Abstract: Background: Although great strides were made in the management of MM, our best chances to eradicate this malignancy may lie in preventing its progression.Most current models to predict risk of transformation in SMM are commonly established at diagnosis and not reevaluated over time, because some parameters such as tumor burden or genetic abnormalities require invasive bone marrow (BM) aspirates. It could be hypothesized that periodic monitoring of tumor biomarkers is needed to improve risk-stratification of SMM patients, and so would be new minimally-invasive methods that can replace those performed in BM samples. Such methods should also monitor immune profiles, to identify patients with stable tumor burden/genetics but at risk of progression due to lost immune surveillance. Aim: Determine the level of concordance between the tumor/immune landscape in BM vs peripheral blood (PB) of SMM patients, as well as to evaluate immune profiles together with circulating tumor cell (CTC) numbers and genetic alterations every 6 months in PB, as minimally-invasive methods for identification of SMM patients at risk of developing active MM. Methods: 300 patients are planned to be enrolled in the iMMunocell study that includes 24 sites across 8 European countries. PB samples are collected every 6 months during three years for next-generation flow (NGF) cytometry monitoring of CTCs and immune profiles. Additionally, CTCs and various immune cells are FACSorted to evaluate, every 6 months, their molecular profile in SMM patients with stable vs progressive disease. BM samples are taken at baseline and every 12 months according to patients' choice, in which the same methods described previously for PB are performed. An interim analysis was preplanned to the moment when 150 patients were enrolled. Results: A total of 170 SMM patients were enrolled and we report here data on the first 150. Thus far, 18/150 (12%) patients progressed to MM and according to 20/20/20 criteria, 1 had low, 7 intermediate and 10 had high risk SMM. Only 7/18 cases who progressed had & gt;20% BM plasma cells (PC) by morphology. CTCs were detectable in 107/150 (71%) patients at baseline (median of 0.001% [0% - 0.42%] and 0.03 [0 - 21] CTCs/µL of PB). There was no correlation (or only modestly-significant) between the percentage of CTCs and BMPC by morphology (r=0.156, p=0.065) or flow cytometry (r=0.293, p=0.02). Median CTC counts were 0.02, 0.03 and 0.11 in SMM patients with low, intermediate and high risk disease according to 20/20/20 criteria, respectively (p=0.002). Median CTC numbers were significantly different between cases with stable vs progressive disease (0.02 vs 0.11, p=0.005). As compared to those with ≤1 CTC/µL of PB, patients with & gt;1 CTC/uL showed significantly higher risk of transformation (8% vs 47%, p & lt;0.001) with a median time to progression of 6 months. In addition to the 150 PB samples analyzed at baseline, another 139 specimens were processed at 6, 12 and 18 months. The fluctuation in CTC numbers every 6 months was generally low (median, 0.03 CTCs/uL; IQR, 0.003 - 0.12), though in 10% of patient-samples the absolute variation was & gt;0.5 CTCs/uL. Data on the genetic landscape of CTCs analyzed every 6 months from baseline to disease progression will be shown at the meeting. Immune monitoring in patient-paired PB and BM samples at baseline (n=50) uncovered that 48 of 74 innate and adaptive immune cell types measured by multidimensional flow cytometry had similar distribution. Furthermore, we found significant differences in the distribution of three CD8 T cell subsets defined by differential expression of CD28, CD127, PD1, TIGIT, in PB of SMM patients with stable vs progressive disease. In patients with longitudinal PB samples from baseline until progression to active MM (n=7), there was a significant decrease in helper effector memory CXCR3+CCR4+ and cytotoxic CD127+TIGIT+PD1+ T cells, together with a significant increase in adaptive NK cells and Tγδ CD69+ T cells. Conclusions: This is the first study performing CTC and immune monitoring every 6 months in PB samples from patients with SMM. Our results show a significant correlation between CTC counts and stable vs progressive disease, and suggest that CTC kinetics could be complementary to the 20/20/20 criteria for real-time identification of individual SMM patients at risk of developing active MM. Beyond CTC numbers, this study is uncovering key immune cell types associated with disease progression. Disclosures Terpos: Amgen: Honoraria, Research Funding; Genesis: Honoraria, Other: travel expenses , Research Funding; Janssen: Honoraria, Other: travel expenses , Research Funding; Takeda: Honoraria, Other: travel expenses , Research Funding; Celgene: Honoraria; Medison: Honoraria. Raab:Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Heidelberg Pharma: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Ocio:Sanofi: Consultancy, Honoraria; Secura-Bio: Consultancy; Oncopeptides: Consultancy; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; MDS: Honoraria; GSK: Consultancy; Takeda: Honoraria; Asofarma: Honoraria. Martinez-Lopez:Novartis: Consultancy; Janssen-cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Janssen: Consultancy, Honoraria. de la Rubia:Amgen: Consultancy, Other: Expert Testimony; Celgene: Consultancy, Other: Expert Testimony; Janssen: Consultancy, Other: Expert Testimony; Ablynx/Sanofi: Consultancy, Other: Expert Testimony. Hajek:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Oncopeptides: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Ludwig:Celgene: Speakers Bureau; Janssen: Other: Advisory Boards, Speakers Bureau; Bristol Myers: Other: Advisory Boards, Speakers Bureau; Sanofi: Other: Advisory Boards, Speakers Bureau; Amgen: Other: Advisory Boards, Research Funding, Speakers Bureau; Takeda: Research Funding; Seattle Genetics: Other: Advisory Boards. Goldschmidt:Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Incyte: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Molecular