Abstract: The long-term impact of intentional weight loss on cardiovascular events remains unknown. We describe 12-month changes in body weight and cardiovascular risk factors in PREvención con DIeta MEDiterránea (PREDIMED)-Plus, a trial designed to evaluate the long-term effectiveness of an intensive weight loss lifestyle intervention on primary cardiovascular prevention. RESEARCH DESIGN AND METHODS Overweight/obese adults with metabolic syndrome aged 55–75 years (n = 626) were randomized to an intensive weight loss lifestyle intervention based on an energy-restricted Mediterranean diet, physical activity promotion, and behavioral support (IG) or a control group (CG). The primary and secondary outcomes were changes in weight and cardiovascular risk markers, respectively. RESULTS Diet and physical activity changes were in the expected direction, with significant improvements in IG versus CG. After 12 months, IG participants lost an average of 3.2 kg vs. 0.7 kg in the CG (P & lt; 0.001), a mean difference of −2.5 kg (95% CI −3.1 to −1.9). Weight loss ≥5% occurred in 33.7% of IG participants compared with 11.9% in the CG (P & lt; 0.001). Compared with the CG, cardiovascular risk factors, including waist circumference, fasting glucose, triglycerides, and HDL cholesterol, significantly improved in IG participants (P & lt; 0.002). Reductions in insulin resistance, HbA1c, and circulating levels of leptin, interleukin-18, and MCP-1 were greater in IG than CG participants (P & lt; 0.05). IG participants with prediabetes/diabetes significantly improved glycemic control and insulin sensitivity, along with triglycerides and HDL cholesterol levels compared with their CG counterparts. CONCLUSIONS PREDIMED-Plus intensive lifestyle intervention for 12 months was effective in decreasing adiposity and improving cardiovascular risk factors in overweight/obese older adults with metabolic syndrome, as well as in individuals with or at risk for diabetes.

Abstract: MRD monitoring is one of the most relevant prognostic factors in elderly MM patients, irrespective of age or cytogenetic risk. Second-generation MFC immune profiling concomitant to MRD monitoring also helped to identify patients with different outcomes.

Abstract: Background: MM and AL are the two most common malignant monoclonal gammopathies. Both diseases result from the accumulation of clonal PCs, but their clinical behavior is significantly different suggesting fundamental differences in disease biology. Previous attempts to identify genetic hallmarks that could explain such differences have been unsuccessful. Furthermore, it is unknown if MM and AL arise from the same or different normal PC counterparts. Aim: To define a transcriptional atlas of the normal PC development in peripheral blood (PB) and bone marrow (BM) for comparison with the transcriptional programs of clonal PCs in MM and AL. Methods: A total of 93 subjects were studied. In 7 healthy adults (HA), PB PCs were phenotypically sorted according to heavy-chain isotypes (IgG, IgA and IgM). In addition, 5 different BM PCs subsets were isolated based on the differential expression of CD19, CD39, CD81 and CD56, due to their ascribed role in dissecting unique BM PC differentiation states. Clonal PCs from patients with MM (n=38) and AL (n=41) were isolated by FACS according to patient-specific aberrant phenotypes. Due to small numbers of PCs sorted from each subset in HA and clonal PCs in AL patients, we used an RNAseq method optimized for limited cell numbers. Differential expression across all pairwise comparisons between groups was analyzed with Deseq2 R package followed by k-means clustering of genes in R. Single-cell RNAseq (scRNAseq, 10xGenomics) was performed in a total of 35,910 PCs from 3 HA, 2 MM and 2 AL. We used Seurat R package to