon” (GRS 2080/A/19, 2019) and (GRS COVID 70/A/20, 2020).

Allogeneic hematopoietic stem-cell transplantation and solid-organ transplantation are associated with an increased risk of secondary neoplasms. Indeterminate cell histiocytosis (ICH) is a rare disease composed of so-called indeterminate cells, an alleged cutaneous dendritic cell subset displaying histological and some ultrastructural and immunophenotypic features of Langerhans cells but lacking Birbeck granules. We report a case of cutaneous ICH occurring after allogeneic hematopoietic stem-cell transplantation for a myelodysplastic syndrome in a 56-year-old man. Microsatellite analysis demonstrated that the neoplastic cells were derived from the donor's hematopoietic system. This case broadens the spectrum of complications after stem-cell transplantation and demonstrates that cutaneous ICH in the setting of myelodysplastic syndromes may have a nonrelated origin to dysplastic myeloid cells.

Key Clinical Message Sixty-seven-year-old patient, diagnosed with multiple myeloma who had received autologous stem cell transplantation, following bortezomib, dexamethasone and thalidomide conventional regimen, achieving complete response, developed rash, diarrhea, and severe respiratory failure, 80 days after the transplantation procedure. He was diagnosed with graft-versus-host disease and bronchiolitis obliterans syndrome.

Immunoglobulin M (IgM) monoclonal gammopathies show considerable variability, involving three different stages of presentation: IgM monoclonal gammopathy of undetermined significance (IgM-MGUS), asymptomatic Waldenstrom's macroglobulinemia (AWM), and symptomatic WM (SWM). Despite recent findings about the genomic and transcriptomic characteristics of such disorders, we know little about the causes of this clinical heterogeneity or the mechanisms involved in the progression from indolent to symptomatic forms. To clarify these matters, we have performed a gene expression and mutational study in a well-characterized cohort of 69 patients, distinguishing between the three disease presentations in an attempt to establish the relationship with the clinical and biological features of the patients. Results showed that the frequency of genetic alterations progressively increased from IgM-MGUS to AWM and SWM. This means that, in contrast to MYD88 p.L265P and CXCR4 WHIM mutations, present from the beginning of the pathogenesis, most of them would be acquired during the course of the disease. Moreover, the expression study revealed a higher level of expression of genes belonging to the Toll-like receptor (TLR) signaling pathway in symptomatic versus indolent forms, which was also reflected in the disease presentation and prognosis. In conclusion, our findings showed that IgM monoclonal gammopathies present higher mutational burden as the disease progresses, in parallel to the upregulation of relevant pathogenic pathways. This study provides a translational view of the genomic basis of WM pathogenesis.

Transformation of Waldenström’s macroglobulinemia (WM) to diffuse large B-cell lymphoma (DLBCL) occurs in up to 10% of patients and is associated with an adverse outcome. Here we performed the first whole-exome sequencing study of WM patients who evolved to DLBCL and report the genetic alterations that may drive this process. Our results demonstrate that transformation depends on the frequency and specificity of acquired variants, rather than on the duration of its evolution. We did not find a common pattern of mutations at diagnosis or transformation; however, there were certain abnormalities that were present in a high proportion of clonal tumor cells and conserved during this transition, suggesting that they have a key role as early drivers. In addition, recurrent mutations gained in some genes at transformation (for example, PIM1, FRYL and HNF1B) represent cooperating events in the selection of the clones responsible for disease progression. Detailed comparison reveals the gene abnormalities at diagnosis and transformation to be consistent with a branching model of evolution. Finally, the frequent mutation observed in the CD79B gene in this specific subset of patients implies that it is a potential biomarker predicting transformation in WM.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease influenced by genetic and environmental factors. The role of the HLA system in tumor antigen presentation could be involved in susceptibility and disease control. We analyzed the phenotypic frequencies of HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 in 250 DLBCLs, comparing them with 1940 healthy individuals. We also evaluated the influence of HLA polymorphisms on survival in those patients treated with curative intention using cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP)-like regimen without (n = 64, 26%) or with (n = 153, 61%) rituximab. DLBCL patients have a higher phenotypic frequency of HLA-DRB1*01 (29% vs 19.5%, P = .0008, Pc = .0104) and a lower frequency of HLA-C*03 (6.4% vs 17.9%, P < .0005, Pc = .007) compared with healthy individuals. Irrespective of the age-adjusted International Prognostic Index, those patients receiving a CHOP-like plus rituximab regimen and carrying the HLA-B44 supertype had worse 5-year progression-free (54% vs 71%, P = .019) and 5-year overall (71% vs 92%, P = .001) survival compared with patients without this supertype. Our data suggest that some HLA polymorphisms influence the development and outcome of DLBCL, allowing the identification of an extremely good-risk prognostic subgroup. However, these results are preliminary and need to be validated in order to exclude a possible population effect.

The pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains partially unknown. The analysis of the B-cell receptor of the malignant cells could contribute to a better understanding of the DLBCL biology. We studied the molecular features of the immunoglobulin heavy chain (IGH) rearrangements in 165 patients diagnosed with DLBCL not otherwise specified. Clonal IGH rearrangements were amplified according to the BIOMED-2 protocol and PCR products were sequenced directly. We also analyzed the criteria for stereotyped patterns in all complete IGHV-IGHD-IGHJ (V-D-J) sequences. Complete V-D-J rearrangements were identified in 130 of 165 patients. Most cases (89%) were highly mutated, but 12 sequences were truly unmutated or minimally mutated. Three genes, IGHV4-34 , IGHV3-23 , and IGHV4-39 , accounted for one third of the whole cohort, including an overrepresentation of IGHV4-34 (15.5% overall). Interestingly, all IGHV4-34 rearrangements and all unmutated sequences belonged to the nongerminal center B-cell–like (non-GCB) subtype. Overall, we found three cases following the current criteria for stereotyped heavy chain VH CDR3 sequences, two of them belonging to subsets previously described in CLL. IGHV gene repertoire is remarkably biased, implying an antigen-driven origin in DLBCL. The particular features in the sequence of the immunoglobulins suggest the existence of particular subgroups within the non-GCB subtype.

The human leukocyte antigen (HLA) system could play an essential role in multiple myeloma (MM) disease control. This report describes the results comparing HLA-DRB1 phenotypic frequencies in 181 MM patients (53 smoldering/indolent MM and 128 symptomatic MM patients) and healthy individuals. Higher DRB1*01 phenotypic frequencies were found in the smoldering patients compared with symptomatic MM patients (38% vs 14%, P = 0.001) and with the healthy individuals (38% vs 22%, P = 0.01). Additionally, higher DRB1*07 phenotypic frequencies were found in symptomatic MM compared with control population (38% vs 28%, P = 0.01). The present data suggest that HLA-DRB1*01 individuals may have a better ability to efficiently present myeloma-related antigens to immunocompetent cells, which could favor a better immune response against the tumor. This would translate into a more appropriate disease control associated with more indolent disease and prolonged survival.

[Purpose]: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3′UTR in CCND2 overexpression in multiple myeloma. [Experimental Design]: Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3′UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3′ rapid amplification of cDNA ends (RACE)-PCR. [Results]: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3′RACE experiments revealed that changes in CCND2 3′UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3′UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction. [Conclusions]: Overall, these results highlight the impact of CCND2 3′UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma., This study was partially supported by Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias: PI08/0568 and PI13/00111), Asociacion Española Contra el Cancer (AECC, GCB120981SAN), >Gerencia Regional de Salud, Junta de Castilla y Leon> (GRS 702/A/11, BIO/SA57/13 and BIO/SA35/14) grants, the Spanish Myeloma Network Program (RD12/0036/0058) and the INNOCAMPUS Program (CEI10-1-0010). I. Misiewicz-Krzeminska was supported by Instituto de Salud Carlos III (PS09/01897). M.E. Sarasquete was supported by Contrato Miguel Servet (CP13/00080). Research in the I. Sanchez-García group was supported by FEDER and MINECO (SAF2012-32810, and Red de Excelencia Consolider OncoBIO SAF2014-57791-REDC), Instituto de Salud Carlos III (PIE14/00066), NIH (R01 CA109335-04A1), >Junta de Castilla y Leon> (BIO/SA32/14 and CSI001U14) grants, the Fundacion Inocente Inocente, the German Carreras Foundation (DJCLS R13/26) and by the ARIMMORA project (European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 282891). Research in the C.V. Duenas group was supported by >Miguel Servet> Grant (CP14/00082 AES 2013-2016) from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad). I. Sanchez-García is an API lab of the EuroSyStem project and a partner within the Marie Curie Initial Training Network DECIDE.

