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The surface expression of CD117 on plasma cells (PCs) from normal individuals and patients with multiple myeloma (MM) has been analysed using triple-stained cells for flow cytometry. In addition, the clinical significance of CD117 expression in MM patients and its possible value for the evaluation of minimal residual disease was explored. A total of 11 healthy volunteers and 56 untreated MM patients were included in the study. The expression of CD117 was analysed by flow cytometry, using simultaneous staining with the MAbs BB4, CD117 and CD38. Cell acquisition was performed in two consecutive steps using a live gate drawn on SSC/CD38 cells and a total of 300,000 events were acquired. For data analysis, the Paint-a-Gate Plus software (Becton Dickinson) was used. PCs were identified according to their strong reactivity for CD38 and their positivity for BB4, as well as by their light scatter distribution. Dilution experiments of CD117+ myelomatous PCs with normal bone marrow (BM) cells were performed in order to assess the sensitivity level of the technique for detection of CD117+ residual PCs. None of the PCs from normal BM samples showed reactivity for the CD117 antigen. In contrast, CD117 antigen was present in 18/56 MM patients (32%), the proportion of positive cells in these cases being as high as 92.1 +/- 9%. Therefore, within PC lineage the c-Kit antigen would be restricted to the myelomatous population and thus could be considered as a 'tumour-associated marker' for monitoring minimal residual disease in about one third of MM patients. Dilution experiments indicate that the detection limit with this marker would be 10(-4) (one myelomatous PC/10(4) normal BM cells). Upon comparing the clinical and haematological disease characteristics of CD117-positive and CD117-negative cases, no significant differences were found.

The aim of this study was to analyse the expression of NK-associated antigens in both peripheral blood and bone marrow lymphocytes from a large series of newly diagnosed multiple myeloma patients. 112 patients with untreated multiple myeloma (MM) were included in the study. 36 sex- and age-matched healthy volunteers were used as controls for peripheral blood (PB) studies and 14 for the bone marrow (BM) studies. Simultaneous stainings with the CD3/CD56, CD2/CD16 and CD8/CD57 monoclonal antibodies were systematically performed in PB and CD3/CD56 and CD2/CD16 in BM in order to analyse their relationship with the clinical and biological characteristics of the disease and survival. The expression of NK-associated antigens (CD56, CD16 and CD57) assessed within the lymphoid gate, was significantly increased (P < 0.001) in the PB of MM patients both in relative and absolute numbers. In the BM a significant increase in the percentage of CD56+ lymphocytes (P < 0.001) was also observed; in contrast, the proportion of CD16+ cells did not differ significantly from that of normal BM samples. The number of CD56+CD3- lymphocytes increased significantly within high-risk patients (869 +/- 671) as compared to intermediate (388 +/- 212) and low-risk patients (274 +/- 199) (P = 0.04). Moreover, patients with high values of CD56+CD3- lymphocytes showed a statistically significant association with several adverse prognostic factors including anaemia, hypoalbuminaemia, renal failure, high beta 2M, DNA diploidy and high S-phase plasma cells. In addition, patients with higher absolute numbers of PB CD56+CD3-lymphocytes displayed a poorer prognosis, whereas patients with higher values of CD57+CD8- cells had a better outcome.

In acute non-lymphoblastic leukemia (ANLL) progenitor cells frequently display a certain degree of autonomous growth. The aim of the present work was to analyze the autonomous proliferative capacity of leukemic progenitors in both de novo and secondary to myeloproliferative disorders (MPD) and myelodysplastic syndromes (MDS), acute myeloid leukemias and to correlate with clinical and biological characteristics of the disease. Clonogenic assays with and without leukocyte conditioned medium with PHA (LCM-PHA) were performed and the autonomous proliferation index (API) calculated in a series of 50 patients (34 de novo ANLL, eight secondary to MPD and eight secondary to MDS). Patients were divided into two groups according to their API, low (or = 0.4) or high (0.4). Autonomous growth was observed in 84% of cases studied (82% in de novo ANLL, 75% secondary to MDS and 100% secondary to MPD). The group with the highest API (29 patients) had increased levels of hemoglobin (P = 0.006) and platelets (P = 0.01). A high API was also associated with an immature phenotype of blast cells (P = 0.02). Upon analyzing the de novo ANLL separately we observed that a high API correlated with high Hb values (P = 0.02), a lower rate of complete remission (42% vs 61%) and a lower survival rate (medium of 3 vs 10 months). These findings suggest that the capacity for autonomous proliferation can condition the clinical and biological profile of the disease.

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 2967 Zoledronic Acid (ZOL) has demonstrated up to half a year improvement in survival of multiple myeloma patients. This conjoins with prior experimental studies to support a potential anti-myeloma effect for bisphosphonates. However, the in vivo effect can be confounded with the concomitant use of chemotherapy and new drugs, as well as with competing risks that emerge from the beneficial effect of bisphosphonates on the skeletal related events and its relationship with survival. Accordingly, the real anti-tumor effect of ZOL remains to be elucidated in clinical practice, for example in patients free of the disturbing effect of chemotherapy. New therapies provide an 80–90% response rate in MM, although most patients ultimately relapse. During relapse, many patients show an initial re-positivization or re-growth of the M-component (biochemical relapse) that is usually not accompanied by clinical symptoms. The usual decision at this point is not to treat such patients. Thus, these asymptomatic patients represent a perfect group to explore the antitumor benefit of ZOL in the absence of any other cytotoxic therapy. Aim: To analyze the anti-tumor effect of ZOL in the absence of any other cytotoxic drug in MM patients under asymptomatic biochemical relapse. Primary end-point was Symptomatic Progression Free Survival (sPFS). Secondary evaluation variables were response rate, skeletal related events and time to next chemotherapy. Patients and methods: 192 patients are calculated to be recruited in a randomized, prospective, open label phase IV trial in which a group of patients receive ZOL (4 mg iv./4 wk, 12 doses) and Best Supportive Care (BSC) and the rest only BSC. All patients are monitorized every 4 wk. Results: This is an interim analysis corresponding to the first 93 patients included in the trial: 49 treated with ZOL and 44 without ZOL. Asymptomatic Biochemical Relapse was confirmed in all patients who had a median age of 68 yr (40–87) and a male female distribution of 50/43. M-component distribution was IgG (69%), IgA (29%) and only light chain (2%). Relapse had presented after 1, 2 or 33 lines of therapy in 67%, 22% and 11% of cases, respectively. Prior treatment had always included transplant (65%), bortezomib (33%), IMiDs (33%), or a combination of them. Lytic bone lesions were present in 66% of patients and one or two skeletal related events (SRE) had presented prior to the inclusion in the trial in 31% of cases. FISH/cytogenetics was abnormal in 49% of cases: t(11;14) 18%, Rb deletion (alone) 16%, del(p53) 9%, t(4;14) 6% and t(14;x) 4%. After randomization, both groups of patients were well balanced in terms of prognostic features, prior response, and time from diagnosis and relapse to the inclusion in the trial. 25 patients have completed the program and four terminated before completion due to patient refusal (n=2) and development of other diseases (n=2). 31 patients are still ongoing and 38 have progressed before 12 mo of treatment, with a median sPFS of 287 days (9.4 months) with similar results between the two arms (271 vs. 308 days for patients receiving or not receiving ZA, respectively; 12-month projected sPFS was 47% vs. 42%, respectively). Interestingly, patients not treated with ZOL progressed with more advanced bone disease (8 cases of new bone lesions or re-growth of prior lesions, 1 spinal cord compression, and 2 cases of hypercalcemia) vs. patients treated with ZOL (two cases re-growth of bone lesions, p Conclusions: Zoledronic Acid therapy in MM with asymptomatic relapse seems to reduce the risk of progression with symptomatic bone disease and SREs. The possible antitumor effect of Zoledronic Acid alone in biochemical relapses cannot be elucidated yet. This interim analysis supports the continuation of the trial to reach a higher number of patients and longer follow-up. Disclosures: García-Sanz: Novartis Oncology: Grant Support Other. De La Rubia:Celgene, Janssen: Consultancy, Speakers Bureau. Granell:Janssen: Honoraria; Celgene: Honoraria.

Abstract 2914 Background: Based on clinical and biological data, a multistep model of disease progression starting in monoclonal gammopathy of undetermined significance (MGUS) continuing through MM, sometimes with an intermediate entity called smoldering MM (SMM), and ending in extramedullary disease, has been proposed. Material and Methods: To gain further insights into the role of the transcriptome deregulation in the transition from normal plasma cells (NPC) to clonal PC and from indolent clonal PC to malignant PC, we performed gene expression profiling (GEP) by Human Gene 1.0 ST Array (Affymetrix) in purified PC from 41 patients with MM, 33 with high-risk SMM and 20 with MGUS. In addition, 5 healthy donors were also included in order to relate the deregulation of GEP of clonal populations to normal condition. Results: Initially, we investigated whether the selected PC population from monoclonal gammopathy patients displayed specific GEP that were clearly distinguishable from NPC. 126 common genes were differentially expressed in MGUS, SMM and MM as compared to NPC (q-value < 10−5). The two most significant molecular and cellular functional categories were cell cycle and cell death (P < 0.01). The top 3 canonical pathways were EIF2 signaling, regulation of EIF4 and p70S6K signaling, and mTOR signaling (P < 0.001). Interestingly, 17 and 9 out of the 126 significant deregulated genes were small nucleolar RNA molecules and zinc finger proteins. GADD45A was the most significant up-regulated gene in clonal PC vs NPC. Subsequently, we looked for genes deregulated in MGUS vs MM, SMM vs MM and MGUS vs SMM, in order to identify gene categories and molecular pathways involved in MM development. A total of 1,184 genes were differentially expressed between MGUS and MM (q-value < 10−5) with an imbalance in favor of antiapoptotic state in MM. Thus, BAG3, GADD45B, AKT1 and AKT2 were overexpressed in MM while APAF1 and BCL2L1 were underexpressed in MM. The molecular and cellular function most significantly affected in this comparison was cell death with 106 genes involved (P < 0.01). One of the top 10 genes upregulated in MM vs MGUS was TERC. In the SMM vs MM comparison, 1,163 genes were deregulated (q-value < 10−5). DNA replication, recombination and repair (ATM, RAD17, RAD50) was the most significant deregulated molecular and cellular function. Telomere extension by telomerase and Myc mediated apoptosis were included within the most significant canonical pathways. The analysis showed that a set of 627 differentially expressed genes was able to differentiate MGUS from SMM (q-value < 10−5). Cell death was identified as the most significant molecular function affected (P < 0.01). The RAC signaling pathway deregulation (P < 0.01) was included within the five most significant canonical pathways. In addition, NFKB signaling was also in the top canonical pathways (P < 0.01). Finally, we also looked for those genes significantly deregulated among the three entities, which in turn were progressively up or downregulated from MGUS to SMM and to MM. We reasoned that these genes could be the most significant for promoting multistep transformation. Surprisingly, only eight out of the 2,974 significant genes exhibited a progressive deregulation (P < 0.0001) in the evolving stages of monoclonal gammopathies (Figure 1). All the genes with a progressive increase from MGUS to SMM and to MM were snoRNA. APAF1, VCAN and MEGF9 showed a progressive downregulation in the transition from MGUS to SMM and to MM. Conclusion: Our data show that although MGUS, SMM and MM are not clearly distinguishable groups according to their GEP, several signaling pathways and genes were significant deregulated in the different steps of transformation process. Disclosures: Lahuerta: Janssen: Honoraria; Celgene: Honoraria. Oriol:Janssen: Honoraria; Celgene: Honoraria.