remove batch effect followed by canonical correlation to perform an integrated analysis of all single PCs from HA, MM and AL subjects. Results: Principal component analysis of RNAseq data unveiled two major clusters of normal PCs: those in PB and those in BM (with some transcriptional diversity between CD19+ and CD19- PCs), whereas the CD19+CD39+CD81+CD56- BM subset co-localized with PB and CD39- BM PCs (Panel A). Clonal PCs from MM and AL patients clustered together, and both displayed some transcriptional variance related to the spatial location of normal PCs (i.e. PB or BM). In total, 2174 genes were found significantly deregulated after cross-comparing the 10 PC groups (adj.p-value 〈 0.01, logFC 〉 1) and semi-supervised k-means clustering unveiled 8 transcriptional modules (Panel B). Namely, the transition from PB into BM PCs was characterized by genes related to proliferation (clusters 1 & 2), whereas CD39+ and CD39- BM PC subsets differed on the expression of genes associated with proliferation, homing, and metabolism (1, 2, 4 & 6). Thus, CD19+CD39+CD81+CD56- BM PCs emerged as a novel subset that bridges new-born PB with long-lived (CD39-) BM PCs. Interestingly, clonal PCs from MM and AL shared transcriptional programs related to quiescence (5 & 6) with long-lived BM PCs; however, skewing of polyclonal immunoglobulin gene expression (3) and active gene transcription (8) emerged as hallmarks of the neoplastic transformation from normal, long-lived PCs into clonal PCs. That notwithstanding, the later displayed expression levels of the proliferation and homing transcriptional modules (1 & 4) similar to new-born PB and CD39+ BM PCs. Of note, a small transcriptional cluster of genes related to ribosome biogenesis (7) was significantly more expressed in MM than AL. These findings led us to integrate scRNAseq profiles of normal and clonal BM PCs from MM and AL patients, to define PC clusters based on their transcriptional program rather than their normal vs malignant status (Panel C). This strategy unveiled 11 different PC clusters with unequal distribution between groups. Thus, more than half of clonal PCs in MM and AL were assigned to a cluster that is also predominant in normal PCs (1). By contrast, other clusters with a transcriptional program similar to that of new-born PCs (2 & 5) became rarer in MM and AL. Furthermore, a cluster of PCs with an immature-like phenotype (6) was detectable in MM but almost absent in AL. Conclusions: This is the first integrated analysis of the transcriptional programs of normal PC subsets and clonal PCs in MM and AL, both at the bulk and single-cell levels. Our results unveil shared and exclusive transcriptional states in normal and clonal PCs, together with unique differences between clonal PCs in MM and AL. Thus, we provide here a fundamental resource to understand normal PC development and the cellular origin of both malignant monoclonal gammopathies. Figure Figure. Disclosures Puig: Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Ocio:Pharmamar: Consultancy; AbbVie: Consultancy; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy; BMS: Consultancy; Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding. Oriol:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Martinez Lopez:Bristol Myers Squibb: Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau. Mateos:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lahuerta:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: 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; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel:Sanofi: Consultancy; Takeda: Consultancy; Novartis: Consultancy; MSD: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Brystol-Myers Squibb: Consultancy; Amgen: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees.