Copy number analysis can be useful for assessing prognosis in diffuse large B cell lymphoma (DLBCL). We analyzed copy number data from tumor samples of 60 patients diagnosed with DLBCL de novo and their matched normal samples. We detected 63 recurrent copy number alterations (CNAs), including 33 gains, 30 losses, and nine recurrent acquired copy number neutral loss of heterozygosity (CNN-LOH). Interestingly, 20 % of cases acquired CNN-LOH of 6p21 locus, which involves the HLA region. In normal cells, there were no CNAs but we observed CNN-LOH involving some key lymphoma regions such as 6p21 and 9p24.1 (5 %) and 17p13.1 (2.5 %) in DLBCL patients. Furthermore, a model with some specific CNA was able to predict the subtype of DLBCL, 1p36.32 and 10q23.31 losses being restricted to germinal center B cell-like (GCB) DLBCL. In contrast, 8p23.3 losses and 11q24.3 gains were strongly associated with the non-GCB subtype. A poor prognosis was associated with biallelic inactivation of TP53 or 18p11.32 losses, while prognosis was better in cases carrying 11q24.3 gains. In summary, CNA abnormalities identify specific DLBCL groups, and we describe CNN-LOH in germline cells from DLBCL patients that are associated with genes that probably play a key role in DLBCL development., This work was supported by research funding from the Health Council of Castilla y León (GRS265/A/08), the Health Research Program (PS09/01382), and the Red Temática de Investigación Cooperativa en Cáncer (RTICC) grant RD12/0036 (groups 0069, 0029, 0036, 0058, and 0060) included in the National Plan I+D+I supported by the Instituto Carlos III and the Fondo Europeo de Desarrollo Regional (FEDER), the Spanish Ministry of Economy and Competitiveness, and the European Regional Development Fund (ERDF) 'Una manera de hacer Europa' (Innocampus; CEI-2010-1-0010). ES was supported by CM10/00078-Río Hortega, an ISCIII contract, FEHH grant 2013–2014 and JR14/00025-Juan Rodés, an ISCIII contract. IS was supported by the Subprograma Juan de la Cierva (JCI-2011-10232) and a Miguel Servet contract (CP13/00159).

[Context]: MiR-155 plays a critical role in the development of B-cell malignancies. Previous studies have shown a deregulation of miR-155 in specific cytogenetic subtypes of multiple myeloma (MM). However, the mechanisms that regulate miR-155 expression in MM are not fully understood. [Objective]: In the present study, we explored the regulation of miRNA-155 in MM by DNA methylation mechanisms and the impact of miR-155 expression in survival of MM patients. [Method]: Primary samples were obtained from 95 patients with newly diagnosed myeloma. Methylation was analyzed by Methylation Specific PCR, sequencing of bisulfite treated DNA and luciferase assay. [Results]: qRT-PCR analysis revealed that miR-155 was differentially expressed in MM and its upregulation was associated with longer survival. DNA methylation of CpG island present in the first exon of miR-155 host gene was associated with its low expression in MM cell lines and patient samples. Our results showed for the first time that in vitro methylation of part of the promoter and first exon abrogated the miR-155 expression. We further showed that miR-155 expression in MM cell lines was increased by demethylating 5-aza-dC treatment and decreased by RNA-directed DNA methylation. Additionally, we found that LPS >immunological challenge> was insufficient to induce miR-155 expression in MM cell lines with methylated DNA around transcription start site (TSS). [Conclusion]: This study provides evidence that DNA methylation contributes to miR-155 expression in myeloma cells. Interestingly, the survival data showed an association between miR-155 expression and outcome of MM., This work was supported by The Fund for Health of Spain (FIS) [PI080568 and PS0901897], the “Gerencia Regional de Salud, Junta de Castilla y León” [GRS 702/A/11] and the Spanish Myeloma Network Program [RD12/0036/0058], “Asociación Española Contra el Cancer” [AECC, GCB120981SAN]. M.E. Sarasquete was supported by Contrato Miguel Servet (CP13/00080).

