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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.

Introduction VDJH usage description in B-cell hematological malignancies has brought new insights into the clonal differentiation and clinical implications for certain pathologies, improving patients' care, although no correlation has been made in MM. We present the largest-to-date VDJH gene repertoire analysis in MM, consisting in biological and clinical data from 413 patients. This database was used to comprehensively investigate the characteristics of VDJH rearrangements: gene segment usage, somatic hypermutation (SHM), CDR3 characterization, and immunoglobulin stereotyped clusters assessment. We also investigated the potential relationship of these molecular features with the outcome. Methods Newly diagnosed MM patients were included in the study. All were managed according to the recommendations of the GEM-PETHEMA Spanish MM group, including the inclusion of most patients in the GEM2000 and GEM2005 schemes. Monoclonal assessment was carried out with FR1 VH-JH amplification, following the BIOMED-2 methods. Moreover, 113 cases were also analyzed by NGS using the LymphoTrack IGH FR1 assay (Invivoscribe Tech, San Diego, CA). VDJH usage and mutational status were analyzed with IMGT-V-Quest. ClustalX2.0 was used to perform clustering analysis with CDR3 regions from our cohort and 1117 additional sequences obtained from IMGT/LIGM-DB corresponding to unique rearrangements from both normal and tumor human B cells. Molecular and clinical data were used to perform survival studies. Results The overall VDJH detection rate was 92.5% (382/413), including 376 patients with only one detectable rearrangement, five with a biallelic rearrangement, and one with a biclonal rearrangement, also detectable by flow cytometry. Thus, 388 rearrangements where detected: 362 were productive, and 26 unproductive. Gene segments were identified in productive rearrangements. VH repertoire in MM reflected its normal counterpart, with IGHV3 being the predominant selected family, followed by IGHV4, IGHV1, IGHV2 and IGHV5. IGHD and IGHJ segment usage showed a skewed distribution, with IGHD2 and IGHD3 families accounting for 55.5% of cases, and IGHJ4 and IGHJ6 accounting for 70.8%. SHM level could be identified in 349/362 productive sequences (mean: 9.16%, SD 3.95) with statistically significant differences between certain IGHV families, namely IGHV2 was less hypermutated than IGHV1 or IGHV4. We could also collect other rearrangement features: nucleotide addition by TdT appeared in 92.5% for N1 and 88.6% for N2, with evidence of exonuclease activity in all cases. CDR3 mean length was 16±4 aminoacids (median 15, range 6-29), with preference for Gly, Ala, Asp, Tyr (~10% each one). The R/S mutation ratio was 2.39 for CDRs versus 1.74 for FWRs, showing the high influence of antigen selection over the former. No stereotyped clusters were found in our cohort. Relationship between these finding and clinical evolution was done through univariate and multivariate analyses. A shorter Progression-Free Survival (PFS) related to well-known clinical factors: presence of plasmacytomas, poor performance status or ISS stages, response to therapy, and poor-risk cytogenetics (p Conclusions VDJH usage somatic hypermutation levels and CDR3 composition in multiple myeloma resembles the normal mature B cell repertoire. In contrast to Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma or Marginal Zone Lymphoma, stereotyped receptors were absent in MM, which shows no evidence of autoantigen-driven selection. Cases with SHM levels below 7% were associated with aggressive outcome that, in the same line of CLL, would reflect the expansion of more immature clones in these cases. More studies are needed to deepen in the clinical meaning of these new findings. Disclosures Puig: Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria. Ocio:Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy; Pharmamar: Consultancy; AbbVie: Consultancy; Novartis: Consultancy, Honoraria; Sanofi: Research Funding; BMS: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding. Mateos:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Lahuerta:Amgen: Honoraria; Janssen: Honoraria; Celgene: Honoraria. San-Miguel:Celgene: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; MSD: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Brystol-Myers Squibb: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees. García-Sanz:Incyte: Consultancy; Amgen Inc.: Research Funding; Gilead: Research Funding; Spanish Government: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; BMS: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses.

Monoclonal TCRαβ+/CD4+ T-large granular lymphocyte (T-LGL) lymphocytosis is a T-cell disorder with a restricted TCR-Vβ repertoire. In the present study we explored the potential association between the expanded TCR-Vβ families, the CDR3 sequences of the TCR-Vβ gene, and the HLA genotype of patients with monoclonal TCRαβ+/CD4+ T-LGL lymphocytosis. For that purpose, 36 patients with monoclonal TCRαβ+/CD4 + T-LGL lymphocytosis (15 TCR-Vβ13.1 versus 21 non-TCR-Vβ13.1) were selected. For each patient, both the HLA (class I and II) genotype and the DNA sequences of the VDJ-rearranged TCR-Vβ were analyzed. Our results show a clear association between the TCR-Vβ repertoire and the HLA genotype, all TCR-Vβ13.1+ cases being HLADRB1* 0701 (P = .004). Interestingly, the HLA-DR7/TCR-Vβ13.1- restricted T-cell expansions displayed a highly homogeneous and strikingly similar TCR arising from the use of common TCR-Vβ gene segments, which shared (1) unique CDR3 structural features with a constantly short length, (2) similar combinatorial gene rearrangements with frequent usage of the Jβ1.1 gene, and (3) a homolog consensus protein sequence at recombination junctions. Overall, these findings strongly support the existence of a common antigendriven origin for monoclonal CD4+ T-LGL lymphocytosis, with the identification of the exact peptides presented to the expanded T cells deserving further inves tigations., This work has been partially supported by the following grants: FIS 02/1244 and FIS 05/0399, from the Ministerio de Sanidad y Consumo, Madrid, Spain; RETICC RD06/0020/0035 from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Madrid, Spain; SA 103/03, from the Consejería de Educación y Cultura, Junta de Castillo y León, Valladolid, Spain; and 05/287, from the Consejería de Salud, Junta de Andalucía, Sevilla, Spain. P.B. is supported by a grant from the University of Salamanca (Reg. N. 430). A.C.G.-M. is supported by a grant from FIS (CP03/00035).