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 936 Demonstration of bone marrow (BM) infiltration by lymphoplasmacytic lymphoma is essential to the diagnosis of WM, and a trephine biopsy is considered mandatory for this assessment. Multiparameter flow cytometry (MFC) has demonstrated its clinical relevance in MGUS and myeloma; however, immunophenotypic studies on IgM monoclonal gammopathies are scanty, and focus only in patients with WM. Herein, MFC immunophenotyping was performed on BM samples from 244 patients, including 67 IgM MGUS, 77 smoldering, and 100 symptomatic WM newly diagnosed patients according to the Second International Workshop. A four color panel that systematically allowed the identification of B cells and plasma cells (PC), and their phenotypic characterization for a total of 24 antigens was used. We first analyzed the percentage of B cells and PC in BM and the percentage of light chain restricted cells in both compartments. Our results show a progressive increment of B cells from IgM MGUS to smoldering and symptomatic WM (medians of 2%, 9% and 12%; P10% B cells, in contrast to 34% and 55% of smoldering and symptomatic WM (P10% vs ≤10%: median TTP of 47m vs 145m; P=.016) and light chain restricted B cells (100% vs 10% B cells showed a trend for inferior OS (P=.080), and significant differences emerged when comparing patients with 100% vs Focusing on the antigenic profiles of B cells and PC, we noted that within the B-cell compartment there was a progressive increment of CD22dim (69%, 92% and 88%; P50% of WM patients, which raises the possibility that the WM clone may arise, at least in some cases, before antigenic stimulation; subsequent maturation of the clone into PC would explain the typical presence of somatic hypermutations. On the other hand, B-cells from IgM MGUS and WM patients were negative in ≥90% of cases for CD5, CD10, CD11c and CD103, which can be useful to differentiate between WM and other B-NHL. Finally, the antigenic profile of PC in IgM MGUS and WM was similar to that of normal PC, and different from myeloma PC by consistently showing a CD27+ and CD56- phenotype, in addition to sIgM+ expression in ≥87% of all cases. Similarly to B-cells, we also noted that within the PC compartment there was a progressive increment of CD19+, CD45+ and sIgM+ CD20+ PC from IgM MGUS to smoldering and symptomatic WM. This underlies that this transition is asssociated with an accumulation of light chain restricted clonal PC displaying an immature/plasmablastic phenotype. In summary, our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenström's clone, as well as for the differential diagnosis, risk of progression and survival in WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 3941 Multiple Myeloma (MM) is a neoplastic proliferation of aberrant plasma cells that has well defined clinical steps including MGUS, SMM, MM and PCL. These clinical stages represent a good model in which to study the multistep acquisition of genetic events underlying cancer progression. The transition of normal plasma cells to myelomatous cells is thought to be the result of the sequential acquisition of “genetic hits” causing the classical hallmarks of cancer. Underlying this transition, the existence and competition between different tumour clones, also termed intra-clonal heterogeneity, has been recently highlighted in a number of human cancers. In this work we have analysed the Darwinian intra-clonal population dynamics of MM in order to better understand disease progression, development of treatment resistance, and the effectiveness of targeted therapies. Next generation sequencing (NGS) is an excellent tool which can identify complex intraclonal relationships in MM allowing the identification of acquired mutations as well as being able to place them within a Darwinian context. This type of model incorporates not only the competition between already existent clones, but also the emergence of new clones during disease progression via the acquisition of novel mutations. Single cell analysis ensures the firm identification of the different clonal populations harbouring common and distinct mutations effectively validating the NGS data. In this work, we have extended our sequence analysis of MM as well as studying paired patient samples at different stages of the disease process including relapses or transitions to later stages of disease. Samples include 6 MGUS patients, 4 high-risk SMM patients that later developed symptomatic MM, 2 MM patients at presentation and relapse, and 1 MM patient that later evolved to PCL. Additional paired patient samples will be added to the final analysis. CD138-positive bone marrow plasma cells were selected to a purity >95%. For SMM-MM paired samples, we used 100 ng of tumour and non-tumour cell DNA to undergo whole genome sequencing, using 120 bp paired-end reads on a GAIIx (Illumina) to a median depth of 32x, with 98% at 1x and 84% at 20x coverage. For the rest of the samples, we used 50 ng of DNA and performed whole exome sequencing using 76 bp paired-end reads on a GAIIx (Illumina) to a median depth of 61x, with 99% at 1x and 85% at 20x exomic coverage. Data were aligned to the human genome (hg19) using Stampy and BWA and acquired SNVs and indels called using GATK. Single cells from a subset of patients were sorted into lysis buffer on 96 well plates using a FACSAria cell sorter. Specific target amplification (STA) was performed before quantitative PCR using genotyping assays specific for the mutations of interest. The STA product was diluted before qPCR interrogation for mutations using the 96.96 dynamic microfluidic array and the BioMark HD (Fluidigm). The results of this analysis show that: The mutational gene spectrum found in MM is consistent with our previous sequencing analysis of t(4;14) and t(11;14) MM cases.The mutational landscape and intraclonal diversity seen in paired samples exist at the earliest stages of myeloma including asymptomatic stages such as MGUS or SMM; however, clonal size varies during progression. The number of SNVs in High Rish SMM does not significantly differ from MM with clinical symptoms requiring treatment. Hence, these data suggest that all of the genetic deregulation necessary to give rise to an aggressive clinical state is already present in asymptomatic stages of the disease, but is masked by the existence of a predominant clone with a more benign pattern of behaviour that may compete for access to a putative myeloma niche.Different patterns of “clonal tides” are seen at presentation-relapse pairs and following the transition to PCL, consistent with a Darwinian process of tumour evolution. Their high genetic complexity is displayed by the accumulation of multiple mutations in various genes involving diverse biological functions, but that may eventually supply the hallmarks of cancer. These tumour cell populations may be either genetically stable, linearly evolving, or heterogeneous clones with changing predominance. The emergence of new clones, which could be driven by mutations in key genes such as KRAS or BRAF, increases the complexity and effectiveness of targeted therapies. Disclosures: No relevant conflicts of interest to declare.

1630 The management of recurrent or refractory Hodgkin's lymphoma (HL) remains challenging. Previous published data have suggested that infiltrating normal B lymphocytes in classic HL lesions may contribute to the survival of Hodgkin and Reed-Sternberg cells in vivo. The objective of this prospective, multicenter, phase II trial was to investigate the activity of an anti-CD20 monoclonal antibody, ofatumumab, in combination to a standard platinum-based salvage regimen, ESHAP (O-ESHAP) followed by high-dose chemotherapy and autologous stem cell transplantation (ASCT) for patients with classical HL failing to first line chemotherapy. Forty- five patients (25 M / 21 F, median age 34 years, range 18–66) were enrolled in the study between June 2010 and June 2012. Treatment consisted on three cycles of ESHAP plus ofatumumab 1,000 mg days 1 and 8 on first cycle and day 1 on second and third cycles. At the time of study entry, 66% of patients had III-IV Ann Arbor stage, 16% bulky disease, 18% B symptoms, 40% extranodal HL and 52% ≥3 involved nodal areas. We respect to response to first-line therapy, 46% patients had achieved a completed response (CR) and then relapsed, 6% had a partial remission (PR), whereas the remaining 48% were primary refractory. Eighty-one percent patients have received 3 cycles of O-ESHAP as scheduled, three patients 2, and five 1 cycle (1 patient due to toxicity, 1 patient's decision, 2 HL progression, and 4 treatments ongoing). Grade 3–4 WHO hematological toxicity was observed in 16%, 19%, and 20% after cycles 1, 2, and 3, respectively. Non-hematological toxicity was reported in 32%, 10%, and 20%, respectively. Overall response (OR) rate was 63% (49% CR and 14% PR). Response to O-ESHAP according to prior response to first-line chemotherapy is shown in [table 1][1]. Adequate PBSCs collection was achieved in 94% mobilized patients. Twenty-six out of 33 patients have already proceeded to ASCT. Two patients died of neutropenic sepsis after ASCT and HL progression, respectively. Preliminary results of this ongoing trial suggest that addition of ofatumumab to ESHAP is safe and has a promising clinical activity in patients with relapsed/refractory HL candidates to ASCT. | | Response to first-line chemotherapy | |:---------------------- | ----------------------------------- | ----------------- | | | Relapsed or partial response (n=17) | Refractory (n=16) | | Response after O-ESHAP | | | | OR | 16 (94%) | 7 (44%) | | CR | 14 (82%) | 3 (22%) | | PR | 2 (12%) | 4 (22%) | | Refractory | 1 (6%) | 9 (56%) | Table 1. Response to O-ESHAP according to previous response to first-line treatment Disclosures: No relevant conflicts of interest to declare. [1]: #T1

Abstract 4566 Cytogenetic abnormalities are present in virtually all cases of Waldenström's macroglobulinemia (WM). Among them, deletion of the long arm of chromosome 6 (6q-) is the most frequently reported in the literature, but the potential impact on disease evolution has not been completely elucidated. In this study we have analyzed the prevalence of this aberration in 203 patients with B-cell lymphoproliferative disorders (B-LPD) producing a monoclonal IgM component. Detection was evaluated by In Situ Fluorescence Hybridization in CD19+ separated cells and results were correlated with disease characteristics. The frequency of 6q21 deletion varied according to the type of B-LPD: 1/42 (2%) in IgM-MGUS, 5/69 (7%) in asymptomatic WM (aWM), 25/83 (30%) in symptomatic WM (sWM) and 0/9 in other IgM B-LPD. Within MGUS and aWM, patients with deletion of 6q displayed a high frequency of adverse prognostic features and required treatment earlier than patients with normal 6q (37 mo vs. +120 mo, p Disclosures: Ocio: PharmaMar: Patents & Royalties, Research Funding. San Miguel:Celgene: Honoraria; Millenium: Honoraria; Janssen: Honoraria.