Multiple myeloma (MM) pathogenesis has been explained for many years by the cancer biology dogma introduced in 1976 by Peter Nowell:1 first, a single plasma cell would be immortalized by an error in the immunoglobulin genes rearrangement process; then, a progressive stepwise acquisition of somatic cell mutations would induce a sequential selection, leading to domination by the fittest clone.2 In line with this idea of ‘myeloma stability’, single-nucleotide polymorphism arrays studies in diagnostic-relapse paired samples have revealed the presence of common clonal characteristics.3 Biologically, the M-protein remains usually constant across MM evolution, to the point that it is conventionally used to monitor treatment response and disease progress. Further, the variable domain of the rearranged immunoglobulin heavy-chain genes (or CDR3 region) has been used as a patient-specific myeloma fingerprint in minimal residual disease (MRD) studies.

MYD88 L265P mutation has been reported in ∼90% of Waldenstrom's Macroglobulinemia (WM) patients and immunoglobulin M (IgM) monoclonal gammopathies of uncertain significance (MGUS), as well as in some cases of lymphoma and chronic lymphocytic leukemia. The present study aimed to develop a real-time allele-specific oligonucleotide PCR (ASO-RQ-PCR) to detect the MYD88 L265P mutation. We first evaluated the reproducibility and sensitivity of the technique with a diluting experiment of a previously known positive sample. Then, we evaluated the applicability of the methodology by analyzing 30 selected patients (10 asymptomatic WM, 10 symptomatic WM, and 10 IgM MGUS) as well as 10 healthy donors. The quantitative ASO-PCR assay could detect the MYD88 L265P mutation at a dilution of 0.25%, showing an inverse correlation between the tumor cell percentage and the cycle threshold (CT) value, thus allowing for tumor burden quantitation. In addition, mutated cases were distinguished from the unmutated by >10 cycles of difference between CTs. To sum up, ASO-RQ-PCR is an inexpensive, robust, and optimized method for the detection of MYD88 L265P mutation, which could be considered as a useful molecular tool during the diagnostic work-up of B-cell lymphoproliferative disorders.

We evaluated the MYD88 L265P mutation in Waldenström's macroglobulinemia (WM) and B-cell lymphoproliferative disorders by specific polymerase chain reaction (PCR) (sensitivity ∼10(-3)). No mutation was seen in normal donors, while it was present in 101/117 (86%) WM patients, 27/31 (87%) IgM monoclonal gammapathies of uncertain significance (MGUS), 3/14 (21%) splenic marginal zone lymphomas and 9/48 (19%) non-germinal center (GC) diffuse large B-cell lymphomas (DLBCLs). The mutation was absent in all 28 GC-DLBCLs, 13 DLBCLs not subclassified, 35 hairy cell leukemias, 39 chronic lymphocytic leukemias (16 with M-component), 25 IgA or IgG-MGUS, 24 multiple myeloma (3 with an IgM isotype), 6 amyloidosis, 9 lymphoplasmacytic lymphomas and 1 IgM-related neuropathy. Among WM and IgM-MGUS, MYD88 L265P mutation was associated with some differences in clinical and biological characteristics, although usually minor; wild-type MYD88 cases had smaller M-component (1.77 vs 2.72 g/dl, P=0.022), more lymphocytosis (24 vs 5%, P=0.006), higher lactate dehydrogenase level (371 vs 265 UI/L, P=0.002), atypical immunophenotype (CD23-CD27+ +FMC7+ +), less Immunoglobulin Heavy Chain Variable gene (IGHV) somatic hypermutation (57 vs 97%, P=0.012) and less IGHV3-23 gene selection (9 vs 27%, P=0.014). These small differences did not lead to different time to first therapy, response to treatment or progression-free or overall survival.

The pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains partially unknown. The analysis of the B-cell receptor of the malignant cells could contribute to a better understanding of the DLBCL biology. We studied the molecular features of the immunoglobulin heavy chain (IGH) rearrangements in 165 patients diagnosed with DLBCL not otherwise specified. Clonal IGH rearrangements were amplified according to the BIOMED-2 protocol and PCR products were sequenced directly. We also analyzed the criteria for stereotyped patterns in all complete IGHV-IGHD-IGHJ (V-D-J) sequences. Complete V-D-J rearrangements were identified in 130 of 165 patients. Most cases (89%) were highly mutated, but 12 sequences were truly unmutated or minimally mutated. Three genes, IGHV4-34, IGHV3-23, and IGHV4-39, accounted for one third of the whole cohort, including an overrepresentation of IGHV4-34 (15.5% overall). Interestingly, all IGHV4-34 rearrangements and all unmutated sequences belonged to the nongerminal center B-cell-like (non-GCB) subtype. Overall, we found three cases following the current criteria for stereotyped heavy chain VH CDR3 sequences, two of them belonging to subsets previously described in CLL. IGHV gene repertoire is remarkably biased, implying an antigen-driven origin in DLBCL. The particular features in the sequence of the immunoglobulins suggest the existence of particular subgroups within the non-GCB subtype.

The frequencies of human leukocyte antigen (HLA) class I and class II specificities and haplotypic associations were determined in 1940 unrelated donors from Castilla y Leon and compared with other Iberian, Mediterranean and European populations. Specificities were determined using polymerase chain reaction reverse sequence-specific oligonucleotide or polymerase chain reaction sequence-specific primer techniques. In the analysis, 19, 29 and 13 specificities were found for HLA-A, -B and -DRB1, respectively, with HLA-A*02 (26%), -A*01 (11%), -B*44 (16%), -B*35 (10%), -DRB1*07 (16%) and -DRB1*13 (14%) showing the highest frequencies. In addition, 10 common HLA-A-B-DRB1 haplotypic associations were observed, A*01-B*08-DRB1*03 (3%) and A*29-B*44-DRB1*07 (3%) being the most frequent ones. These findings indicate that the population of Castilla y Leon is genetically equidistant from the Portuguese and other Spanish populations and shares a common origin with other Iberian populations, in which European, Mediterranean and North African genetic components are present; this is in agreement with the historical and genetic background of the population. These data contribute to a better understanding of the genetic structure of the Iberian Peninsula and provide a healthy control population from our region that should be useful for the study of disease associations.