Leukemic B-chronic lymphoproliferative disorders (B-CLPDs) are generally believed to derive from a monoclonal B cell; biclonality has only occasionally been reported. In this study, we have explored the incidence of B-CLPD cases with 2 or more B-cell clones and established both the phenotypic differences between the coexisting clones and the clinicobiologic features of these patients. In total, 53 B-CLPD cases with 2 or more B-cell clones were studied. Presence of 2 or more B-cell clones was suspected by immunophenotype and confirmed by molecular/genetic techniques in leukemic samples (n = 42) and purified B-cell subpopulations (n = 10). Overall, 4.8% of 477 consecutive B-CLPDs had 2 or more B-cell clones, their incidence being especially higher among hairy cell leukemia (3 of 13), large cell lymphoma (2 of 10), and atypical chronic lymphocytic leukemia (CLL) (4 of 29). In most cases the 2 B-cell subsets displayed either different surface immunoglobulin (sIg) light chain (n = 37 of 53) or different levels of the same sIg (n = 9 of 53), usually associated with other phenotypic differences. Compared with monoclonal cases, B-CLL patients with 2 or more clones had lower white blood cell (WBC) and lymphocyte counts, more frequently displayed splenomegaly, and required early treatment. Among these, the cases in which a CLL clone coexisted with a non-CLL clone were older and more often displayed B symptoms, a monoclonal component, and diffuse infiltration of bone marrow and required early treatment more frequently than cases with monoclonal CLL or 2 CLL clones.

Recent studies suggest the potential involvement of common antigenic stimuli on the ontogeny of monoclonal T-cell receptor (TCR)- αβ+/CD4+/NKa+/CD8-/+dim T-large granular lymphocyte (LGL) lymphocytosis. Because healthy persons show (oligo)clonal expansions of human cytomegalovirus (hCMV)-specific TCRVβ+/ CD4+/cytotoxic/memory T cells, we investigate the potential involvement of hCMV in the origin and/or expansion of monoclonal CD4+ T-LGL. Peripheral blood samples from patients with monoclonal TCR-αβ+/ CD4+ T-LGL lymphocytosis and other T-chronic lymphoproliferative disorders were evaluated for the specific functional response against hCMV and hEBV whole lysates as well as the >MQLIPDDYSNTHSTRYVTVK> hCMV peptide, which is specifically loaded in HLADRB1*0701 molecules. A detailed characterization of those genes that underwent changes in T-LGL cells responding to hCMV was performed by microarray gene expression profile analysis. Patients with TCR-αβ+/ CD4+ T-LGL displayed a strong and characteristic hCMV-specific functional response, reproduced by the hCMV peptide in a subset of HLA-DRB1*0701+ patients bearing TCRVβ13.1+ clonal T cells. Gene expression profile showed that the hCMV-induced response affects genes involved in inflammatory and immune responses, cell cycle progression, resistance to apoptosis, and genetic instability. This is the first study providing evidence for the involvement of hCMV in the ontogeny of CD4+ T-LGL, emerging as a model disorder to determine the potential implications of quite a focused CD4+/ cytotoxic immune response. © 2008 by The American Society of Hematology., This work has been partially supported by the following grants: FIS 05/0399, from the Ministerio de Sanidad y Consumo, Madrid, Spain; RTICC RD06/0020/0035 from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Madrid, Spain and 05/287, from the Consejería de Salud, Junta de Andalucía, Sevilla, Spain. AC. G-M. is supported by a grant of FIS (CP03/00035).

We have explored the potential role of genetics in the development of osteonecrosis of the jaw (ONJ) in multiple myeloma (MM) patients under bisphosphonate therapy. A genome-wide association study was performed using 500 568 single nucleotide polymorphisms (SNPs) in 2 series of homogeneously treated MM patients, one with ONJ (22 MM cases) and another without ONJ (65 matched MM controls). Four SNPs (rs1934951. rs1934980, rs1341162. and rs17110453) mapped within the cytochrome P450-2C gene (CYP2C8) showed a different distribution between cases and controls with statistically significant differences (P=1.07 × 10-6, P= 4.231 × 10-6, P= 6.22 × 10-6, and P=2.15 × 10-6, respectively). SNP rs1934951 was significantly associated with a higher risk of ONJ development even after Bonferroni correction (P corrected value=.02). Genotyping results displayed an overrepresentation of the T allele in cases compared with controls (48% vs 12%). Thus, individuals homozygous for the T allele had an increased likelihood of developing ONJ (odds ratio 12.75, 95% confidence interval 3.7-43.5) © 2008 by The American Society of Hematology., This work has been partially supported by grants PI06-1354 from the Spanish Fondo de Investigaciones Sanitarias de la Seguridad Social, Red Española de Cancer RD06/0020/0006, and with the support of the Hemato-Oncology Institut, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clinic, Barcelona, Spain.