Abstract 296 A well defined model of disease progression has been established in myeloma based on the transition of normal plasma cells to monoclonal gammopathy of undetermined significance (MGUS), smouldering myeloma (SMM), myeloma (MM) and finally to plasma cell leukaemia. The most likely mechanism underlying the initiation and progression of myeloma is that the primary genetic events, such as IGH translocations or hyperdiploidy confer a proliferative/survival advantage but not a truly malignant phenotype. Subsequently, these cells acquire secondary genetic aberrations that are associated with uncontrolled proliferation and invasion which manifest as bone lesions and myelosuppression. We have addressed the hypothesis that we can gain critical understandings of the mechanisms leading to disease progression, by defining the different genetic events present in the early pre-clinical stages of the disease (SMM) compared to those present in the clinical stages of the disease (MM). One of the essential components of disease progression is the acquisition of novel genetic hits and clonal selection based on the selection and expansion of the subclone most able to survive. We hypothesise that within the malignant plasma cells there is substantial clonal heterogeneity and that at different stages of the disease the extent of this is variable, with progression occurring in an oligoclonal fashion by acquisition of multiple genetic hits rather than as a result of the linear acquisition of sequential genetic hits. In this study we utilized massively parallel sequencing to study paired plasma cells from 2 patients who had progressed from SMM to MM and compared them to the patients non-involved DNA. Patients had been diagnosed with SMM, and had a bone marrow aspirate taken, at least 18 months prior to being diagnosed with MM. Both samples were taken before the patient underwent any treatment. The 2 patients were analyzed at the two time points and compared to the germline DNA obtain from the peripheral blood sample, using whole genome sequencing to identify acquired single nucleotide variants (SNVs), indels and translocations in the SMM and MM samples. Additionally, changes acquired at the transition from SMM to MM were examined along with frequency of abnormal reads at these sites. 100 ng genomic DNA from CD138 MACsorted cells and normal white cells was sequenced using 75 bp paired-end reads on a GAIIx (Illumina) to a median depth of 32x and 98% at 1x and 84% at 20x coverage. Data were aligned to the human genome (hg19) using ELAND v2e and acquired SNVs and indels called using CASAVA 1.8. In both samples the majority of SNVs were found in intergenic (range 64–71%) or intronic (25–31%) regions. The number of acquired SNVs within exons (including UTRs) accounted for only 0.5–1.1% of all variants. There was no statistical difference in the genomic distribution of SNVs between SMM and MM samples. Analysis of the SMM and MM samples shows that SNVs in the MM sample, which are acquired in the tumour, are all present in the SMM sample albeit at lower levels, indicating that the dominant MM clone was always present before transformation from SMM. Additionally, there are SNVs present in the SMM sample which are not found in the MM sample from the same individual. For example, variant calls can be detected at an incidence of 11–15% in the SMM sample and are undetected in the MM sample, at this sequencing depth. Acquired indels were found almost exclusively (>99%) within intergenic and intronic regions in both SMM and MM samples. As with the SNVs, all indels found in the MM samples were also found in the SMM sample from the same patient, but not all indels found in the SMM sample were found in the MM sample. The results presented indicate that during the progression from SMM to MM there is an oligoclonal selection process which results in the emergence of a dominant clone, and other sub-clones containing non-advantageous passenger mutations are not selected. Any of these SMM clones can clearly and rapidly transform to MM with full malignant potential. Therefore, targeted therapeutic approaches aimed at mutations in the MM dominant clone may not be curative, but may allow the propagation of minor clones without these mutations. It is therefore imperative to determine which mutations are present in pre-malignant clones to establish a clinical course of action in treatment. Disclosures: Humphray: Illumina: Employment. Murray:Illumina: Employment. Ross:Illumina: Employment. Bentley:Illumina: Employment.

Abstract 810FN2 Increasing evidence shows that a small fraction of MM patients (pts) treated with high-dose therapy followed by autologous stem cell transplantation achieve long-term remission. Interestingly, this is not restricted to pts in complete response (CR), since those that revert to a monoclonal gammopathy of undetermined significance (MGUS) profile may also achieve long-term remission, despite the persistence of residual myeloma plasma cells (PCs). These results suggest that in addition to the anti-myeloma therapy, other factors may play a role in the control of the disease. Herein, we used 8-color MFC for detailed characterization of the structural components of the immune system and hematopoietic precursor cells (HPC) in paired bone marrow (BM) and peripheral blood (PB) samples from 26 MM patients in long-term disease control (LTDC): 9 in continuous CR and 17 who reverted to an MGUS profile and that subsequently showed stable disease without treatment for ≥5 years (median of 9 years; range, 5–19). As controls, paired BM and PB samples from 23 newly-diagnosed MGUS and 16 MM pts, together with 10 healthy adults (HA), were studied in parallel. In all BM and PB samples the distribution of the major T- (CD4, CD8, Tregs and γδ), NK- (CD56dim and CD56bright) and B-cell subsets (Pro-B, Pre-B, naïve and memory), in addition to normal PCs, dendritic cell (DC) subsets (plasmacytoid, myeloid and monocytic), monocytes, and CD34+ HPC (myeloid and lymphoid), were studied. The percentage and absolute count of each cell population was analysed in the BM and PB, respectively. Comparison of the two groups of MM pts with LTDC (9 CR vs. 17 MGUS-like) showed similar (p>.05) cellular profiles in PB and BM, except for an increased number of BM and PB normal PCs in CR patients (P≤.04). Consequently, for all subsequent analyses, LTDC myeloma pts were pooled together. When compared to HA, patients with LTDC had increased numbers of CD8 T-cells and CD56dim NK-cells in both the BM and PB (p≤.03 and p≤.01, respectively). Despite this, the distribution of BM and PB CD4, CD8 and γδ T-cells among LTDC patients was similar (p>.05) to that of both newly-diagnosed MM and MGUS cases; in contrast, BM and PB Tregs were significantly decreased vs newly-diagnosed MM (P=.03) and MGUS (P=.04). Regarding B-cells and normal PCs, LTDC patients showed increased numbers of BM B-cell precursors (both Pro-B and Pre-B cells) and normal PCs vs. newly diagnosed MM (P≤.05), but not MGUS, together with increased numbers of naïve B-cells vs. both MM and MGUS pts (P≤.01); all such cell populations returned to levels similar (p>.05) to those of HA. As expected, this also included the number of CD34+ B-cell HPC which was increased among patients who achieved LTDC vs MM (p=.02), at levels similar (p>.05) to those of MGUS and HA. Regarding DC, LTDC patients showed normal DC numbers in PB (but with higher PB myeloid-DC numbers vs. MM; p=.02), in association with decreased numbers of plasmacytoid DC and increased monocytic-DC in the BM vs. HA (p≤.04). No differences were found for the numbers of BM and PB monocytes. In summary, here we investigated for the first time the immune cell profile of MM patients who achieve long-term disease control. Our results show that, as newly-diagnosed MM, patients that achieve long-term disease control also show increased numbers of cytotoxic T-cells and CD56dim NK-cells; however, in contrast to newly-diagnosed MM, among LTDC patients such increase is associated with lower numbers of T-regs and an almost complete recovery of the normal PC, B-cell precursor and naïve B-cell compartments both in BM and PB. Further investigations on the activation and functional status of these cell populations are warranted.MO (%)/SP (cels./μl)HA N= 10MGUS N= 23MM N= 16LTDC-MM N= 26T cells9.588110.6117313113711926 CD4+4.85004.6624^6*5085463 CD8+3.7∼216∼4.63865.32645.3431 TCR γδ.2426.3230.2428.3421 Treg.4137.4141^.54*38.3432NK cells.7∼87∼1.51982.11721.6212 CD56 dim.65∼79∼1.41922.21681.6202B cells2.81471.8104.97*68*1.9160 Pro B.11—.06—.02*—.07— Pre B.6—.4—.08—.23— Naive SP—80—57^—36*—118 Normal-PCS.18.9.11.7.008.72*.11.84DCs.3449.3653.6848.558 Monocytes2.22472.42853.43023.1315 m-DC SP—11—14—8*—12 MO-DC.11∼29.2036.434.2837 p-DC.2∼4.1.145.112.8.123.8CD34+.9∼1.46.61.1.261.4.431.4 Mie-HPC.8∼—.53—.26—.36— Linfo-HPC.1—.07—.03*—.05—*p≤.05 LTDC vs MM: ^ p≤.05 LTDC vs MGUS; ∼ p≤.05 LTDC vs HA Disclosures: Paiva: Jansen-Cillag: Honoraria; Celgene: Honoraria. Martinez:Janssen: Honoraria; Celgene: Honoraria. Maiolino:Centocor Ortho Biotech Research & Development: Research Funding.