CCND2 is highly expressed in most of multiple myeloma (MM) samples without CCND1 or CCND3 overexpression. D-type cyclins are highly homologous proteins and there is a growing body of evidence that the functions of the D cyclins are mostly exchangeable. During mouse development, the three D-type cyclins are expressed following an often mutually exclusive pattern and their function may be tissue-specific. Likewise in MM CCND1 or CCND2 expression is commonly mutually exclusive. The mechanisms by which CCND2 is upregulated in a set of MMs are not completely deciphered. In this study, we investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at3'UTR in CCND2 overexpression in MM. First, we observed that ectopic transfection of MM cell lines with several miRNAs, directly targeting CCND2 3'UTR according to luciferase reporter assay, decreased the level of cyclin D2, although not in all the cell lines. This fact suggested the possible disruption of miRNA target sites. Indeed, we detected the presence of short CCND2 mRNA, both in MM cell lines and primary cells, using four different methodological approaches: qRT-PCR, Northern blot, mRNA FISH and 3’RACE PCR with product sequencing. The short CCND2 isoform was observed by qRT-PCR in the majority of patients and in all MM cell lines expressing CCND2. This finding was confirmed by Northern blot results. The abundance of each CCND2 mRNA isoform was also assessed by two-color mRNA FISH designed to discriminate the two mRNA different in length. This approach also enabled us to notice that no subpopulation of cells distinguishable by the load of one isoform with respect to another was present. The results obtained by RACE experiments in MM cell lines support the idea that changes in CCND2 3’UTR length are explained by alternative polyadenylation (APA). The functional consequences of 3’UTR shortening is the mRNA stabilization due to the loss of miRNA sites and regulatory elements located in the 3’UTR. Accordingly, the luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. The short 3’UTRs lacking miRNA-binding sites have been associated with increased expression of different genes at both the mRNA and the protein level. Here, we observed significant higher level of overall CCND2 mRNA expression in those MMs with greater abundance of the shorter 3'UTR isoform. The previous intriguing observation showing that the level of cyclin D2 increased in U266 when cyclin D1 was silenced, was confirmed by our experiments. Moreover, functional analysis showed significant mRNA shortening after CCND1 silencing, suggesting that cyclin D1 could downregulate CCND2 level by modification of polyadenylation/clavage reaction. Since CCND2 expression is undetectable in myeloma cells with t(11;14), we extended our investigation to explore if DNA methylation might play a role in abolishing CCND2 expression. We observed that the CpG island more proximal to CCND2 TSS was highly methylated in MM cell lines with t(11;14). In summary, our results reveal that CCND2 expression in MM is mainly regulated by post-transcriptional mechanisms. Downregulation of specific miRNAs directly targeting CCND2 contributes to overexpression of CCND2 in a set of MM. Moreover, the shortening of CCND2 3'UTR by alternative polyadenylation with the consequent loss of miRNA binding sites is also participating in CCND2 upregulation. In fact, this mechanism seems to play a decisive role in the regulatory network between CCND1 and CCND2 in MM. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma (MM) pathogenesis has been explained for many years by the cancer biology dogma introduced by Peter Nowell: first, a single plasma cell would be immortalized by an error in the immunoglobulin genes rearrangement process; then, a progressive stepwise acquisition of somatic cell mutations would induce a sequential selection and domination by the fittest clone. In line with this idea of “myeloma stability”, SNP arrays studies in diagnostic-relapse paired samples have revealed the presence of common clonal characteristics. Biologically, the M-protein remains usually constant across MM evolution and further, the variable domain of the rearranged immunoglobulin heavy chain genes (or CDR3 region) has been used as a patient-specific myeloma fingerprint in minimal residual disease (MRD) studies. However, massive genome studies with Next Generation Sequencing (NGS) have challenged this concept, showing a significant intraclonal heterogeneity at diagnosis with the possible presence of several clonal progenitors or tumor-initiating cells. In this study, we have characterized and compared the CDR3 region in 52-paired samples from 26 MM patients aiming: 1) to assess mono-clonality in MM evolution through the analysis of the CDR3 sequence and, 2) to validate ASO RQ-PCR approaches for MRD in MM, based on the constancy and specificity of the CDR3 region. Samples were obtained at diagnosis and progression (19 pairs) or at 2 different timepoints of progressive disease (7 pairs). Median time between sampling was 2 years. M-protein subtype remained stable in all pairs but 1, associated with a light-chain escape phenomenon. All samples proceeded from bone marrow (BM) except for 2 pairs, composed by BM and extramedullary disease (spleen and testes). Two major cytogenetic changes were identified: increased 13q14 deletion (from 7 to 54%) in 1 pair and increased 17p (p53) deletion (from 5 to 87%) in a further one. Treatments administered between sampling included most of the current approaches used in MM (data not shown). Genomic DNA isolation, PCR amplification and sequencing were performed following conventional methods. Germline VH, DH and JH segments were identified by comparison with public databases. CDR3 region was first identified in all samples and then compared between the two samples in the 26 pairs: the sequence of nucleotides was constantly identical in each pair, including those associated with major cytogenetic changes, a light-chain escape, extramedullar vs. BM infiltration and relapsed (and therefore, treatment selected) vs. refractory disease. Therefore, we can first conclude that the main tumor clone in MM retains a specific signature across all stages of disease evolution that allows the identification of samples as evolutionary related. This major clone signature is not modified by clinical or biological changes in the disease nor under different treatment pressures and would thus identify disease relapse and progression. Our results have also a clear impact on the validity of molecular MRD techniques. The high rate of complete responses (up to 50-60%) currently achieved in MM has prompted the use of new techniques for disease assessment. Today, ASO RQ-PCR, based on the use of specific primers and probes complementary of the VDJH rearrangement, continues to be the most sensitive approach. One pitfall of this technique would be the potential instability of PCR targets over time, which would induce false negative results. In B-cell precursor ALL, this is estimated to happen in 30-40% of cases but has not been deeply evaluated in MM yet. With the present study, we can also conclude that the junction region of the VDJH rearrangement in MM constantly identifies the myeloma cells responsible for relapse and therefore can be used as a reliable target for MRD assessment by ASO RQ-PCR and more recently, by NGS methods. If the CDR3 region remains stable, the novel concept of clonal tiding in MM should not be interpreted as a poly- or oligoclonal but subclonal. In MM, tides can be subclonal, but the ocean remains monoclonal. Disclosures No relevant conflicts of interest to declare.