Background: Waldenström's macroglobulinemia (WM) is a rare immunoproliferative neoplasia with indolent characteristics that shows important variability, involving three different stages of presentation: IgM Monoclonal Gammopathy of Undetermined Significance (IgM-MGUS), asymptomatic WM (AWM), and Symptomatic WM (SWM). Whole-genome sequencing and some specific approaches have identified MYD88 L265P (90%) and CXCR4 (29%) mutations as the most recurrent somatic mutations in WM. However, other genetic abnormalities under as well as the mechanisms responsible for this clinical heterogeneity still remain to be clarified. Therefore, our aim was to analyze the genomic landscape of WM, distinguishing between the three stages of the disease, by using a targeted next generation sequencing (NGS) strategy. Methods: In this study, we performed a comprehensive mutation analysis of genes previously described as frequently involved in Waldenstrom Macroglobulinemia in a large and well characterized cohort of WM patients with the aim to dissect relationships between genotype and clinical and biological characteristics to integrate somatic mutations into a clinical/molecular prognostic model. Twelve genes of interest (ARID1A, CD79A, CD79B, TP53, MYBBP1A, TRAF2, TRAF3, RAG2, HIST1H1B, HIST1H1C, HIST1H1D, and HIST1H1E) were analyzed by high throughput sequencing (Illumina MiSeq, San Diego, CA) with a novel custom amplicon-based panel in a cohort of 61 patients (pts) diagnosed according to WHO classification as follows: 14 MGUS, 23 AWM and 24 SWM. DNA was extracted from bone marrow separated CD19+ B-cells and sequenced in a MiSeq (Illumina) using 150-bp paired-end reads and a mean depth of 2000X. Bioinformatics analysis was carried out with Illumina VariantStudio 2.2. Results were correlated with biological and clinical data of the patients. MYD88 and CXCR4 mutation status, available in all cases, was assessed by ASO-PCR and Sanger Sequencing, respectively. Results: Apart from MYD88 L265P mutations (present in 90% of cases) and CXCR4WHIM (21% of cases), 23 non-synonymous mutations were found, corresponding to 18/61 (30%) patients. Only one patient with MGUS demonstrated one additional mutation (7%), while seven of the AWM (30%), and 10 of the SWM (42%) demonstrated additional mutations (p Apart from the clinical diagnosis and the requirement of therapy, no relevant correlations between the presence of mutations and the final clinical behavior was found in any patient, although the patient with a mutated TP53 corresponded to a very high resistant form of the disease. Finally, no relevant differences in progression free and overall survival were seen in this series based on the presence or absence of somatic mutations. Conclusion: Our data reveal an increased incidence of mutations along the different steps of evolution in WM: IgM MGUS, asymptomatic WM and symptomatic WM. Thus, this would mean that in contrast to MYD88 L265P, present from the beginning of the pathogenesis, most of these mutations would be acquired during the evolution of the disease, and before therapy initiation. Finally, CD79B, which is part of the B-cell receptor pathway, was frequently mutated gene in our series, emerging as an interesting therapeutic target. This confirms the relevance of the BCR signaling pathway, reinforcing the use of biological agents blocking this pathway in the treatment of these patients. Disclosures Mateos: Celgene: Consultancy, Honoraria; Takeda: Consultancy; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria. Ocio:Array BioPharma: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy; Mundipharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; MSD: Research Funding; Pharmamar: Consultancy, Research Funding; Janssen: Honoraria. Puig:Janssen: Consultancy; The Binding Site: Consultancy.

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.