Abstract: Introduction: Several studies have shown the role of the immune system in the development of MM, but there is no systematic description of normal B-cell regeneration during treatment points. Recently, EuroFlow consortium has developed NGF panel with high sensitivity to evaluated MRD and potentially, to assess of the normal B-cell compartment of patients with MM. Aims: Here we evaluated the B cell regeneration pattern of bone marrow (BM) from patients with MM by Next Generation Flow (NGF) cytometry at diagnosis and at minimal residual disease (MRD) time points of treatment. Material and Methods: Overall 190 samples of MM patients (45% female and 55% male, median age of 65 years - 37 to 87 years, of which: 16 at diagnosis, 30 post-induction, 76 D+100, 59 maintenance/consolidation and 9 out of treatment, and 8 samples of BM healthy donor - HD (≥ 50 years). At the moment treatment, D+100 were included samples from two research centers: HCS/USAL (n=64) and HUCFF/UFRJ (n=12). Induction regimens were composed of triple protocols (PI + IMIDs + Steroids); followed by high doses of melphalan with ASCT, while patients in maintenance/consolidation followed second line treatment regimens - IP+IMIDs and/or PIs+Steroids and/or monoclonal antibodies or INF or IPs. All samples of BM were subjected to bulk cell lysis with ammonium chloride solution for 〉 107 cell acquisition and labeled with a combination of 10 antibodies - Panel EuroFlow MM MRD.Results: Of the 174 post treatment samples, 36% presented MRD- and 64% MRD+. At diagnosis, patients exhibit a significant reduction of precursors B cells and normal plasma cells (nPC) relative to HD, probably a reflection of the occupation of their binding sites by cPC. At post-induction, there was an increase in precursors B, compared to MM patients at diagnosis, associated with a reduction of mature B-cell (transitional/naïve and memory), regardless of MRD status. Concerning the nPC compartment, a reduction was observed, relative to HD. During treatment, reduction of the tumor burden leaves these sites free for precursors B, which rapidly recover while leads to a drastic decrease in mature B cells and regeneration of the precursors to mature B-cells is slower. On the other hand, in D+100, independent of the MRD status, there was a post treatment medullary recovery, with an increase in B precursors and transitional/naïve B-cells, in contrast, with a reduction of memory B-cells. Out of treatment, we observed a recovery of precursors B, mature B-cells and increase of nPC, but the immune recovery of these nPC is not sufficient to reach the levels of a healthy individual. Conclusion: NGF emerges as an optimal approach for simultaneous assessment of the BM regeneration profile of B-cells and the MRD status. After starting therapy, MM patients re-establish the compartments of B-cell precursors and transitional/naïve B-lymphocytes; however, memory B cells and nPC do not recovery until the end of treatment. This study is a starting point for exploring the importance of the B cells of the medullary microenvironment in the MM. Its potential impact on patient outcome deserving further investigations Disclosures Puig: Takeda: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Mateos:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Abstract: Background Transformation into aggressive lymphoma (AL) is a rare complication of indolent lymphoproliferative disorders (LPDs) and is characterized by poor outcome. Immunoglobulin M (IgM) gammopathies are a spectrum of conditions, from monoclonal gammopathy of undetermined significance (MGUS) to Asymptomatic Waldenstroem Macroglobulinemia (AWM) and Symptomatic WM (SWM) that can eventually evolve to transformed WM (tWM). Actually, tWM represents a clinical challenge, mainly because of its poor characterization. Aims This registry study aims to better characterize tWM, focusing on prognostic factors heralding transformation to AL. Methods Two registries of IgM-MGUS, AWM and SWM [Owen, Semin Oncol 2003] based in Salamanca and in the region of Castilla and Leon (Spain) were investigated to identify cases with histological transformation. IgM-secreting patients with other LPDs (e.g. chronic