Abstract 1589 Introduction: Acquired potentially N-glycosylation sites are produced by somatic hypermutation (SHM) in the immunoglobulin (Ig) variable region. This phenomenon is produced in ∼9% of normal B-cells and seems to be related to certain B-cell lymphoproliferative disorders (B-LPDs) such as follicular lymphoma (FL, 79%), endemic Burkitt lymphoma (BL, 82%) and diffuse large B-cell lymphoma (DLBCL, 41%). These data suggest that new potential N-glycosylation sites could be related to germinal center B (GCB)-LPDs. By contrast, in other B-LPDs, such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), MALT lymphoma, Waldenström macroglobulinemia (WM) or multiple myeloma (MM), these modifications have not been analyzed in deep. Aims: To evaluate the acquisition of potential N-glycosylation sites in B-LPDs, including immunohystochemical DLBCL subtypes (GCB and non-GCB) and specific non-GCB-LPDs, such as hairy cell leukemia (HCL), splenic marginal-zone lymphoma (SMZL), CLL, MCL, ocular extranodal marginal zone lymphoma (OAEMZL), MM and WM. Patients: A total of 953 sequences (203 from our group and 750 previously published sequences) of B-LPDs were included. Diagnosis distribution was as follows: DLBCL (n=235), MCL (n=235), CLL (n=166), MM (n=96), OAEMZL (n=82), SMZL (n=68), WM (n=38) and HCL (n=33). Methods: Acquired N-glycosylation sites were counted according to the sequence Asn-X-Ser/Thr, where X could be any amino acid except Pro. Natural motifs in germline sequences of IGHV1–08, IGHV4–34 e IGHV-5a were not considered. Fisher test was used to perform comparisons between groups. To distinguish DLBCL biological subtypes (GCB and non-GCB DLBCL), Hans' algorithm was used. Results: A total of 83 out of the 235 DLBCL cases acquired at least a new N-glycosylation site, a higher value than in normal B-cells (35% vs. 9%, p Conclusions: We described for the first time the pattern of N-glycosylation in HCL, SMZL, OAEMZL and in the immunohystochemical DLBCL subtypes, where the GCB-DLBCL showed a higher number of new N-glycosylation sites with respect to non-GCB DLBCL and other non-GCB-LPDs. The presence of novel N-glycosylation sites in FL, BL and in GCB-DLBCL strongly suggests that these motifs are characteristic of the germinal center B-LPDs. Disclosures: No relevant conflicts of interest to declare.

Abstract 2680 Introduction: The Human Leukocyte Antigen (HLA) system plays an important role in antitumor immune response. In the last years, different genome-wide association studies have suggested that 6p21.3 (which include HLA system), is a risk region for lymphoma susceptibility. It has been also reported that specific alleles could modify the prognosis in patients diagnosed of lymphoma, including diffuse large B cell lymphoma (DLBCL), treated without Rituximab. Aim: Our hypothesis is that HLA system could play an essential role in disease control of DLBCL. Here, we have evaluated the effect of HLA Class I (A, B and C) and II (DRB1 and DQB1) alleles in DLBCL incidence and survival. Patients and Methods: A total of 251 patients diagnosed of DLBCL according to the 2008 WHO classification were analyzed (68% of them received Rituximab-based regimens). Control population consisted in 1940 healthy donor individuals analyzed for HLA-A, B, and DRB1 alleles and 200 for HLA-C and HLA-DQB1. Overall survival (OS) was calculated from diagnosis until death for any cause. Event-free survival (EFS) was calculated from diagnosis until event defined as death for any cause, relapse or progression of the disease. Allele frequencies and Hardy-Weinberg equilibrium were estimated using the Arlequin software package, version 3.5.1.2. Comparison of allele and phenotype frequencies between populations was performed with the two-sided Fisher's exact test or χ2 test using GraphPad Prism 4.0 (GraphPad Software, Inc. San Diego, CA, USA) or SPSS software (SPSS 15.0, Inc. Chicago, IL, USA). P-value was adjusted applying a Bonferroni correction (Pc). The effect of biological and clinical variables on OS and EFS at five years was analyzed by univariate (two-sided log-rank test) and multivariate (Cox multivariate analysis) methods. Differences were considered to be statistically significant when P< 0.05. Results: HLA specificities frequencies in patients and controls (Alcoceba et al, Tissue Antigens 2011) were consistent in all cases with the Hardy-Weinberg equilibrium. Phenotypic frequencies showed significant differences in DLBCL patients compared to control population. DRB1*01 frequency was increased in DLBCL patients (28.9% vs. 19.5%, p=0.0009, Pc=0.0117, OR: 1.68; 95% CI: 1.24–2.26). By contrast, DLBCL patients showed a lower frequency in C*03 allele as compared to the control population (6.4% vs. 18.3, p=0.0005, Pc=0.007, OR: 3.2, 95% CI: 1.62–6.3). As far as the outcome of patients was concerned, the presence of B*18 and/or B*44 was significantly associated with a worse EFS (32% vs. 60%, p Conclusions: Our data revealed that HLA specificities could influence the incidence (DRB1*01 and C*03) and outcome (B*18 and B*44) of DLBCL. These results suggest a role for HLA in the immune-surveillance of DLBCL and, also open possibilities for future investigations to answer how a specific HLA allele could affect in DLBCL. Disclosures: No relevant conflicts of interest to declare.