Abstract 1620 Poster Board I-646 Background So far, none of the high-density microarray studies in acute myeloid leukemia (AML) have provided a useful approach with relevant diagnostic and prognostic value for the routine clinical practice. We aimed to assess the clinical utility of a small combination of genes and fusion genes included in a microfluidic card by means of real-time quantitative PCR (RQ-PCR) in AML patients. Patients and methods Ninety-six clinically relevant markers were analyzed simultaneously using TaqMan® Low Density Arrays (TLDAs) (Applied Biosystems, Foster City, CA). This plataform includes: 3 control genes (ABL1, GADPH and GUS), 36 AML specific fusion transcripts described, NPM1 mutations and 55 genes which RNA expression levels have been associated with prognosis or pathogenic process in AML. To assess the feasibility of the designed TLDA we evaluated the gene expression profile of 191 leukemia patients at diagnosis: 167 AML (inv(16): n=11, t(8;21): n=9, t(15;17): n=7, with 11q23 abnormalities: n=27, with other cytogenetic abnormalities: n=20 and normal karyotype n=93), 16 pre-B-ALL, 5 T-ALL and 3 CML with rare BCR-ABL fusions (b2a3, b3a3 and e19a2). Results We observed a high correlation between TLDA results for fusion transcripts and those obtained by gold standard techniques (RQ-PCR based on Europe against Cancer protocols). Indeed, we identified 89 patients with different fusion transcripts, including 18 cases previously reported as negative by cytogenetics or FISH approach. Patients with a favorable karyotype showed a distinctive gene expression profile. Thus, inv(16) AML overexpressed: MYH11, CLIP3, CTNNB1, SPARC, SNAI1 and MN1; t(8;21) AML expressed high levels of ETO, POU4F1, CAV1, CD34, FOXO3A, PRAME and BAALC, and t(15;17) AML showed upregulation of HGF, FGF13, WT1 and PRAME. Further, the 51 NPM1 mutated patients (38 A, 5 B, 6 D and 2 DD1, confirmed by sequencing) also showed a specific gene profile, showing high NPM1, CD34, ABCB1, ABCG2, BAALC, PROM1 or BCL2 RNA levels and low HOXA7, HOXA9 and MEIS1 RNA levels. In contrast, AML with 11q23 abnormalities or with FLT3-ITD mutations did not exhibit a characteristic pattern. Several genes (EVI1, BAALC, ERG, PRAME or PIM1) showed prognostic significance in AML with normal karyotype and AML with NPM1 mutations, thus confirming TLDA as a useful approach for risk assessment in AML. Interestingly, PIM1 overexpression was an independent predictor for shorter relapse-free (RFS) and overall survival (OS) in patients with normal karyotype (p=0.012 and p Conclusions We showed the diagnostic and prognostic value of the designed TLDA platform for evaluation of AML patients. Furthermore, in our series PIM1 has been identified as the most important prognostic marker and thus this oncogene could be used for a more accurate risk classification of AML patients. Disclosures Gutiérrez: Janssen Cilag: Honoraria; Celgene: Honoraria. Díaz-Mediavilla:Janssen-Cilag: Honoraria; Celgene: Honoraria.

Several mutations and aberrant gene expression have been proposed as prognostic factors in cytogenetically normal acute myeloid leukemia (CN-AML), but few studies have analyzed the clinical value of several genes in a large group of CN-AML patients. In 121 de novo CN-AML included into the Spanish PETHEMA therapeutic protocols, we evaluated FLT3 and NPM1 mutations and BAALC , ERG , EVI1 , MLL -PTD, MN1 , PRAME , RHAMM and WT1 RNA levels. Ninety-one patients achieved CR with standard induction therapy while 20/111 (16.5%) were refractory to treatment. This latter subgroup showed higher ERG (media 1.0±0.8 vs. 0.6±0.6;p=0.012) and lower PRAME (media 28.8±52.9 vs. 1640.6±6102.0;p=0.014) levels than no-refractory patients. Moreover, high ERG expression (defined by median value) was associated with shorter OS at 2-years (38% vs. 61%;p=0.012) and RFS at 2-years (38% vs. 60%;p=0.005). By contrast, high PRAME levels (defined by 75 th percentile) was related to longer OS (61% vs. 48%;p=0.029) and RFS (74% vs. 43%;p=0.016). Additionally, high EVI expression (defined by 75 th percentile) was associated with a poorer OS (37% vs. 56%;p=0.049). The same results in OS were observed in patients with high BAALC RNA levels (median value; 41% vs. 62%; p=0.049). Interestingly, ERG , EVI1 and PRAME markers improved the current prognostic classification of CN-AML based on FLT3 and NPM1 status. Therefore, intermediate risk patients ( FLT3-ITD/NPM1mut and FLT3wt/NMP1wt ) with low ERG , low EVI1 or high PRAME achieved long OS and RFS, similar to the favorable FLT3 wt/ NPM1 mut subgroup. By contrast, the opposite expression subgroup (high ERG , high EVI1 or low PRAME ) showed OS and RFS similar to the adverse FLT3 -ITD/ NPM1 wt subset. These results allowed identify clearly two risk-subgroups for OS and RFS, based on three molecular markers ( FLT3 , NPM and one of the 3 proposed genes). In conclusion, we demonstrated that ERG , EVI1 and PRAME are relevant prognostics markers in CN-AML and they could improve their risk-stratification.

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.