Abstract 5014 Exposure to toxic compounds and pesticides leads to an increased risk to develop Multiple Myeloma (MM). The metabolism and the excretion of xenobiotics are mediated by the enzymes and transporters acting in the detoxifying/elimination process. The nuclear receptors NR1I2 (or PXR) and NR1I3 (or CAR) act as xenosensor activating the detoxifying/elimination process in response to the intracellular levels of xenobiotics. It has been hypothesized that part of the individual variability in drug metabolism efficiency could be due to the genetic variations within these regulator genes affecting their expression and/or function. To investigate the impact of genetic variation within these genes on MM susceptibility, we selected and genotyped 10 tag Single Nucleotide Polymorphisms (SNPs) in the PXR gene and 7 tag SNPs in the CAR gene in 627 MM cases (320 males and 307 females) and 883 (459 males and 424 females) controls from different European populations. All the SNPs were in Hardy-Weinberg equilibrium (p>0.001), with the exception of the PXR SNP rs2461818 that was therefore excluded from the analysis. We found no association of any of the genotyped SNPs with MM risk. In the same way, haplotype distribution showed no differences between cases and controls. This was the first comprehensive investigation of genetic variation in xenobiotic regulators genes PXR and CAR in relation to MM risk and our data suggest that common variants in these genes have no impact in modifying MM risk. Table I. Genotype distribution of the PXR and CAR SNPs among MM cases and controls. SNP (rs) Cases (%) Controls (%) OR* 95%C.I. p-value p-trend PXR C/C 429 (69.5) 623 (70.7) 1.00 Ref 0.423 rs10511395 A/C 160 (25.9) 228 (25.9) 1.02 0.81 – 1.30 0.851 A/A 28 (4.6) 30 (3.4) 1.38 0.81 – 2.35 0.232 PXR C/C 452 (74.0) 656 (74.8) 1.00 Ref 0.451 rs1054190 C/T 137 (22.4) 200 (22.8) 1.00 0.78 – 1.28 0.993 T/T 22 (3.6) 21 (2.4) 1.56 0.84 – 2.88 0.155 PXR C/C 412 (65.9) 591 (67.2) 1.00 Ref 0.819 rs11917714 C/T 190 (30.4) 250 (28.5) 1.07 0.85 – 1.35 0.535 T/T 23 (3.7) 38 (4.3) 0.84 0.49 – 1.44 0.536 PXR C/C 223 (36.3) 296 (33.7) 1.00 Ref 0.126 rs12488820 C/T 289 (47.0) 407 (46.4) 0.93 0.74 – 1.18 0.574 T/T 103 (16.7) 175 (19.9) 0.79 0.58 – 1.06 0.119 PXR G/G 430 (69.6) 593 (67.4) 1.00 Ref 0.807 rs13071341 A/G 166 (26.9) 269 (30.6) 0.85 0.67 – 1.07 0.158 A/A 22 (3.5) 18 (2.0) 1.70 0.90 – 3.22 0.102 PXR A/A 352 (58.7) 516 (39.4) 1.00 Ref 0.981 rs3237359 A/G 209 (34.8) 291 (33.5) 1.04 0.83 – 1.30 0.720 G/G 39 (6.5) 62 (7.1) 0.90 0.59 – 1.37 0.619 PXR C/C 255 (41.2) 383 (43.6) 1.00 Ref 0.815 rs13059232 C/T 299 (48.3) 390 (44.4) 1.16 0.93 – 1.44 0.192 T/T 65 (10.5) 106 (12.0) 0.94 0.66 – 1.33 0.711 PXR A/A 300 (48.7) 437 (49.7) 1.00 Ref 0.258 rs3732357 A/G 240 (39.0) 361 (41.0) 0.94 0.75 – 1.17 0.589 G/G 76 (12.3) 82 (9.3) 1.31 0.92 – 1.85 0.130 PXR T/T 328 (53.6) 463 (52.9) 1.00 Ref 0.424 rs1357459 C/T 249 (40.7) 345 (39.4) 1.02 0.82 – 1.27 0.850 C/C 35 (5.7) 67 (7.7) 0.75 0.49 – 1.17 0.206 CAR A/A 218 (35.4) 335 (38.1) 1.00 Ref 0.571 rs3003596 A/G 296 (48.0) 393 (44.7) 1.16 0.93 – 1.46 0.191 G/G 102 (16.6) 151 (17.2) 1.04 0.77 – 1.41 0.799 CAR G/G 264 (42.7) 371 (42.0) 1.00 Ref 0.642 rs3813627 G/T 276 (44.7) 392 (44.4) 0.98 0.79 – 1.23 0.882 T/T 78 (12.6) 120 (13.6) 0.91 0.66 – 1.26 0.581 CAR A/A 441 (73.1) 635 (73.5) 1.00 Ref 0.911 rs11265571 A/T 147 (24.4) 207 (24.0) 1.01 0.79 – 1.29 0.911 T/T 15 (2.5) 22 (2.5) 0.97 0.49 – 1.89 0.921 CAR T/T 404 (64.2) 575 (65.7) 1.00 Ref 0.836 rs2307418 G/T 193 (31.1) 268 (30.6) 1.02 0.81 – 1.27 0.879 G/G 23 (3.7) 32 (3.7) 1.05 0.60 – 1.83 0.863 CAR C/C 348 (56.6) 508 (57.7) 1.00 Ref 0.527 rs2502805 C/T 220 (35.8) 313 (35.6) 1.05 0.84 – 1.30 0.693 T/T 47 (7.6) 59 (6.7) 1.16 0.77 – 1.74 0.484 CAR A/A 245 (39.8) 346 (39.4) 1.00 Ref 0.770 rs4073054 A/C 291 (47.2) 412 (46.9) 0.98 0.78 – 1.22 0.855 C/C 80 (13.0) 120 (13.7) 0.94 0.68 – 1.31 0.720 CAR C/C 360 (57.6) 524 (59.8) 1.00 Ref 0.391 rs4233368 A/C 225 (36.0) 302 (34.4) 1.09 0.88 – 1.36 0.439 A/A 40 (6.4) 51 (5.8) 1.15 0.74 – 1.78 0.538 Genotype distribution among MM cases and controls in the overall population. * OR are adjusted for age, gender and region of origin. Differences in samples numbers are due to failures in genotyping. Disclosures: No relevant conflicts of interest to declare.

Abstract 4051 Multiparameter flow cytometry (MFC) immunophenotyping has shown to be of value for differential diagnosis and minimal residual disease assessment in multiple myeloma. However, the clinical value of MFC immunophenotyping in other plasma cell disorders (PCD) remains largely unexplored. Systemic light chain (AL) amyloidosis is a rare PCD characterized by the accumulation of monoclonal light chain fragments leading to end-organ damage and short survival. Bone marrow (BM) plasma cell (PC) infiltration in AL is usually low and thus the identification of clonal PC can be often difficult by immunohistochemistry and/or immunofluorescence. In the present study we focused on 34 BM samples sent to our institution with a suspected diagnosis of AL. MFC immunophenotypic studies were performed using the following 4-color combinations of MoAbs (FITC/PE/PerCP-Cy5.5/APC): CD38/CD56/CD19/CD45 (n=34); in addition cy-Kappa/cy-Lambda/CD19/CD38 staining was add to confirm the clonal or polyclonal nature of BMPC in equivocal cases. Ploidy and cell cycle analysis were additionally performed in a subset of cases (n=12/34). From the total 34 cases included in the present study, 28 had a confirmed diagnosis of AL. The remaining 6 cases were finally diagnosed with localized - amyloidoma - (n=2) and familial (n=1) forms of amyloidosis, multiple myeloma-associated amyloid (n=2) and congestive pericarditis (n=1). Interestingly, the presence of clonal PC was detected by MFC in 27 of the 28 (96%) patients with AL; in turn, clonal PC were undetectable in the BM of all cases with localized and familial forms of amyloidosis. The median overall level of PC (M-PC plus N-PC) seen in MFC immunophenotypic analyses of BM samples of the 28 patients with AL was 1.9% (range: 0.1% - 15%), with a significant positive correlation between PC enumerated by MFC and conventional morphology (r=0.5; p=.01). Within the BMPC compartment, the median proportion of clonal PC was of 94% (mean 81% ± 29%); in 6 cases all BMPC were clonal while in the remaining 22 patients residual normal PC persisted (median of normal PC/BMPC 13% ± 31%). The most common aberrant phenotypes were down-regulation of CD19 (92%) and CD45 (83%), followed by overexpression of CD56 (56%) and infra-expression of CD38 (42%). Aneuploidy was only found in 18% of cases, all of them hyperdiploid. Cell cycle analysis showed a median % of S-phase and G2-Mitosis PC of 0.7% and 3.5%, respectively. Concerning patients' outcome, cases with undetectable normal PC (6/28, 21%) had a significantly decreased overall survival (OS) compared to patients with persistent BM normal PC at diagnosis (22/28, 79%) with 3-year OS rates of 0% vs. 59%, respectively (p=.001). In summary, these preliminary data suggests that MFC immunophenotyping investigations may be clinically relevant in patients with suspected amyloidosis for i) differential diagnosis between AL and other forms of amyloidosis and, ii) prognostication of patients with AL according to the presence or absence of baseline persistent normal PC. Disclosures: No relevant conflicts of interest to declare.

Abstract 1916 IgM monoclonal gammopathies are a heterogeneous group of clonal B-cell disorders that include indolent forms - IgM monoclonal gammopathy of undetermined significance (IgM-MGUS) and asymptomatic Waldenström macroglobulinemia (aWM) - as well as symptomatic WM. Similarly to what has been hypothesized in plasma cell disorders, the mechanism leading to the malignant transformation of indolent IgM-MGUS and a-WM to symptomatic WM could underlie evolving cytogenetic abnormalities. However, cytogenetic abnormalities have been mainly explored in patients with WM, while this information in IgM-MGUS and aWM patients is still scanty. In the present study we have analyzed the frequency and prognostic impact of genetic changes as detected by FISH in a series of 161 newly diagnosed patients with an IgM monoclonal gammopathy. Median follow-up in the overall series was of 31 months. Patients were diagnosed with an IgM-MGUS (n=31), a-WM (n=50) and WM (n=80) according to the 2002 WM consensus group criteria. FISH analysis was performed on immunomagnetically enriched bone marrow B-cells to ascertain the following chromosomal abnormalities: 6q and retinoblastoma (Rb) deletions, as well as the IGH translocations t(4;14), t(11;14) and t(14;16). Table 1summarizes the frequency of cytogenetic abnormalities detected in the overall series of patients. Interestingly, the proportion of patients with 6q deletion significantly increased (p=.002) from IgM-MGUS (0%) to a-WM (12%) and WM (28%). The frequency of Rb deletion was generally low, and only detected in a-WM (4%) and WM (2%) patients, while absent in IgM-MGUS cases (0%). In turn, the frequency of IGH translocations was superior in IgM-MGUS (6%) and a-WM (8%) compared to WM (1%) patients. Interestingly, t(11;14) represented 50% of all translocations in IgM-MGUS and a-WM but not in WM. By contrast t(4;14) and t(14;16) were undetectable in the overall series of patients. According to the frequency of 6q deletions and its evolving pattern, we further explored if this specific cytogenetic abnormality had an impact on patients outcome. In patients with symptomatic WM the presence of 6q deletion do not showed an impact on patients progression-free survival (p=.5) and overall survival (p=.8). However, in the group of patients with a-WM those harboring a 6q deletion vs. no 6q deletion showed a significantly increased risk of progression to symptomatic disease: median time to progression - TTP- of 37 vs. 145 months, respectively (p=.002) and 3-year TTP rates of 67% vs. 100%, respectively (p=.002). In summary, these results suggest that the presence of 6q deletion may be a secondary event associated with the malignant transformation of indolent IgM-MGUS to a-WM and symptomatic WM. Accordingly, routine screening of 6q deletion in patients with a-WM may help to identify a subgroup with higher risk of progression. Disclosures: No relevant conflicts of interest to declare.