INTRODUCTION: Several studies have reported the presence of expanded T-cell clones in patients with multiple myeloma (MM), which could be involved in an anti-tumour response and extended survival. The Human Leukocyte Antigen (HLA) system seems to play an essential role in MM control and could influence disease control. This feature has been poorly studied and there are only few data favouring higher incidence of some HLA specifities such as B18 and B5 in myeloma patients. AIM: To compare HLA-DRB1 phenotypic frequencies in smoldering MM versus symptomatic MM patients and control individuals. PATIENTS: A total of 181 patients with a diagnosis of MM were analysed. According to their behaviour patients were classified into two subsets: 128 symptomatic MM who were homogeneously treated according to the GEM-2000 protocol (Spanish Myeloma Group/PETHEMA protocol) and 53 patients with the diagnosis of smoldering MM according to the criteria of the International Myeloma Working Group and who were free of therapy for at least 1 year following diagnosis. Additionally, 1818 healthy donor individuals from the Castilla y Leon registry for hematopoietic stem cell-transplantation were included as control population. All three populations involved Caucasian individuals. METHODS: After genomic DNA extraction, HLA-DRB1 typing at low-resolution level (two digits) was carried out using the PCR-rSSO methodology according to the standards of the European Federation of Immunogenetics. Allele frequencies were estimated by direct counting. Comparisons of allele and phenotype frequencies between populations were performed with the two-sided Fisher’s exact test using GraphPad Prism 4.0 Software. The strength of associations was estimated by the odds ratio (OR), and their 95% confidence intervals (CI) were calculated by Cornfield methods (values of p < 0.05 were considered statistically significant). P-value was corrected (Pc) for the number of valid comparisons made (Bonferroni correction). RESULTS: DRB1 phenotypic frequencies were not significantly different among MM patients and healthy control individual. In contrast, when the two MM cohorts were analyzed, DRB1*01 phenotypic frequencies were significantly higher in the smoldering patients as compared to symptomatic MM patients (37.7% vs. 14.1, p=0.0011, Pc=0.0143, OR: 3.7, 95% CI: 1.76–7.81). Furthermore, DRB1*01 phenotypic frequencies were significantly higher in the smoldering patients as compared to the healthy control individuals (37.7% vs. 21.7%, p=0.0106, Pc>0.05, OR: 2.19, 95% CI: 1.24–3.86). In addition, symptomatic MM patients showed a higher incidence in DRB1*07 phenotypic frequencies as compared to control population (38.3% vs. 27.6%, p=0.0111, Pc>0.05, OR: 1.63, 95% CI: 1.12–2.36). CONCLUSIONS: The present data suggest that HLA-DRB1*01 phenotype is associated with indolent MM and this may reflect a better ability to efficiently present myeloma-related antigens to immunocompetent cells.