Waldenström Macroglobulinemia (WM) is characterized by monoclonal IgM paraprotein and bone marrow (BM) infiltration by lymphoplasmacytic lymphoma. The normal counterpart of WM malignant cell seems to be a post germinal centre IgM B-cell, which tumoral transformation occurs after cessation of somatic mutation (SM) but prior to Class switch recombination (CSR). However, recently has been reported that CSR can be possible “ex-vivo”, since clonotypic transcripts encoding post-switch isotypes have been observed in some WM cells cultured with CD40L/IL-40. However, this process has not been shown to occur “in vivo” until now. #3754, a 51-year-old woman, was diagnosed in 2001 of WM with a M-IgM spike (46 g/L), anemia (Hb 9·6 g/dL), 76% lymphoplasmacytic monoclonal B-cells in BM and normal cytogenetics. In April 2005, an important IgG increase was observed (23 g/L). Immunofixation demonstrated an IgG-k paraprotein in the mid g-region and a monoclonal IgM-k paraprotein at the b-region corresponding to the two monoclonal peaks detected on serum electrophoresis. After 6-mercaptoethanol treatment, a single band was seen at the line stained with kappa, suggesting the presence of a single clone. Other causes of IgG monoclonal components were excluded considering clinical factors, immunophenotype analyses (San Miguel et al, 2003), quantity of DNA and cell cycle analyses (Ocio et al, 2005). However, the definitive proof for a unique monoclonal population was provided through molecular analysis. A single clonotypic rearrangement was detected by amplifying the complete VDJH fragment at diagnosis moment, according to the protocol describes in Biomed II (Leukemia2003; 17.2257–2317). Method describes from Billadeu et al (Billadeau et al, 1993) was used for isotype identification. So, cDNA monoclonal amplification was observed at tubes corresponding to Cm, Cd and Cg. All monoclonal PCR products were directly sequenced in an automated ABI 377 DNA sequencer. VH, DH & JH segments identification, as well as SH recognition was made using the V-BASE sequence directory alignment program, and the CH regions were compared at BLAST. All sequences obtained showed the same clonotypic CDR3 sequence (VH4-59/JH6) as well as the same SH (10,75%) pattern that monoclonal amplification at diagnosis, indicating the presence of the same clone at that moment (Figure 1). In conclusion, we report for the first time a WM case in which tumor cells were able to carry out CSR, showing IgG and IgM clonotypic amplification, as well as producing both paraprotein components. This constitutes the first in vivo demonstration that CSR is possible in WM cells, and are able to develop a fully functional isotype class switch recombination not only in vitro but also in vivo. Figure 1: Deduced amino acid sequence of tumor-derived VDJH gene with the three heavy chain isotypes (A: Cμ, B: Cδ C: Cγ). Sequences indicates the somatic mulation pattern. Comparison for WM are made with the closest germline VH gone; uppercase, replacement (R) mutation; lower case, silent (S) mutation. Each mutation was defined by nuclieotide exchanges in a single codon, with successive mutations leading in some cases to 2 or 3 distinct R or S events. These are shown as aligned amino acid changes at specific sites. Figure 1:. Deduced amino acid sequence of tumor-derived VDJH gene with the three heavy chain isotypes (A: Cμ, B: Cδ C: Cγ). Sequences indicates the somatic mulation pattern. Comparison for WM are made with the closest germline VH gone; uppercase, replacement (R) mutation; lower case, silent (S) mutation. Each mutation was defined by nuclieotide exchanges in a single codon, with successive mutations leading in some cases to 2 or 3 distinct R or S events. These are shown as aligned amino acid changes at specific sites.

The detection of PML-RARa by real-time PCR (RQ-PCR) is becoming an important tool for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL) patients; however, the clinical value of using a RQ-PCR approach remains to be determined. Our aim was to analyze the correlation between relapse risk and MRD levels assessed by RQ-PCR at different phases of treatment, and to compare these data with conventional qualitative RT-PCR. Follow-up samples from 145 APL patients treated with the PETHEMA protocols were analyzed by RQ-PCR protocol from Europe Against Cancer program and by RT-PCR method from European BIOMED-1 Concerted Action. After induction therapy, no association was found between the presence of a RQ-PCR positive result and relapse. After the third consolidation course, three cases remained positive by RQ-PCR assays and two of them (67%) relapsed. By contrast, only 16 out of 119 (13%) patients with negative RQ-PCR relapsed. During maintenance therapy and out-of treatment, a significant correlation between a RQ-PCR positive result and a poor relapse-free survival was observed (p < 0.0001). All patients with > 10 PML-RARa normalized number copies (NCN) at maintenance or out-of therapy relapsed (n=19), while all patients with < 1 NCN within out-of treatment group remained in hematological remission (n=75). 8 patients with < 1 NCN during maintenance therapy finally relapsed, but all of them appeared beyond the end of treatment. In addition, in the intermediate group of patients (NCN 1–10) (n=18) relapse probability of 40% at 5 years was observed. All hematological relapses were predicted if a follow-up sample had been obtained within the previous 4 months. Based on the information provided by RQ-PCR in samples obtained after the end of consolidation and subsequently, a relapse risk stratification could be established for APL patients.

p15 and p14/p16 tumor suppressor genes, have been reported to be frequently inactivated by various mechanisms in haematological malignancies such us MM. Alterations of these cell cycle inhibitors in MM display a close correlation with the cell cycle and clinical outcome. We have evaluated by real time quantitative RT-PCR (RQ-PCR) the expression of the p14/p16 and p15 genes in purified bone marrow plasma cells (PBMPC) from MM patients in order to evaluate the possible clinical, biological and prognostic significance of these cell cycle regulators. RNA extracted from purified BMPC from 53 untreated symptomatic MM and a pool of buffy coat from healthy donors (reference value) was analyzed by RQ-PCR using Assays-on-Demand gene expression mixes specific for p14/p16 and p15 genes in an ABI PRISM 7700 SDS (Applied Biosystems, Foster City, CA, USA). Values were corrected with a control gene (ABL). The relative quantification of gene expression was performed through the cycle threshold (CT) increment method. Patients were classified into different groups depending on gene expression values. Thus, according to p15 expression, 29% of patients (n=14) showed higher levels than the control and this group was characterized by the presence of good prognostic markers such us low Lactato dehidrogenase levels (LDH), low b2-microglobulin (B2M) and C-Reactive Protein (CRP) serum levels and absence of monoclonal proteinuria. Similar results were found for p14/p16 expression. Fifteen patients (28%) displayed a high p14/p16 expression and the group was also characterized by good prognostic features: low CRP, B2M and LDH levels. When p14/p16 and p15 genes were simultaneously analyzed, clinical and biological parameters showed a statistically significant correlation with gene expression. Thus patients with low gene expression had a high B2M (≥3 mg/dl) and high C-reactive protein (≥3 mg/dl). As far as survival was concerned, patients with a high p15 expression had a longer overall survival of 100% vs. 58% at 30 months (p=0,01), with the additional value that no deaths have been observed in any such patients. Similar results were observed for the group of patients displaying a high p14/p16 expression since they displayed a much better OS (100% vs. 63% at 30 months, p=0,02). Again, we should note that no deaths have been presented in this group. All these findings were much more evident when the three genes were simultaneously considered. Thus, within the group of 21 patients with at least one of the two genes overexpressed there have been no deaths vs. 11 among the 27 patients with low levels. This resulted in quite different OS curves for the two groups of patients (Figure 1) of 100% vs. 49% at 30 months (p=0,00). In conclusion, these genes significantly determine patients’ outcome thanks to their ability to classify them into different groups with different clinical, biological and outcome characteristics.