Abstract 309 Background: Novel insights into the biology of myeloma cells have led to the identification of relevant prognosis factors. CA has become one of the most important prognostic factors, and the presence of t(4;14), t(14;16) or del(17p) are associated with poor prognosis. DNA ploidy is another important prognostic factor with non-hyperdiploid cases being associated with a poor outcome. There are some controversies about whether or not bortezomib-based combinations are able to overcome the poor prognosis of CA. In the VISTA trial, bortezomib plus melphalan and prednisone (VMP) appeared to overcome the poor prognosis of CA in terms of response rate (RR) and survival; however, the number of patients with CA was rather small. Here we report a subanalysis, in a series of elderly newly diagnosed myeloma patients included in the Spanish GEM05MAS65 trial, in order to evaluate the influence of CA by FISH as well as DNA ploidy status on RR and survival. Patients and methods: Patients included in this study were randomized to receive 6 cycles of VMP vs bortezomib, thalidomide and prednisone (VTP) as induction therapy consisting on one 6-week cycle of bortezomib using a bi-weekly schedule (1·3 mg/m2 on days 1, 4, 8, 11, 22, 25, 29 and 32) plus either melphalan 9 mg/ m2 on days 1 to 4 or oral daily thalidomide 100 mg, and prednisone 60 mg/ m2 on days 1 to 4; this first cycle was followed by five 5-week cycles of once-weekly bortezomib (1·3 mg/ m2 on days 1, 8, 15 and 22) plus the same doses of MP and TP. After induction therapy, patients were subsequently randomised to maintenance therapy with VP or VT, consisting of one convencional 3-week cycle of bortezomib (1·3 mg/ m2 on days 1, 4, 8 and 11) every 3 months, plus either prednisone 50 mg every other day or thalidomide 50 mg/day, for up to 3 years. FISH analysis for del(13q), t(11;14), t(4;14), t(14;16) and del(17p) was performed at diagnosis according to standard procedures using purified plasma cells, and DNA ploidy status was analysed following induction therapy by multiparametric FCM using propidium Iodide and specific markers for discrimination between myelomatous and normal cells. Result: In 231 out of the 260 patients included in the trial, FISH analysis at diagnosis were available and two groups were identified: high-risk group (n= 44 patients with t(4;14) and/or t(14;16) and/or del(17p)) and standard-risk group (n=187 patients without CA, and/or del(13q) and/or t(11;14)). There weren't differences in the rates of CA according to the treatment arm. RR was the same in the high-risk vs standard-risk groups, both after induction (21% vs 27% CR) and maintenance (39% vs 45% CR). However, high-risk patients showed shorter PFS as compared to standard risk both from first (24 versus 33 months, p=0·04, HR 0·6, 95% IC 0·4-0·9) and second randomization (17 versus 27 months, p=0·01, HR 0·5, 95% IC 0·2-0·8). This also translated into shorter OS for high risk patients (3-year OS rate: 55% versus 77% from first randomization, p=0·001, HR 0·4, 95% IC 0·2-0·7) and 60% versus 85% from second randomization, p When we analyzed the influence on survival of the DNA ploidy status (224 patients) by comparing patients with hyperdiploid (132 patients) versus non-hyperdiploid DNA content (92 patients) assessed by FCM, it was found that the former group showed longer OS (77% versus 63% at 3 years, p=0·04), and this difference was more evident in patients treated with VTP (77% versus 53% at 3 years, p=0·02), while no significant differences were observed in the VMP arm. Conclusion: The present schema, particularly after month sixth (using maintenance with bortezomib every 3 months) doesn't overcome the negative prognosis of high-risk cytogenetics in terms of PFS and OS in elderly myeloma patients, and this applied to either patients with t(4;14) or del(17p), and it was independent of the induction treatment arm. Our data also show that non-hyperdiploid cases displayed worse outcome than hyperdiploid patients, particularly under VTP induction treatment. These results suggest that continuous efforts are still required in order to overcome the dismal prognosis of high-risk patients. Disclosures: Mateos: Janssen Cilag: Honoraria; Celgene: Honoraria. Off Label Use: VTP is not approved for the treatment of elderly newly diagnosed myeloma patients. VT and VP are not approved for maintenance therapy. Gutierrez:Janssen Cilag: Honoraria; Celgene: Honoraria. Oriol:Janssen Cilag: Honoraria; Celgene: Honoraria. Martínez-López:Janssen Cilag: Honoraria; Celgene: Honoraria. Garcia-Laraña:Janssen Cilag: Honoraria; Celgene: Honoraria. Palomera:Janssen Cilag: Honoraria. Hernández:Janssen Cilag: Honoraria. García-Sanz:Janssen Cilag: Honoraria; Celgene: Honoraria. Cibeira:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lahuerta:Janssen Cilag: Honoraria; Celgene: Honoraria. Blade:Janssen cilag: Honoraria; Celgene: Honoraria. San Miguel:Janssen Cilag: Honoraria; Celgene: Honoraria.

Abstract 1941 Rituximab and bortezomib are active agents in the treatment of WM. Based on preclinical studies which indicated synergism between bortezomib and rituximab, in 2006 we designed a large phase II multicenter trial to evaluate the combination of these agents in previously untreated patients with WM requiring therapy based on consensus recommendations (Kyle et al, Sem Oncol 2003;30:116). This trial was conducted by the European Myeloma Network in 10 centers. In order to prevent the “IgM flare effect” seen with rituximab-based regimens, one course of single agent bortezomib was first administered at a standard dose of 1.3 mg/m2 IV on days 1, 4, 8 and 11. Ten days later, the patients received four courses of 35 days duration each. In courses 2 to 5, bortezomib was administered weekly at a dose of 1.6 mg/m2 on days 1, 8, 15 and 22. During courses 2 and 5, immediately after the administration of bortezomib, patients received dexamethasone 40 mg IV followed by rituximab 375 mg/m2 IV. Patients received a total of 8 infusions of rituximab. Bortezomib was administered weekly in order to reduce the incidence of neurotoxicity which can be significant in WM patients treated with standard schedule bortezomib (Treon et al, Clin Cancer Res 2007;13:3320). A single dose of dexamethasone was given before each dose or rituximab in order to take advantage of potential synergism with rituximab and to reduce allergic reactions but to avoid steroid-induced complications. During treatment, valacyclovir prophylaxis for herpes zoster was prescribed. After completion of treatment, patients with CR, PR, MR or SD according to consensus criteria (Kimby et al, CLM 2006;3:380) were followed without further therapy until there was evidence of progressive disease. Dose modifications for toxicity were allowed and bortezomib could be reduced from 1.6 mg/m2 to 1.3 mg/m2 to 1.0 mg/m2. The trial was initiated in March 2007, 61 patients were scheduled to be enrolled and the study completed accrual in June 2010. Patients characteristics included: age >65 years in 60% of patients, hemoglobin 3 mg/dL in 63%, serum monoclonal protein >7 g/dL in 3.4%, lymphadenopathy in 42%, splenomegaly in 29%, and B-symptoms in 42% of patients. According to IPSS for WM, 18% of patients were rated as low risk, 23% as intermediate risk and 59% as high risk. The main reasons to start treatment included cytopenias in 43% of patients, hyperviscosity in 22%, presence of B-symptoms in 18% and lymphadenopathy in 8%. So far, 54 patients are evaluable for response. On an intent to treat, response rating include CR in 2 (4%), PR in 33 (61%), MR in 8 (15%), SD in 5 (9%) and PD in 6 (11%) patients. In responding patients, at least MR occurred within 2.3 months of treatment and the median time to best response is 4.8 months. Plasmapheresis was not required in any patient before or after treatment with BDR. An “IgM flare phenomenon” was not seen and this was attributed to the initial course of single agent bortezomib. Median follow up for all patients is 12 months, and so far 10 patients have progressed. Eight patients have died, 5 due to causes unrelated to WM or complications of treatment. Toxicities include: neutropenia (grade ≥3) in 13% of patients; thrombocytopenia (grade ≥3) in 5%; peripheral sensory neuropathy, grade 1,2 in 30%, but grade ≥3 in only 5%; neuropathic pain in 17%, but grade ≥3 in only 2%, gastrointestinal toxicity, grade 3 in 7%; and infections in 17% (grade ≥3 in 7%). One patient died of septic shock in absence of neutropenia. Three patients (5%) experienced pulmonary toxicity (grade 3/4) which was attributed to bortezomib and consisted of dyspnea, decrease of O2 saturation and diffuse pulmonary infiltrates on chest CT scan. This toxicity resolved completely after administration of steroids and 2 of 3 patients continued treatment as per protocol. Only one patient (who had discontinued valacylovir prophylaxis) developed herpes zoster. The dose of bortezomib was reduced in 30% of patients primarily because of peripheral neuropathy. This is the largest trial that has evaluated the role of a bortezomib-containing regimen in the frontline setting of symptomatic patients with WM, most of whom were rated as high risk according to IPSS. We conclude that the BDR regimen is active. An update on response, toxicity and time to progression will be performed in November 2010. Disclosures: Dimopoulos: Ortho-Biotech: Honoraria; Millennium: Honoraria. Off Label Use: Bortezomib for Waldenstrom's Macroglobulinemia. García-Sanz:Ortho-Biotech: Honoraria; Millennium: Honoraria; Celgene: Honoraria. Merlini:Millennium: Honoraria; Ortho-Biotech: Honoraria. Sonneveld:Ortho-Biotech: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

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.

MicroRNAs (miRNAs) are small non-coding RNA that regulate gene expression by inducing RNA degradation or translational inhibition of their mRNA targets. Recently, deregulation of miRNAs has been associated with cancer development. MiRNA expression profiling has been analysed in different hematological malignancies, but the information about miRNA expression in MM is limited. We investigated the expression levels of 368 miRNAs using a quantitative PCR-method (“TaqMan low density arrays-microRNA assay”, Applied Biosystems) in 49 patients with newly diagnosed MM. Moreover, 4 MM cell lines (MM1S, OPM2, U266 and RPMI) and 4 samples of normal human plasma cells (PC) were also included in the study. In all the bone marrow samples a CD138 positive PC isolation was performed. Relative quantification of miRNA expression was calculated with the 2−ΔΔCt method. The data were normalized respect to RNU48 and relative to a calibrator sample (average of normal human PC samples). Hierarchical clustering based on the average-linkage method with the centered correlation metric was used for unsupervised analysis. Differentially expressed miRNAs were identified using “Significant Analysis of Microarrays” (SAM) algorithm, the t test and the nonparametric Wilcoxon rank sum test. Primary myeloma cells displayed a distinctive miRNA expression pattern compared to normal PC, characterized by the downregulation of the miRNAs differentially expressed. Unsupervised analysis of the data revealed that the MM samples segregated mainly into two clusters: one contained 12 out the 14 MM cases with t(4;14), 20 out of the 30 MM cases with RB deletion, and the 3 samples with t(14;16) which were tightly clustered. The other cluster contained 5 out of the 7 MM samples without genetic abnormalities (IGH translocation, RB and P53 deletions and gain on 1q were ruled out by FISH analysis). MM samples with t(11;14) and those with gains on 1q were dispersed along the dendrogram. The comparative analysis of the miRNA expression profile between MM with t(4;14) and the remaining MM samples showed a set of 5 up-regulated miRNA (let-7c, miR-125a, miR-125b, miR-135a, miR-99a) in the t(4;14) group. Although MM samples with t(11;14) were not clustered together, the supervised analysis identified two upregulated miRNAs (miRNA-345 and miRNA-193a) compared to those MM cases without this translocation. None of the differentially expressed miRNA was located in the chromosomal regions involved in the specific translocations. Upon comparing the miRNA expression in cases with or withouth RB deletion it was found that the three most differentially expressed miRNA were miR-145, miRNA-378 and let-7b (downregulated in MM with RB deletion). There was no correlation between the expression level of miRNAs located on chromosome 13 and the RB status by FISH. In the same way, the miRNAs differentially expressed in MM with 1q gains were not located in 1q. In summary, these results indicate that miRNA expression is deregulated in myeloma cells, and what is more important, the miRNA pattern of expression in MM seems to be associated with specific genetic abnormalities, which indicate that they may play a relevant role in MM pathogenesis.

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.