lymphocytic leukemia, marginal zone lymphoma, IgM-multiple myeloma) were excluded from the analysis. All patients provided written informed consent in accordance to Helsinki's declaration. Statistical analysis was carried out using R v 3.3.3. tool; survival analyses were performed with Log-Rank method, while group comparison was performed with t-student for continuous variables and Chi-square tests for categorical variables. Results Data from 903 patients with IgM-secreting disorders diagnosed between 1976 and 2019 were analyzed; 587 cases with confirmed diagnosis of IgM-MGUS, AWM or SWM were selected. Out of 587 IgM-gammopathies, 22 cases with histological transformation to AL were identified. Cumulative incidence of tWM was: 1.4% at 5, 3.4% at 10 and 5.3% at 12 years, respectively (figure 1). Clinical features at first diagnosis of patients subsequently developing tWM where then analyzed: 3/22 tWM evolved from previous IgM-MGUS, while the remaining patients originally presented with AWM (6/22) or SWM (13/22). IPSS-WM prognostic score was LR for 5, IR for 12 and HR for 3/20 patients, respectively [Morel, Blood 2009]. Glancing on distributions between groups according to the outcome, tWM differed from not transformed (NT) cases for: lower median age at diagnosis (66 vs 72 years, p=0.018), lower platelets levels (median 188 vs 235 x 10^9/mmc, p=0.017), higher LDH ratio (0.8 vs 0.67, p=0.015), higher incidence of chromosome 6q deletion by FISH (40 vs 14%, p=0.021) and higher clonal B lymphocytes infiltration on marrow aspirate by flow cytometry (15 vs 4.5%, p= 0.022). Moreover, 13/22 patients received anti-WM treatment within 3 months from initial diagnosis, mainly chlorambucil-based; 5/22 patients received rituximab in first line and 13 in second line. From the whole series, after a median follow-up of 80 months, median transformation-free survival was 61 months from initial diagnosis (range: 0-228). Among these, Only 1/22 of tWM patient is still alive; 19/21 deaths were thus related to AL/WM, with a median survival after transformation of 12 months (0-53). In the whole series (n=587 ), median OS from initial diagnosis of IgM gammopathy was 76 months for the tWM group (6-225), that is shorter than the NT group (128 months, p=0.012, figure 2). Focusing only on patients treated at initial diagnosis, median survival after first treatment (SAFTI) was 62 vs 90 months for tWM vs NT (p=0.011, figure 3), and median time to next treatment was 28 vs 46 months, respectively (p=0.13). Overall, 10/22 tWM patients received ≥3 treatment lines, and median number of lines prior to transformation was 2 (0-3). Finally, in the whole series IPSS-WM score at diagnosis confirmed to impact on survival (median OS=151, 119 and 56 months for LR, IR and HR groups, respectively, p 〈 0.001). However, this was not the case for tWM cases only, where OS was no longer different between groups. Conclusions In this retrospective study, we confirmed dismal outcome for tWM patients; incidence of transformation was comparable to expectations at 5 years, but higher at subsequent follow-up. At initial diagnosis of IgM gammopathy, younger age, low platelets level, high LDH ratio, high B lymphocytes infiltration by flow cytometry and presence of 6q deletion were significantly enriched among patients subsequently developing tWM. IPSS-WM score looked less predictive among tWM patients probably given to the limited numbers of tWM series. Novel prognostic tools are eagerly awaited for tWM patients. Figure Disclosures Cavallo: Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Puig:The Binding Site: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria. Ferrero:Gilead: Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Speakers Bureau; EUSA Pharma: Membership on an entity's Board of Directors or advisory committees. Boccadoro:Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding; Sanofi: Honoraria, Research Funding.

Abstract: Benign (ie, IgM MGUS and smoldering WM) clonal B cells already harbor the phenotypic and molecular signatures of the malignant WM clone. Multistep transformation from benign (ie, IgM MGUS and smoldering WM) to malignant WM may require specific copy number abnormalities.