Introduction: Waldenström’s Macroglobulinemia (WM) is an uncommon lymphoproliferative disorder characterized by bone marrow infiltration of the lymphoplasmocytic cells and IgM monoclonal gammopathy. The normal counterpart of the WM malignant cell is believed to be a post germinal-center B cell. However, the low frequency of WM has hampered investigation into Immunoglobulin Heavy Chain (IgH) rearrangements in large series of patients and whether preferential use of specific genetic segments or non-functional rearrangements exits. Aim: Molecular characterization in a large cohort of WM patients analyzing their functional, complete VDJH, and non-functional, incomplete DJH, IgH rearrangements, the pattern of somatic hypermutation (SHM) and gene segment usage. Patients and methods: 47 patients with unequivocal diagnosis of WM were included in the study. Bone Marrow samples (always with more than 10% of tumor cell) were used for amplification of clonal rearrangements employing the Biomed-2 strategy (Leukemia2003; 17; 2257), followed by direct automated sequencing in ABI 377 DNA sequencer using Big-Dye terminators (Applied Biosystems, Foster City, CA). Results: All patients showed a monoclonal amplification of at least one VDJH or DJH rearrangement. VDJH monoclonal rearrangements were detected in 42/47 (89.3%) patients while DJH were observed in 20 (42.5%) patients. VH, DH and JH gene segment usage: VH3 gene segment was the most commonly represented family (76,2%) while the other gene segments were scarcely detectable, and VH5 was totally absent. In the complete VDJH rearrangements, DH6 was the family most commonly represented while DH2 (45%) and DH4 (30%) were the most common in the incomplete DJH rearrangement. Regarding JH elements, JH4 (38%) was the most represented in the complete rearrangements and JH5 (46%) in the incomplete rearrangements. Somatic hypermutation: SHM were observed in all except two of the cases (94%, 34 of 36 cases), in which the study could be performed. (median 9,38; range2.9–16.6). In the remaining clonal cases (6 cases, 14.3%) no amplification of FR1 region was obtained, also suggesting the presence of SHM. Conclusions: In this series of patients, we observed the presence of incomplete rearrangements with an incidence similar (44%) to that described in multiple myeloma. The preferential usage for determined families of genetic segments, in VDJH and in DJH, suggests the presence of positive and negative selection processes. Finally, the majority, (94 %) of the cases, presented SHM, although the presence of two patients lacking in SHM suggests a pre-follicular origin for some WM cases.

Gene Expression Profiling through RNA arrays has provided new clues to Multiple Myeloma pathogenesis and prognostic pattern evaluation. Recently, ZHX2, CHC1L & RAN expression have been highlighted as key elements in MM. In the present paper, we have evaluated theses genes by real time quantitative RT-PCR (RQ-PCR) in purified plasma cells from 74 patients with plasma cell discrasias. MATERIAL AND METHODS: Purified Bone Marrow cells were obtained from the following patients: 6 MGUS, 7 smoldering MM, 59 newly diagnosed symptomatic MM patients and 2 Plasma cell leukemia (PCL). After RNA extraction, RQ-PCR of CHC1L(C), RAN(R) and ZHX2 (Z) genes was carried out using the standard protocol from TaqMan® gene expression Assays-on-Demand in an ABI-PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Expression levels were normalized with ABL gene and expressed in n-fold times compared to the expression in a pool of RNA from mononuclear cells from healthy donors. The expression level of the different genes was evaluated for correlation with the diagnosis, clinical characteristics and prognosis of the patients. RESULT AND CONCLUSIONS: None of these genes displayed a clear relationship with the different stages of disease pathogenesis, although ZHX2 gene was slightly more expressed in the indolent forms of the proliferative disorders (MGUS and SMM). Within symptomatic MM patients, several interesting associations were observed. Thus, in hyperdiploid MM cases, CHC1L expressions observed were fewer than in those with a normal DNA index, confirming the participation of the gene product in chromosomal condensation during the mitosis. No other important associations were observed for this gene, although patients with the lowest expression displayed a very good prognosis, but without reaching statistically significant differences. As expected, RAN expression was related to S-Phase PC, since patients with high S phase values (>1.8 %) displayed higher levels of RAN transcripts. This, however, only resulted in a marginal impact on survival. ZHX2 provided the most interesting results, whereby decreased levels of ZHX2 were related to unfavorable prognostic indicators such as B2 microglobulin >4 mg/L and Hemoglobin levels SUMMARY: From this study we can confirm that RAN, CHC1L and especially ZHX2 genes play a role in the pathogenesis and behavior of MM, this could be helpful in predicting survival of patients.