Autologous stem cell transplantation (ASCT) has become the standard of care for young MM pts. The analysis of prognostic factors will contribute to identify which pts benefit more from this procedure. However, in this type of analysis it is important that all pts receive uniform treatment, and since primary refractory pts have a different prognosis from pts sensitive to initial chemotherapy, our policy was to use up-front a more intensive treatment approach for the refractory pts. In this report we will focus on the analysis of clinical prognostic factors in sensitive MM pts to induction chemotherapy. The Spanish PETHEMA/GEM2000 trial included untreated MM pts younger than 70 years and consisted on six alternating cycles of chemotherapy VBMCP/VBAD followed by high dose therapy (BuMel or Mel200) supported with ASCT. Pts achieving CR IF- or CR IF+ after the first transplant received maintenance treatment with interferon/prednisone, while pts with a remaining visible M-spike (partial or minimal response) received a second HDT/SCS with CBV (cyclophosphamide, etoposide and BCNU) or a dose-reduced intensity allogeneic transplant if donor is available. Between January 2000 and December 2004, 803 pts were considered to have chemosensitive disease and were transplanted as above described. Median age at time of diagnosis was 59 y (range: 31–70). Baseline characteristics were similar to those previously reported in other series in which young patients were included. Following initial chemotherapy, the response rate was: 13.6% CRIF-, 14% CRIF+ and 60% PR. Response rate increased after HDT/SCS: CRIF-, CRIF+ and PR rates were 38%, 20% and 35%, respectively. Early death rate, occurring during the first two months after ASCT was 3,5% (28 pts). 207 out of 803 pts received a second HDT/SCS. 531 pts (66%) started maintenance therapy after transplant with interpheron and prednisone in most of them (77%). After a median follow-up of 45 months (range: 6–91), the median EFS time for the 803 pts was 45 months (95% CI: 40,5–49,1), defining event as relapse/progression or death; median OS time has not been reached and the estimated 8-year EFS and OS were 30 and 53%, respectively. Univariate analysis identified the following adverse prognostic factors for EFS: age ≥ 59 y, advanced Durie and Salmon stage, advanced ISS stage, Monoclonal component ≥ 30g/l, Hemoglobin (Hb) level < 11 g/dl, creatinine > 1,5 mg/dl, Bone Marrow Plasma cells infiltration ≥ 45%, performance status according to EGOG 3–4, and failure to achieve CR IF- after induction chemotherapy or after the first transplant. The same prognostic factors were identified in the univariate analysis for OS. On multivariate analysis only five variables retained an independent prognostic influence on EFS and OS: age ≥ 59 y (p:0,04), Hb < 11 g/dl p:0,000), advanced ISS stage (p:0,03) and lack of CRIF- after transplant (p:0,000). The three ISS stages could be subdivided into two subgroups using the variables selected in the multivariate analysis. Thus pts who didn’t achieve CR IF- after transplantation and had an additional adverse factor (either age or anemia) showed a significant different outcome within the ISS stage I, II and III.

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.

Introduction: Liver metabolism has two fundamental types of enzymes. Phase two enzymes include GlutathioneS-transferases (GST), with three main variants: GST-M1, GST-T1 and GST-P1. Individual differences in these enzymes could influence their detoxificating capacity. Aim: To study the influence of several genetic GST liver enzymes variants in the development of liver Sinusoidal Obstruction Syndrome (SOS) in patients with multiple myeloma (MM) that received BUMEL as conditioning regimen for Autologous Stem Cell Transplantation (ASCT). Patients: 91 patients with MM - included in Spanish protocol Myeloma 2000 that had received BUMEL as conditioning schedule for ASCT - have been studied; 12 of them had developed SOS. 62 healthy individuals as well as 12 patients with monoclonal gammapathy of uncertain significance (MGUS) were also studied. Methods: Three genotype variants of GST enzymes were studied: presence or absence of GST-M1, GST-T1 and GST-P1 polymorphism Ile105Val by real time PCR in light cycler v2 or ABI PRISM 7900HT thermocyclers. Associations between variables were studied by X2 and exact Fisher test. Logistic regression analysis was performed to calculate Odds ratios and relative risk of SOS development. Results: Significant differences (p>0.0001) were found in the prevalence of homozygous genotype GST-P1 Ile105Val comparing the group of patients with MM and SOS (5 of 12, 41%) and the rest of MM patients without SOS (5 of 79, 6%). This genotype was present in 17% of healthy individuals and MGUS. The genotype absence of GST-M1 was observed in 33% (4/ 12) of MM patients with SOS, 46% (37/79) of MM patients without SOS (p=0.38), 56% of controls and 71% of MGUS. The genotype absence of GST-T1 was observed in 50% (6/ 12) of MM patients with SOS, 27% (22/79) without SOS (p=012), 19% (16/62) of healthy individuals and 21% of MGUS. 33% (4/12) of MM patients with SOS presented the absence of GST-T1 genotype and the presence of homozygous GST-P1 Ile105Val and none of the patients with MM without SOS (p=0,000) presented these genotypes. The only factor associated to development of SOS in regression study was the presence of genotype GST-P1 Ile105Val in homozygosis (OR 10.57 CI 2.45–45.6 p=0.002). Conclusions: The genotype absence of GST-T1 together with the homozygous GST-P1 Ile105Val polymorphism, especially the latter, should be considered as new risk factors in the development of SOS in MM undergoing ASCT conditioning regimen with BUMEL.

Monoclonal Gammopathy of Uncertain Significance (MGUS) is a monoclonal disorder defined by the presence of a serum monoclonal protein

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.

The existence of circulating endothelial progenitor cells (EPC) in adult humans is under intensive investigation due to its potential clinical application. In the present study we have attempted to identify the EPC with colony forming capacity and compare their characteristics with those of the monocytic-macrophage lineage. 42 healthy donors were analysed (7 Bone Marrow, 20 steady-state Peripheral Blood (PB), 4 apheresis products from mobilised PB and 9 buffy-coat products). Median age was 38 years and M/F ratio was 19/23. EPC were obtained by culturing MNC in IMDM with VEGF and beta-FGF. At day 7 the colonies were counted and flow cytometry and immunohistochemistry studies were carried out. Sequential studies were performed on days +21, +28 and +35 of culture with vascular growth factors. Monocytic cells were obtained by adhesion to plastic surface or CD14+ cell selection using magnetic microbeads (Miltenyi, Biotech). Then, monocytic cells were cultured using the same conditions as EPC until day +21 or alternatively with VEGF, beta-FGF and IGF. Von Willebrand gene expression was analysed by RT-PCR and the formation of vascular structures was analyzed by Matrigel assays on both cell sources, EPC and monocytic cells. The mean number of EPC colonies at day 7 was significantly higher in BM (813±695) than in steady-state PB (21.2±2.5), while mobilized PB displayed intermediate values (272±274). By contrast, using the same medium as EPC, monocytic cells did not form colonies at day 7, but cord-like structures could be seen in 7 out 9 cases. However, when the cells were cultured by adding IGF to the medium, a greater number of colonies could be observed. By immunohistochemistry colonies were positive for CD45, CD31 and lysozyme but negative for vWf. Flow cytometry analysis showed that colonies were positive for CD4, CD13, CD14, CD31, CD33, CD45, lysozyme and VE-cadherin, weak positive for CD15 and CD105, and did not express CD16, CD34, CD133 or KDR. This phenotypic profile remained unchanged at all time-points analysed. The immunophenotype of cultured monocytes at day +21 was identical to that of pre-cultured monocytes and both were similar to those obtained in EPC, even for the “specific” markers lysozyme and VE-cadherin. PCR analysis showed small amounts of vWF transcripts in EPC as well as in monocytes but the expression increased after culture with VEGF. Finally, when matrigel assays were carried out, we observed that monocytes formed cord- and tubular-like structures to a higher extent than EPC. Our results in both cell subtypes suggest that the so “called” EPC belongs to the monocytic cell lineage and both populations express some “specific” endothelial antigens such as CD31 or VE-cadherin, as well as monocytic markers such as lysozyme.

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.

The tumoral clone of Waldenström’s macroglobulinemia (WM) shows a wide morphological heterogeneity which ranges from B lymphocytes (BL) to plasma cells (PC), including a lymphoplasmacytic population that defines the disease. The differences between these cell compartments and their cell-counterpart in other lymphoproliferative disorders have not yet been sufficiently explored. We compared the gene expression profiling (GEP) of BL and PC from patients diagnosed with WM, with clonal BL and PC from patients with chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) respectively. Bone marrow samples from 10 WM and 10 MM patients and peripheral blood from 10 CLL patients were used for the analysis. The isolation of the different cell populations was carried out by multiparameter flow cytometry sorting with the following monoclonal antibodies combination: Kappa or Lambda-FITC, CD10-PE, CD38-PerCP-Cy5.5, CD19-PE-Cy7, CD34-APC and CD45-APC-Cy7. Total RNA (100–500 ng) was amplified and labeled using the “GeneChip Two-Cycle cDNA Synthesis Kit” and hybridized to “Human Genome U133A” microarray (Affymetrix). Processing of genechip data was carried out using the Robust Multi-chip Average (RMA) and the Affymetrix Microarray Suite v.5 (MAS5) gene expression algorithms. Two-way hierarchical cluster analysis showed that GEP was able to classify PC from WM and PC from MM in two different groups. In a similar way, BL from WM and CLL were grouped in different clusters. The Significance Analysis of Microarrays (SAM) algorithm identified gene expression changes in a total of 163 genes (103 up and 60 down-regulated) when MW PC were compared with MM PC. Some of these genes were related to regulation of PC development: PAX5 was overexpressed and IRF4 infraexpressed in WM PC. PC from WM displayed high expression levels of MYB and DEK oncogenes while MM PC showed an elevated expression level of v-MAF oncogene. Regarding gene functional categories, “immune response” and “signal transduction” were the biological process more active in WM PC. Comparison between BL from WM and BL from CLL revealed that 31 genes were differentially expressed: IL4 receptor, LEF1 (WNT/b-catenin pathway), fibromodulin (modulator of TGF-b activity) and FGR oncogene (tyrosine kinase protein) showed very low expression levels in WM BL compared to CLL BL. In contrast, the growth factor IL6 was over-expressed in WM BL. These results indicate that both PC and BL from WM are genetically different from the MM and CLL cell-counterpart. The differentially expressed genes have important functions in the B-cell differentiation and oncogenesis. Supported by Spanish Myeloma Network (G03/136) and “Ministerio de Ciencia y Tecnología” (SAF04/06587) and “Junta de Castilla y León” grants (SA032/04)

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.

Fluorescence in situ hybridization (FISH) is an effective alternative for the analysis of cytogenetic abnormalities of low proliferative entities such as Waldenström macroglobulinemia (WM) but, to date, no studies have been carried out to explore the potential impact of molecular cytogenetics on disease evolution. Deletion of the long arm of chromosome 6 (6q-) is the most frequent cytogenetic abnormality in WM. In this study we have analyzed the incidence of this aberration (using both conventional cytogenetics and FISH analysis) in 102 WM patients, and we have correlated it with disease characteristics. Deletion of 6q21 was detected in 6% of patients by conventional cytogenetics, while FISH studies increased this incidence up to 39%. Moreover, in those patients analyzed by cIgM-FISH, the incidence of 6q- was higher (54%). Interestingly, 6q- patients displayed features associated with adverse prognosis (higher levels of ESR, CRP and beta-2 microglobulin), and a greater tendency to display anemia and hypoalbuminemia. Other differentiating variables without reaching statistical significance were a tendency of displaying a higher bone marrow lymphoplasmacytoid infiltration and the amount of monoclonal component in 6q- patients (table). In addition, patients with 6q- displayed shorter survival from time of diagnose (log-rank; p=0.09). In summary, our results show that 6q deletion in WM patients is currently the most frequently found cytogenetic abnormality. Patients with this deletion also shows a trend towards an aggressive disease and poorer outcome in WM. Further studies should focus on possible tumor suppressor genes in the minimal area of deletion at 6q and to determine their role in disease progression. table del6q present del6q absent p-value n 40 (39%) 62 (61%) β2M ≥4mg/dL 48% 16% 0.005 CRP ≥3.5 mg/dL 47% 11% 0.006 Albumin 20 g/L 86% 73% 0.10 BM infiltration >40% 44% 31% 0.20

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.