Abstract: Introduction: The clinical and prognostic utility of MRD monitoring in MM bone marrow (BM) by first generation (4-6-color) flow cytometry (flow-MRD), has been now demostrated for more than a decade. Thereby, flow-MRD is considered to be a well-suited technique for MRD monitoring in MM, due to its high applicability and specificity, and its broad availability in diagnostic laboratories. However, recent results have confirmed that 1st generation flow-MRD has a lower sensitivity than molecular techniques such as allele-specific oligonucleotyde (ASO)-PCR and next generation sequencing (NGS); in addition, the lack of standardization of conventional flow-MRD approaches, also has a negative impact on its reproducibility. Here we report on the validation of the next generation flow (NGF)-MRD approach developed by the EuroFlow Consortium and the International Myeloma Foundation (IMF) for ultrasensitive and standardized detection of MRD in MM. Methods: A total of 275 BM samples were included in the study: 1) a group of 31 normal/reactive and 63 diagnostic MM BM samples were evaluated to identify the most efficient candidate markers for the NGF panel, using a multivariate analysis-based approach; 2) next, a total of 181 BM samples from 15 healthy donors (HD), 36 MGUS, 15 MM and 3 solitary plasmacytoma (SP) patients studied at diagnosis, plus 112 MM follow-up samples, most of which (n=71) corresponded to BM samples from MM patients in very good partial response, complete remission (CR) or stringent CR were analyzed. These samples were evaluated by the EuroFlow-IMF NGF-MRD method. The method was based on a (standardized) lyse-wash-and-stain sample preparation protocol, the measurement of high numbers of BM cells (≥5x106 cells/tube) and an optimized 8-color, 2-tubes, antibody panel, for accurate identification of BM plasma cells (PCs) and discrimination between phenotypically aberrant (aPC) and normal PC (nPC): tube 1: CD138BV421 CD27BV510 CD38FITC CD56PE CD45PerCP Cy5.5 CD19PE Cy7 CD117APC CD81APC C750, and; tube 2: identical to tube 1 except for cytoplasmic (Cy) Immunoglobulin (Ig) kAPC/CyIglAPC C750). Results obtained with the NGF-MRD MM method in the 71 VGPR, CR and sCR samples, were then compared with a 2nd generation (routine) 8-color flow-MRD approach which involved a single 8-color combination staining for the same markers described above for tube 1, but in the absence of full optimization of the positions for the antibody-fluorochrome conjugates and no selection for treatment-independent antibody CD38 clones. In a subset of 16 of these 71 MRD samples in which enough material was available, comparison with NGS was also performed in parallel. Results: In all MGUS, MM and SP cases analyzed at diagnosis, aPC showed aberrant phenotypes vs. nPC from HD BM, based on multivariate analysis of individual cells from each of the patients against a reference data base of normal/reactive PCs (100% applicability). For the MM MRD BM samples, a median of 9.8x106 (range: 2.4-15.3) events were acquired (tube 1 plus 2) vs. 1x106 (range: 0.03-5) events for the 2nd generation flow-MRD approach with an (estimated) 10 times lower limit of detection and 10 times lower limit of quantitation of 3x10-6 and 5x10-6 vs. 3x10-5 and 5x10-5 for the NGF-MRD vs. the 2nd generation flow-MRD approaches, respectively. This led to a higher rate of MRD+ samples with the NGF-MRD method: 14/48 (29%) cases that were flow-MRD-negative with the 2nd generation 8-color flow-MRD method became MRD+ (median percentage of residual aPC of 0.0007%; range: 0.0002 to 0.02%) (Figure 1A). In contrast, 4/38 (11%) samples were negative by NGF, while positive by 2nd generation flow-MRD; one of these four proved to be MRD-negative by cytoplasmic immunoglobulin light chain restriction analysis. Further comparison of NGF vs NGS showed 9 of the 16 samples evaluable were MRD-negative by NGS; from them, one third (3/9) were positive by NGF with a median number of residual aPCs of 0.005% (range: 0.0002-0.006%) (Figure 1B). Conclusions: The EuroFlow-IMF NGF-MRD approach provides a fast, highly applicable, ultrasensitive, standardized and accurate approach for the assessment of MRD in BM samples from MM patients and overcomes the current limitations of both 1st and 2nd generation flow-MRD approaches; preliminary results showed higher sensitivity than NGS. Figure 1. Figure 1. Disclosures Paiva: Sanofi: Consultancy; Binding Site: Consultancy; EngMab AG: Research Funding; BD Bioscience: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Celgene: Consultancy; Janssen: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. Mateos:Janssen-Cilag: Consultancy, Honoraria; Takeda: Consultancy; Celgene: Consultancy, Honoraria; Onyx: Consultancy. San Miguel:Sanofi-Aventis: Honoraria; Novartis: Honoraria; Millennium: Honoraria; Janssen-Cilag: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Onyx: Honoraria. Durie:Celgene: Consultancy; Onyx: Consultancy; Takeda: Consultancy; Johnson & Johnson: Consultancy. van Dongen:Immunostep: Patents & Royalties: Licensing of IP and Patents on immunobead-based dection of fusion proteins in acute leukemias and other tumors. Royalties for Dept. of Immunology, Erasmus MC and for EuroFlow Consortium ; BD Biosciences (cont'd): Other: Laboratory Services in the field of technical validation of EuroFlow-OneFlow antibody tubes in dried format. The Laboratory Services are provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL; Cytognos: Patents & Royalties: Licensing of IP on Infinicyt software, Patents on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies, Patents on MRD diagnostics, and Patents on PID diagnostics.; Cytognos (continued): Patents & Royalties: Royalty income for EuroFlow Consortium. The Infinicyt software is provided to all EuroFlow members free-of-charge. Licensing of Patent on detection of IgE+ B-cells in allergic diseases. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics. Royalty income for EuroClonality-BIOMED-2 Consortium; BD Biosciences: Other: Educational Services: Educational Lectures and Educational Workshops (+ related travelling costs). The lectures and workshops fully focus on the scientific achievements of the EuroFlow Consortium (No advertisement of products of BD Biosciences)., Patents & Royalties: Licensing of IP and Patent on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies; Royalty income for EuroFlow Consortium.; Roche: Consultancy, Other: Laboratory Services in the field of MRD diagnostics, provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL..

Abstract: BACKGROUND High dose therapy followed by autologous stem cell transplantation (ASCT) remains the standard of care, especially in Europe, for young and eligible multiple myeloma patients (usually younger than 65 years old). Immunoparesis is defined as a reduction (below the lower normal limit) in the levels of 1 or 2 uninvolved immunoglobulins (Ig) and it is related to a reversible suppression of B lymphocytes that correlates inversely with disease stage. B Lymphocyte reconstitution begins at 3 months after ASCT, with maximum B lymphocyte levels at 1 year after ASCT. AIMS The goal of the present study was to investigate the role of the immunoparesis recovery after ASCT as predictor of relapse or progression in multiple myeloma (MM). METHODS We reviewed medical records of MM patients who underwent to ASCT at University Hospital of Salamanca between 1992 and 2013. The primary endpoint was time to relapse or progression from ASCT. Ig (Ig G, Ig A e Ig M) were collected at the time of diagnosis, before ASCT, every 3 months during the first year after ASCT, and every year up to 5 years after ASCT among eligible patients until the relapse or disease progression. RESULTS 106 multiple myeloma patients who underwent ASCT were included in the analysis. Conventional chemotherapy was administered as induction regimen in 69 patients (65%), whereas novel agents were used in 37 patients (35%). Most patients had immunoparesis at diagnosis (91%) and at the moment of ASCT as well (94%). After a median follow-up of 62 months, median time to progression or relapse (TTP) from ASCT was 31 months (95 % CI: 24.1 - 37.1 months). MM patients with immunoparesis 1 year after ASCT had a significantly shorter median TTP as compared with patients without immunoparesis (33.5 months vs 94.2 months; HR: 2.14, 95% CI: 1.13-4.05; p=0.019). In the group of patients with reduction of both Igs, median TTP was slightly inferior than in the group with reduction of only one of them(33.5 vs 36.4 months, p=0.03). Presence of ISS 3, high-risk cytogenetics at diagnosis, less than partial response achieved before and three months after ASCT were also identified as predictors of progression. Multivariate analysis selected immunoparesis 1 year after ASCT as an independent variable for relapse or progression (HR: 5.97, 95% CI: 1.63-21.88; P=0.007). CONCLUSIONS The lack of immunoparesis recovery at 1 year after ASCT in MM patients is associated with significantly higher risk of relapse or progression and this group of patients could potentially benefit of continuous treatment after ASCT to enhance the immune recovery. Disclosures Ocio: Array