Introduction: Detection of disparity in microsatellite DNA regions (STR - Short Tandem Repeats) between recipient and donor allows for sensitive and specific monitorization of the degree of haematopoietic chimerism. It is well known that disparities between donor and recipient in various polymorphic systems (mainly MHC) are associated with an increased incidence of graft-versus-host disease (GVHD). However, it is still unknown whether or not STR disparities could have a similar biological effect. Aim: To study the relationship between STR disparities and frequency of GVHD, overall survival and event free survival in patients who have received allogeneic transplantation. Patients: 161 consecutive patients transplanted with peripheral blood stem cells from identical MHC sibling donor at a single center were included in the study. Their characteristics were: median age 44 (17–69); Male/Female: 94/67; Sex disparity: 46%; Diagnosis: 39 AML, 26 ALL, 24 MDS, 19 MM, 17 CML, 14 NHL, 10 CLL, 10 HD, 1 CMPD,U, and 1 Hypereosinophilic Syndrome. The conditioning regimen was reduced intensity in 81 patients and myeloablative in the remainly 80 pts. All 161 patients engrafted and were evaluable for acute GVHD (aGVHD), while 128 were included in the analysis of chronic GVHD (cGVHD), according to the available follow-up. Methods: After genomic DNA extraction, PowerPlex®16 System kit (Promega Corporation, Madison, WI) was used to amplify 16 STR regions (15 plus gender marker, Amilogenin). The amplified products were analysed using GeneScan 2.1 (Applied Biosystems, Foster City, CA) after electrophoresis in the ABIPrism 377 (Applied Biosystems). The chi-square and y t-Student tests were used for statistical analysis. Log-rank analysis was applied for comparing differences in survival. Multivariate analysis was carried out according to the cox-regression method. Results: The number of STR disparities between recipient and donor ranged from 4 to 15 (median: 9). Discordances in D13S317, D18S51 and TPOX were associated with higher grades of aGVHD severity (p=0.024, p=0.027 and p=0.034, respectively). Disparities in D16S539 was associated with cGVHD (p=0.043). The number of loci discrepancies was not related to any clinical parameter included in the analysis (aGVHD, cGVHD, EFS y OS). However, when patients were grouped according to STR mismatches (11, n=127 and 17, respectively), shorter OS was associated in patients with >11 disparities (p=0.021). Conclusions: The presence of STR disparities could be associated with the development of complications during sibling allogeneic transplantation, including presentation of aGVHD. The data available only shows a marginal association and must be considered as preliminary.

Introduction: Currently, the origin of the HCL tumor clone is believed to be marginal zone B-cell that has previously contacted the antigen, and has therefore undergone somatic hypermutation (SHM). However, the low frequency of HCL has hamper the investigation of IgH rearrangements in large series of patients and whether preferential uses of specific VDJ segments or non-functional rearrangements exist. Aims: To characterize IgH gene rearrangements in 25 HCL patients in order to ascertain the frequency and characteristics of both incomplete DJH and complete VDJH rearrangements and to determine their somatic mutations. Patients and methods: 25 patients with unequivocal diagnosis of HCL (CD19+CD5-CD22+FMC7+CD23-CD103+) were included in the study. Amplification of clonal rearrangements was carried out using the Biomed-2 strategy (Leukemia2003; 17:2257), followed by direct automated sequencing. Results: All 25 patients displayed a clonal rearrangement, including 21 VDJH rearrangements (84%) and 14 DJH rearrangements (56%). Families VH3 & VH1 were over-represented, while families VH5, VH6 and VH7 were completely absent. The most frequently used VH specific segment was VH3–30 (3/21), by contrast segments used in normal mature B-cells were not found in any of the HLC cases (i.e. VH3–20 & VH3–49). As far as the DH segments is concerned, DH2 family was the most common in both complete and incomplete rearrangements, followed by DH3 in the complete and DH5 in incomplete fusions. Finally, JH6 and JH4 segments were the most frequently observed JH segments in both complete (52 & 42%, respectively) and incomplete (30 & 46%, respectively) rearrangements. Interestingly, JH3 was over-represented in DJH rearrangements three times more frequent than in VDJH rearrangements. Sequence analysis showed that HLC cases displaying a complete VDJH rearrangement were mutated (76%, average deviation to germline sequence of 4.12%). By contrast, none of the 14 cases with incomplete rearrangements showed SHM, (11 exactly matched the germline sequence and three had some deviation, but always below the 2%, which is the consensus cut-off point to define the presence of SHM). Conclusions: Although HCL seems to be a homogeneous disease, molecular analysis of IgH rearrangements of tumor cells shows an important biological heterogeneity. Thus, incomplete rearrangements are frequent, and there is a preferential usage in some VH, DH and JH segments. Furthermore, although the majority of cases seem to have a post-follicular origin, a quarter of them may have originated in cells that have not undergone the SHM process. This is only possible if they have not crossed the germinal center or if their SHM machinery is damaged. These observations suggest that the post-follicular origin (marginal zone B-cell) of HCL should be reviewed.