To assess the value of bone marrow (BM) assessment by flow cytometry FCM after therapy in the clinical outcome of WM patients, we analyzed 42 WM patients who were evaluated before and after therapy. Patients were studied with a panel that always included the CD19, CD22, CD25, and κ/λ light chain immunoglobulin monoclonal antibodies. The mean of abnormal B-cells in the pre-therapeutic BM was 17.8% ± 12.1%, which decreased was after therapy to 5.4% ± 0.7% (P = .049). A linear correlation was seen between the better quality of response and the reduction in the tumor B-lymphocyte counts at the BM, since the ratio of abnormal B cells between pre and posttherapy BM was 1172.17, 221.64, 3.37, 1.03, and 0.56 for responses complete, partial, minor, stable disease and progression, respectively (P < .001). Intensive and rituximab-containing therapies correlated with deeper tumor cell reductions. Finally, the B-cell decrease correlated with the better overall and progression-free survival.

In the present article, two new types of PML/RARA junctions are described. Both were identified in diagnostic samples from two t(15;17)(q22;q21)-positive acute promyelocytic leukemia (APL) patients who failed to achieve complete remission. By using different sets of primers, reverse transcriptase polymerase chain reaction (RT-PCR) of PML/RARA junctions showed atypical larger bands compared with those generated from the three classical PML breakpoints already described. Sequence analysis of the fusion region of the amplified cDNAs allowed us to determine the specificity of these fragments in both patients. This analysis showed two new hybrid transcripts that were 53 and 306 base pairs (bp) longer than that expressed by the NB4 cell line (PML breakpoint within intron 6), and are the result of the direct joining of RARA exon 3 with PML exon 7a (patient 2) or the 5′ portion of PML exon 7b (patient 1), respectively. In patient 1, RT-PCR analysis of the reciprocal RARA/PML junction showed a smaller transcript than that expected in bcr1 cases, while in patient 2 no amplified fragment was obtained. Cytogenetic analysis and/or fluorescence in situ hybridization (FISH) showed that both patients had the t(15;17) translocation. The clinical and hematological profiles expressed by the two patients carrying these unexpected types of PML/RARA rearrangement did not differ significantly from that commonly seen in other APLs with the exception of the poor outcome. Genes Chromosomes Cancer 27:35–43, 2000. © 2000 Wiley-Liss, Inc.

The p16 gene competes with cyclin D for binding to CDK4/CDK6 and therefore inhibits CDK4/6 complex kinase activity, resulting in dephosphorylation of pRb and related G1 growth arrest. Inactivation of this gene has been involved in a variety of tumors by different mechanisms: homozygous/hemyzygous deletions, point mutations and methylation of a 5' CpG island into exon E1alpha of the p16 gene. Homozygous deletions have been rarely found in multiple myeloma (MM) and no point mutations have been reported. Two recent studies have reported a high prevalence of methylation in the exon E1alpha of the p16 gene, but included only a small number of cases. We have analyzed the methylation pattern of exon E1alpha of the p16 gene in 101 untreated MM and five primary plasma cell leukemias (PCL). A PCR assay, relying on the inability of some restriction enzymes to digest methylated sequences, was used to analyze the methylation status. Southern blot analysis was used to confirm these results. Forty-one of 101 MM patients (40.5%) as well as four of the five (80%) primary PCL patients had shown methylation of the exon E1alpha. Our study confirms that hypermethylation of the p16 gene is a frequent event in MM. Leukemia (2000) 14, 183-187.

8512 Background: The GEM-CESAR trial is a potentially curative strategy for high-risk smoldering multiple myeloma (HRsMM) patients (pts) in which the primary endpoint is the achievement of sustained minimal residual disease (MRD) negativity in the bone marrow (BM) by next generation flow (NGF). The value of BM MRD assessment in MM is proven, but alternative, non-invasive methods, accurately reflecting disease burden are needed. Methods: Pts received six 4-week cycles of KRd as induction (K:36mg/m2 twice weekly, R: lenalidomide 25mg po od days 1-21 and d:dexamethasone 40mg po weekly) followed by melphalan 200mg/m2, two further cycles of KRd as consolidation and up to 2 years of Rd (R:10mg/d, d:20mg/week). Efficacy was analyzed in parallel in BM samples by NGF and in serum by SPEP/IFE and QIP-MS/QIP-FLC-MS in 52 out of the 90 pts enrolled in the trial. Standard and MRD responses were carried out as per the IMWG guidelines. For QIP-MS serum immunoglobulins were purified using polyclonal antibodies (anti-IgG, -IgA, -IgM, -total κ and -total λ light chain, -free κ and -free λ light chain). Mass spectra were generated on a MALDI-TOF-MS system. Results: Overall response rate (ORR) was 98% post-induction, 98% post-ASCT, and 100% post-consolidation; 38.4%, 61.5% and 68.6% of pts reached ≥ complete response at each time-point and, among them, 23%, 44% and 55% achieved flow MRD-negativity. Using the combination of QIP-MS/QIP-FLC-MS, the percentage of pts without detectable disease at each timepoint lowered to 12%, 27% and 38% reflecting the higher sensitivity of the method. Against NGF, QIP-MS/QIP-FLC-MS provided negative predictive values of 67%, 92% and 89% (p = 0,0206; p < 0,001; p = 0,003) and identified disease in 95%, 97% and 92% of pts that were positive by NGF-MRD at each respective timepoint. Three pts from this cohort have progressed so far: two were NGF+/MS+ at the three timepoints whilst 1 remained NGF- but QIP-MS/FLC-MS+ throughout. Conclusions: The GEM-CESAR treatment strategy induces a high ORR in HRsMM pts, and the % of cases achieving flow-MRD negativity post-ASCT meets the primary endpoint of the trial. The combined use of QIP-MS and FLC-MS offers higher sensitivity relative to standard methods and may provide relevant information about the right timing for performing a BM aspirate/biopsy. Clinical trial information: NCT02415413 .

In the present study, we analyzed the cell cycle distribution of bone marrow (BM) cells in 120 untreated multiple myeloma patients using a DNA/CD38 double-staining technique at flow cytometry in which plasma cells (PCs) can be clearly discriminated from residual BM cells based on their CD38 expression. This approach allows us to determine the proliferative activity of both PCs and residual normal BM cells. The percentage of S-phase cells in the myelomatous population was found to be significantly lower than that of the residual normal BM cells (P < .001). Regarding the proliferative activity of myelomatous cells, patients with a high number of S-phase PCs (> 3%) showed a significantly (P < .05) increased incidence of anemia and hypercalcemia; higher values of beta 2-microglobulin (beta 2M), urea, and creatinine; and higher numbers of peripheral blood natural killer cells, as well as a poor prognosis as assessed both by response duration and overall survival. With respect to the residual BM normal fraction, a low proliferative activity was significantly (P < .05) associated with the presence of anemia and neutropenia together with increased numbers of BM PCs, a higher incidence of Bence Jones myelomas, and DNA diploidy. Multivariate analysis showed that the number of S-phase PCs was the most important independent prognostic factor, allowing us to discriminate two subgroups of patients with different prognoses, even within the same clinical stage. Moreover, the S-phase PCs, together with beta 2M, age, and performance status, represent the best combination of disease characteristics for stratifying patients according to prognosis and allow the establishment of a simple and powerful staging system for multiple myeloma patients. In addition, this classification can be used for planning treatment in patients who are candidates for transplantation.

8511 Background: Most multiple myeloma (MM) patients will relapse due to persistence of residual tumor cells, or MRD. We compared the prognostic value of traditional response criteria and MRD measurement by a sequencing-based method, LymphoSIGHT, and multiparameter flow cytometry (MFC) in a cohort of 68 uniformly-treated MM patients from the Spanish Myeloma Group trials. Methods: Bone marrow samples were obtained from 68 patients at diagnostic and post-treatment time points on GEM clinical trials (GEM00 and GEM05). All patients were in CR or VGPR at the post-treatment time point. Using sequencing, we identified clonal rearrangements of immunoglobulin (IGH-VDJ, IGH-DJ, and IGK) genes in diagnostic samples. We assessed MRD in follow-up samples, analyzed concordance between sequencing and MFC MRD results, and compared the prognostic value of each method with traditional response criteria. Results: The sequencing assay detected a myeloma-specific gene rearrangement in diagnostic samples from 59 of 68 (87%) patients. We tested MRD in follow-up time points in 56 of the 59 patients. We observed high correlation between MFC and sequencing MRD results (r2=0.86), with MFC underestimating the myeloma burden (Slope=0.4). Of the 56 patients, 45 were positive by sequencing at MRD levels of 10-5 or higher and 11 were MRD negative. There was significantly improved overall survival (OS) in the MRD negative group versus the MRD positive group (median not reached vs. 86 mos, p=0.026). Similar differences were found in progression free survival. When limiting the analysis to the 35 patients in conventional CR, 24 of 35 patients were positive by sequencing at MRD levels at 10-5 and higher and 10 were MRD negative. There was significantly improved OS in the MRD negative group versus the MRD positive group (median not reached vs. 80.92 mos, p=0.041). Conclusions: Our data shows high correlation between MFC and sequencing MRD levels in MM patients. For patients in CR by traditional response criteria, the presence or absence of MRD by sequencing delineated 2 groups of patients with significantly different OS. MRD negativity by sequencing may be a better prognostic indicator than CR by traditional response criteria.