BioPharma: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy; Mundipharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; MSD: Research Funding; Pharmamar: Consultancy, Research Funding; Janssen: Honoraria. Puig:The Binding Site: Consultancy; Janssen: Consultancy. Mateos:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy; BMS: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Abstract: INTRODUCTION Waldenström Macroglobulinemia (WM) has an indolent clinical course. However, as in other indolent lymphoproliferative disorders, transformation into aggressive histologies can occur in up to 10% of WM patients. Recent progress about WM mutational profile has allowed the identification of recurrent mutations in the MYD88, CXCR4 and ARID1A genes. In turn, mechanisms involved in the transformation to aggressive lymphoma have not been yet described. These findings might be of great interest in the search of new therapeutic strategies for these patients whose prognosis remains extremely poor. Also, the integration of WM transformation genomics with available data regarding transformation in other indolent lymphomas would help to understand the whole process of transformation, and try to define biologic risk criteria, investigate new therapies, and develop preventive strategies in order to improve the outcome of our patients. Here we describe the major mutations found at the time of diagnosis and transformation, those that were exclusively found at transformation, and the evolutionary pattern observed in four transformed WM by using whole-exome sequencing. PATIENTS AND METHODS Patients Four patients diagnosed with WM and transformed into diffuse large B-cell lymphoma (DLBCL) were included. Tumor DNA samples at diagnosis and transformation, and germline DNA were available in three out the four patients. The diagnostic sample of the fourth patient was unavailable due to bad quality. We added an extra WM relapse sample prior to transformation in one patient. In all cases, sample tumor infiltration by flow cytometry (FCM) was available. DNA extraction, quantification and quality control DNA was isolated using DNAzol reagent or Maxwell® 16 System. DNA quality controls prior to enrichment and generation of libraries were performed using fluorometry for DNA quantification, and gel electrophoresis for quality evaluation. Enrichment and generation of libraries was performed using Agilent SureSelect XT2 kit V5. Paired-end sequencingwas carried out using the Illumina HiSeq 2000 platform. The number of reads was set up according to each sample tumor infiltration defined by FCM (depth of coverage range: 150x-250x). Bioinformatics analysis DreamGenics® (Oviedo, Spain) supervised the biological procedure and performed bioinformatics analysis. Algorithms and non-commercial pipelines were used to call variants, analyze and compare them. Since our disposal of at least two samples in the four patients, we performed a pair-wise comparative analysis. Since we knew the selected tumor samples' infiltration, we could estimate the percentage of corresponding tumor cells in each sample. MYD88 mutations (known to be constantly mutated in WM) were used to normalize the frequency of the variants. RESULTS The most frequently mutated genes at diagnosis were constantly present along disease evolution, as it was MYD88, whose role in WM is well known. Mutations in CD79B were present in two of three patients (67%), and three of four at transformation (75%), a frequency much higher than it should be expected in conventional WM (10%). Mutations in this gene affected the first tyrosine ITAM kinase domain of the receptor, as it has been frequently described in DLBCL. At transformation, the most frequently acquired mutations affected TP53, CARD11, PIM1 and KMT2D (MLL2). It should be noted that KMT2D was found mutated in all patients at transformation. A linear evolution was observed in two out of three patients, with cumulative acquisition of new mutations at transformation and maintenance of those detected at diagnosis. However, the patient who had also a relapse sample demonstrated a "branching" model of evolution, sharing variants in all tumor samples (diagnosis, relapse, and transformation), while other variants were only detected at relapse. CONCLUSIONS Our study suggests that some genes could be more frequently mutated in this specific subset of patients (i.e. CD79B), which could be considered as potential biomarkers for predicting the risk of transformation in WM patients. In addition, different models of clonal evolution can be found in transformed WM (linear and branching). Disclosures Mateos: Janssen, Celgene, Amgen, Takeda, BMS: Honoraria.