Acute promyelocytic leukaemia (APL) is characterised by the t(15;17) leading to the fusion of the PML gene with the retinoic acid receptor a (RARa) gene. APL is associated with favourable prognosis, however, approximately 15–20% of patients ultimately relapse. With regards to pre-treatment characteristics, is now clear that long-established prognostic factors such as age and leukocyte count have a major impact on outcome. However, none of these parameters is robust enough, in order to adjust treatments strategies according to the risk of relapse. The quantification of PML-RARa fusion gene transcripts (leukemia-specific chimeric mRNA) can add important prognostic information useful for risk group stratification. However, few clinical studies have been carried out and there are discrepancies concerning the prognostic significance of the quantification of fusion gene transcripts of newly diagnosed patients. In this study we tested the PML- RARa fusion gene transcripts level in 126 newly diagnosed patients with t (15;17) in order to determine their correlation with disease characteristics at presentation and to identify a subset of patients at high risk of relapse. The presence of the PML- RARa fusion gene transcripts was analysed from 1mg of RNA by real-time quantitative RT-PCR (RQ-PCR) based on TaqMan probes and the 7700 Sequence Detector, performed according to the “Europe Against Cancer Program” (Leukemia2003, 17:2323; 2318–2357). Results: In 121 out of 126 patients, evaluable data were obtained: 73 (60,3%) cases had bcr-1, 4 (3,3%) cases had bcr-2 and 44 (36,4%) cases had bcr-3 fusion types. The expression of the fusion gene was reported as the PML- RARa normalized quantities (NQ, number of PML- RARa copies per 1x104 ABL copies as gene control). The median of NQ was 3030 and the expression was highly variable (range of 826 to 9605). The NQ was not significantly associated with disease characteristic at diagnosis, such as age, percentage of blasts cells, fusion types and platelet counts. Nevertheless, patients with NQ above 3000 had lower white cell counts (7,4±12,1 vs 24,2±39,3x109/L, p=0,03). Therefore, the fusion transcript copy number per leukemic cell was higher in patients with lower leukocyte count. Moreover, in survival analysis, the NQ did not correlate with disease free survival or overall survival. Conclusions: Our data confirms the wide range of differences in expression activity between individual patients. The NQ did not correlate with the clinical and biological disease characteristics except for white cell counts. Neither did it correlate with principal prognostic parameters (response and survival).

Multiple myeloma (MM) remains as an incurable disease although new therapies can achieve a high rate of complete remissions (CR). Unfortunately, most patients ultimately relapse due to the persistence of minimal residual disease (MRD), and only a minority could be cured. Detection and quantification of these cells is an important tool for monitoring these patients and predicting a potential relapse. Here we analyze by RQ-PCR the MRD in MM patients achieving CR in order to classify them into different risk categories. MATERIAL AND METHODS: 38 MM patients uniformly treated according to the GEM-2000 (Spanish group for Myeloma) protocol, and that have achieved CR following PBSCT were included in the study. 22 were IgG, 9 IgA, 6 B-J and 1 non-secretory (κ/λ 21/16). 27 were male & 11 female with a median age of 58 (range 48–65). Bone marrow samples obtained at diagnosis and 3 months after transplant were analyzed. Complete (VDJH) and incomplete (DJH) Ig rearrangements were amplified with the Biomed-2 strategy (Leukemia2003;17:2257). PCR clonal products were sequenced on an ABI Prism 377 Sequence detector. VH, DH and JH segments were identified by comparing with germinal sequences on V-Base and BLAST databases. An ASO primer at the N-region was designed for each patient with the OLIGO 6.0 software. RQ-PCR was then performed on an ABI Prism 7700 using the ASO specific forward primer, a JH reverse intronic primer (JH1–6) and a TaqMan probe (JH1,2,4,5, JH3 or JH6) to amplify the patient specific rearrangement. Sample quality and quantity was controlled evaluating the standard curve of the albumin gene amplification. MRD was calculated according to ΔCT method. RESULTS: In 14 out of the cases included in the study, MRD investigation was not possible because the N-region was not longer enough to design the ASO primer (n=3), poor quality in the diagnostic sample to obtain the standard curve (n=8) or low plasma cell infiltration at diagnosis to obtain correct dilutions (n=3). The remaining 24 patients were classified into different risk groups according to the MRD level obtained 3 months after transplantation with a cut-off point of 0.01% tumor cells. Thus, progression free survival (PFS) was longer in those patients with MRD< 10−4 (p=0.03, figure 1A). By contrast, upon comparing the impact on PFS of immofixation (IFX) in these 24 patients that were in CR (defined by conventional electrophoresis criteria), it was observed that patients with IFX (−) didn’t showed a different outcome from those IFX (+) (figure 1B). CONCLUSION: In summary, although RQ-PCR is a labor and time-consuming technique, it is an useful tool for monitoring MRD in MM. The level of 10−4 can contribute to classify patients into 2 groups (high and low MRD) with different risk of relapse that can be used to design specific therapies.