Abstract 295 Genetic events mediating transformation from the pre-malignant monoclonal gammopathies (MG) to myeloma (MM) are unknown. Previous FISH analyses have highlighted that most genetic lesions typical of MM are already present in MGUS. However, the genetic abnormalities characteristic of each evolving stage of MG have not been elucidated. To obtain a comprehensive genomic profile of MG cases from the early to the late stages we performed for the first time high resolution analysis on purified plasma cells from 20 MGUS, 20 Smoldering MM (SMM) and 34 MM by high density 6.0 SNP-array. Ten matched non-tumor DNA samples were also included in the analysis. We examined DNA copy number alterations (CNA), copy number neutral loss of heterozygosity (CNN-LOH) and the spectrum of minimally altered regions which could contain relevant genes. Moreover, visual inspection allowed us to detect intermediate situations which corresponded to imbalances present in minor populations (less than 50%) coexisting with the major diploid population. CNA were identified in 69 (93%) of the 74 patients analyzed with a median of 8 imbalances per abnormal case. The only 5 cases with no CNA corresponded to asymptomatic entities. We observed a progressive increase in the incidence of genomic imbalances from MGUS (median, 5/case) to SMM (media, 7.5/case) to MM (median, 12/case) (P=0.006; MGUS vs MM). In particular, gains on 1q, 3p, 6p, 9p, 11q, 19, 21q together with losses on 1p, 16q and 22q may be important genetic events associated with MGUS-MM transition, as they were less frequent in MGUS than in MM (P The subclones analysis allowed us to illustrate that PC bearing CNA increased from MGUS to SMM and to MM. Thus, we observed at least one minor subclone ( Overall, a total of 65 CNN-LOH were detected and 7 locus had a uniparental trisomy with a median number of genes of 188 per CNN-LOH. The frequency of CNN-LOH was higher in active MM as compared to the asymptomatic entities (MM, 53% vs SMM, 25% vs MGUS 25%; p=0.047). Most of the cases showed more than one region affected by this phenomenom. Of note, there were two identical interstitial CNN-LOH in one MGUS and one MM at 8q11.21-q11.23. We identified 12 homozygous deletions (HZD) corresponding to 5 MGUS (25%), 1 SMM (5%) and 3 MM (9%) patients, some of them containing relevant genes such as PRAME, BIRC2, BIRC3, MMPs 7 and 20. Interestingly, two couples of MGUS patients showed the same HZD at 2p22.3 and 8p11.23-p11.22. The latest contained only the gene ADAM 3A, whose function remains unknown, but its HZD has been recently reported in gliomas (Figure 2).Figure 2HZD at 8p11.23-p11.22 in two MGUS (CHAS software).Figure 2. HZD at 8p11.23-p11.22 in two MGUS (CHAS software). A strong association between genetic lesions and fragile sites (FRA) was also detected as more than one third of the focal-recurrent CNA and HZD, and more than a half of the minimal common regions and interstitial CNN-LOH overlapped with known FRA. In summary, our study shows an evolving cytogenetic profile of increasing complexity from MGUS to SMM and to MM. However, although MM display more CNA and CNN-LOH than early steps, MGUS are as genetically aberrant as MM, and the transition from MGUS to MM is not associated to a particular chromosomal imbalance but rather to an expansion of altered clones already present in MGUS. The analysis of sequential samples from the same individual evolving from MGUS and SMM to active MM is essential to confirm these results. In addition, our study reveals new chromosomal regions involved in CNA, HZD and CNN-LOH. A comprehensive investigation of the genes contained in these regions may provide new insights in MM pathogenesis. Disclosures: No relevant conflicts of interest to declare.

We established the IMMEnSE (International Multiple Myeloma rESEarch) consortium, to increase our understanding of the genetic determinants of multiple myeloma (MM) risk, response to therapy and survival. At present we have DNA samples of over 3200 MM cases and 3000 healthy controls from 10 countries, mainly in Europe. For the majority of the cases clinical data on known prognostic factors, therapy outcome and survival have been also collected. We already performed several association studies in the context of the IMMEnSE consortium. In particular, associations were found between MM risk and SNPs in the ABCB1 gene, which encodes for an efflux pump that has a key role in protecting cells from chemical damage (rs10264990: odds ratio (OR) =0.79, 95% confidence interval (CI) 0.68-0.91; p=0.001, and rs17327442: OR=1.99; 95%CI 1.32-3.02; p=0.001). We also investigated the 8q24 region, which has been shown to harbor multiple loci of susceptibility to various cancers, and found an association between a SNP mapping in this region and MM risk (rs2456449: OR=1.37; 95%CI 1.12-1.68; p=0.0022). In addition, IMMEnSE cases and controls were also genotyped for three MM risk SNPs from the first genome-wide association study (GWAS), and the association of two of them (rs4487645 on chromosome 7p15.3 and rs6746082 on chromosome 2p23.3) was confirmed. Finally, SNPs of key telomere-related genes were genotyped and telomere length was measured in MM cases and controls, and a pleiotropic and functional variant of the TERT gene was found to be associated with reduced MM risk (rs2242652: OR=0.81; 95%CI 0.72-0.92; p=0.001). A suggestive association between longer telomeres and increased MM risk was also found (ptrend=0.01). We will study new SNPs emerging as promising candidates from ongoing GWAS on MM risk and survival, as well as SNPs of key genes involved in the pathogenesis of MM. Finally, we plan to study methylation status of key genes involved in MM etiology, and mitochondrial copy number. The role of all these factors will be investigated in relation to MM risk and prognosis. Collection of samples and data of MM cases and controls is ongoing, as well as of subjects with monoclonal gammopathy of undetermined significance (MGUS). Table 1. MM cases and healthy controls collected in the IMMEnSE consortium Country Cases Median age(5th-95th percentile) Controls Median age(5th-95th percentile) Control type Italy 232 63 (46-78) 237 59 (42-76) General population Poland 1,286 63 (42-82) 200 68 (43-79) Blood donors Spain 322 64 (46-82) 1,131 66 (43-84) Hospitalized subjects France 642 57 (37-68) 191 48 (18-63) Blood donors Portugal 70 68 (45-82) 100 58 (53-79) Blood donors Hungary 148 68 (34-90) 105 74 (55-87) Hospitalized subjects Denmark 348 56 (43-65) 1,000 63 (52-73) Blood donors Israel 109 60 (41-77) 95 - Blood donors Canada 62 58 (42-70) - - - Japan 51 66 (47-84) - - - Total 3,270 63 (37-84) 3,059 63 (18-92) Figure 1 Centers involved in IMMEnSE Figure 1. Centers involved in IMMEnSE Disclosures No relevant conflicts of interest to declare.

Introduction: Thalidomide has recently proven to be a useful drug for treatment of refractory and relapsed multiple myeloma patients, up to 35% of whom achieve remission. However, little is known about the potential additive or synergistic eAect upon its association with other drugs with proven eAcacy in MM. Material and methods: The present pilot study was designed to evaluate the toxicity and response rate of the association of thalidomide, cyclophosphamide and dexamethasone (ThaCyDex) in 22 refractory or relapsed MM patients. The protocol scheduled the administration of thalidomide at escalating doses (200 to 800 mg/day), daily oral cyclophosphamide (CTX) (50 mg/day) and pulsed dexamethasone (40 mg/day, four days every three weeks). Results: Adverse eAects were moderate (grade 42) with only two patients in whom treatment was withdrawn due to neuropathy and severe somnolence. Infections were recorded in six patients, four requiring hospitalization for intravenous antibiotic therapy. No cases of thrombocytopenia grade52 werenoted.OthersideeAectsincludedgrade42 constipation (29%),somnolence (35%) or dizziness (12%). In addition, one case of meralgia paresthetica and one with a deep venous thrombosis were noted. Two cases displayed hyperglycemia and myopathy attributed to dexamethasone, which was solved upon changing to prednisone. With a median follow-up of 12 months, 17 patients were evaluable for response; 13 (77%) responded to the therapy, including nine cases (53%) with a 450% M-component reduction (two of them with a complete remission). Only two responders have already progressed, with a projected event free survival of 51% at 12 months. Seven patients have died due to disease progression (n=5), sudden death (n=1) and infection (n=1). Conclusion: This study shows that ThaCyDex is a feasible and promising therapeutic approach for patients with relapsed/refractory MM. The Hematology Journal (2002) 3, 43‐48. DOI: 10.1038/sj/thj/6200150

Abstract 3660 Poster Board III-596 Introduction Despite the major advances in the treatment of classical Hodgkin Lymphoma (cHL) patients, around 30% to 40% of cases in advanced stages may relapse or die as result of the disease. Current predictive systems, based on clinical and analytical parameters, fail to identify accurately this significant fraction of patients with short failure-free survival (FFS). Transcriptional analysis has identified genes and pathways associated with clinical failure, but the biological relevance and clinical applicability of these data await further development. Robust molecular techniques for the identification of biological processes associated with treatment response are necessary for developing new predictive tools. Patients and Methods We used a multistep approach to design a quantitative RT-PCR-based assay to be applied to routine formalin-fixed, paraffin-embedded samples (FFPEs), integrating genes known to be expressed either by the tumor cells and their reactive microenvironment, and related with clinical response to adriamycin-based chemotherapy. First, analysis of 29 patient samples allowed the identification of gene expression signatures related to treatment response and outcome and the design of an initial RT-PCR assay tested in 52 patient samples. This initial model included 60 genes from pathways related to cHL outcome that had been previously identified using Gene Set Enrichment Analysis (GSEA). Second, we selected the best candidate genes from the initial assay based on amplification efficiency, biological significance and treatment response correlation to set up a novel assay of 30 genes that was applied to a large series of 282 samples that were randomly split and assigned to either estimation (194) or validation series (88). The results of this assay were used to design an algorithm, based on the expression levels of the best predictive genes grouped in pathways, and a molecular risk score was calculated for each tumor sample. Results Adequate RT-PCR profiles were obtained in 264 of 282 (93,6%) cases. Normalized expression levels (DCt) of individual genes vary considerably among samples. The strongest predictor genes were selected and included in a multivariate 10-gene model integrating four gene expression pathway signatures, termed CellCycle, Apoptosis, NF-KB and Monocyte, which are able to predict treatment response with an overall accuracy of 68.5% and 73.4% in the estimation and validation sets, respectively. Patients were stratified by their molecular risk score and predicted probabilities identified two distinct risk groups associated with clinical outcome in the estimation (5-year FFS probabilities 75.6% vs. 45.9%, log rank statistic p≈0.000) and validation sets (5-year FFS probabilities 71.4% vs. 43.5%, log rank statistic p Conclusions We have developed a molecular risk algorithm that includes genes expressed by tumoral cells and their reactive microenvironment. This makes it possible to classify advanced cHL patients with different risk of treatment failure using a method that could be applied to routinely prepared tumor blocks. These results could pave the way for more individualized and risk-adapted treatment strategies of cHL patients, enabling subsets of patients to be identified who might benefit from alternative approaches Disclosures: No relevant conflicts of interest to declare.

Here the current studies in cell DNA content of plasma cells (PC), from multiple myeloma (MM) patients is reviewed, focusing on two complementary aspects the detection of clonal abnormalities and the identification of the proliferative rate of PC. There is accumulating evidence that the measurement of cell DNA content by flow cytometry (FCM) is a useful parameter in the clinical evaluation of MM patients. Between 50 and 70% of MM patients display DNA aneuploidy, the majority of them being hyperdiploid. Comparing hyperdiploid with diploid patients, the former seem to display a better prognosis. Fluorescence in situ hybridization studies have confirmed that there is a high incidence of numerical chromosome abnormalities in MM and that trisomies are significantly more common than monosomies (84% vs 14%). The most frequent gains can be seen in chromosome 9 and 15 while the most common monosomies are those of chromosome 13 and X in females. The possibility of analysing the cell cycle distribution by using a propidium iodide (PI)/CD38 double staining technique may be an alternative to other more laborious methods of assessing the PC labelling index. Thus, patients with3% S-phase PC detected by FCM have an adverse prognosis and this parameter is one of the most important independent prognostic criteria for predicting survival in MM patients. Moreover, the number of S-phase PC, together with other prognostic factors, such as beta 2microglobulin, age and performance status can be a very useful tool for stratifying patients into groups in order to establish risk-directed therapeutic protocols.