Partners: Research Funding; Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Mundipharma GmbH: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; GlaxoSmithKline (GSK): Honoraria; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Merck Sharp and Dohme (MSD): Research Funding. Roccaro:European Hematology Association: Research Funding; AstraZeneca: Research Funding; Transcan2-ERANET: Research Funding; Italian Association for Cancer Research (AIRC): Research Funding; Janssen: Other; Celgene: Other; Amgen: Other. San-Miguel:Amgen, BMS, Celgene, Janssen, MSD, Novartis, Takeda, Sanofi, Roche, Abbvie, GlaxoSmithKline and Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees. Paiva:SkylineDx: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Roche: Research Funding; Adaptive: Honoraria; Amgen: Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Kite: Consultancy; Sanofi: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract: Disease control at 5 years would be a desirable endpoint for elderly multiple myeloma (MM) patients, but biomarkers predicting this are not defined. Therefore, to gain further insights in this endpoint, a population of 498 newly diagnosed transplant-ineligible patients enrolled in two Spanish trials (GEM2005MAS65 and GEM2010MAS65), has been analyzed. Among the 435 patients included in this post-hoc study, 18.6% remained alive and progression free after 5 years of treatment initiation. In these patients, overall survival (OS) rate at 10 years was 60.8% as compared with 11.8% for those progressing within the first 5 years. Hemoglobin (Hb) ≥ 12 g/dl (OR 2.74, p  = 0.001) and MGUS-like profile (OR 4.18, p  = 0.005) were the two baseline variables associated with long-term disease-free survival. Upon including depth of response (and MRD), Hb ≥ 12 g/dl (OR 2.27) and MGUS-like signature (OR 7.48) retained their predictive value along with MRD negativity (OR 5.18). This study shows that despite the use of novel agents, the probability of disease control at 5 years is still restricted to a small fraction (18.6%) of elderly MM patients. Since this endpoint is associated with higher rates of OS, this study provides important information about diagnostic and post-treatment biomarkers helpful in predicting the likelihood of disease control at 5 years.

Abstract: Background and Aims: Although recent therapeutic advances have led to an improvement in the outcome of Multiple Myeloma (MM), it still remains an incurable disease, and therefore, new drugs with novel mechanisms of action are needed for myeloma patients. Zalypsis is a new synthetic alkaloid derived from certain marine compounds which has demonstrated significant in vitro and in vivo antitumor activity in different malignancies. It is currently under late Phase I development in solid tumours, with preliminary evidence of activity. In this study, we have analysed the preclinical activity and mechanism of action of Zalypsis in MM. Material and methods: Nine different MM cell lines and BM samples from MM patients and normal donors were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and gene expression profile analysis. The in vivo activity was explored in a human subcutaneous plasmocytoma model and immunohistochemistry was performed in selected tumours. Results: Zalypsis turned out to be the most potent antimyeloma agent we have tested so far in our laboratory, with IC50s in picomolar or low nanomolar ranges depending on the cell lines studied. Interestingly, the sensitivity to Zalypsis was independent of the pattern of resistance of the cell lines to conventional antimyeloma agents such as Dexamethasone or Melphalan. It also showed remarkable ex vivo potency in freshly isolated plasma cells from six patients (including two with plasma cell leukemia) and synergized with many other antimyeloma compounds, being the combination of Zalypsis + Lenalidomide + Dexamethasone particularly attractive. Regarding toxicity, Zalypsis preserved the CD34+ hematopoietic progenitor cells from MM and normal donor BM samples. This remarkable activity prompted us to investigate the mechanism of action of the drug. Besides the induction of apoptosis and cell cycle arrest, Zalypsis provoked DNA double strand breaks, which were evidenced by an increase in phospho Histone H2AX and phospho CHK2, followed by a striking overexpression of p53 in MM cell lines bearing wild type forms of this protein. Of note, no other compound currently used in the MM clinic induced such an increase in p53 protein levels. In addition, in a subset of MM cell lines in which p53 was mutated, Zalypsis also provoked DNA double strand breaks and induced cell death, although higher concentrations were required. Changes in the gene expression profile of MM cells treated with Zalypsis were concordant with these results, since important genes involved in DNA damage response were deregulated. This include genes implicated in the ATM repair pathway, such as TLK2, ATR, CHEK2, RAD5 and BRIP1 and other mRNAs related to DNA repair, such as RAD23B, XPC, XRCC1, XRCC5 and GADD45A. These results were confirmed in vivo in a model of human subcutaneous plasmocytoma in SCID mice. Zalypsis (0.8 and 1 mg/Kg) decreased tumour growth and improved survival of mice implanted with MM1S (wild type p53) and OPM-1 (mutated p53) plasmocytomas. Immunohistochemical studies in tumours from treated animals also demonstrated DNA damage with H2AX phosphorylation and p53 overexpression. Conclusion: The potent in vitro and in vivo antimyeloma activity and the singular mechanism of action of Zalypsis uncovers the high sensitivity of tumour plasma cells to double strand breaks, and strongly supports the potential use of this compound in multiple myeloma patients.

Abstract: Introduction: Disease control at five years would be a desirable endpoint for elderly multiple myeloma (MM) patients; however, the percentage of cases reaching this objective as well as the biomarkers to predict it, are not well defined. Objective and design: In order to gain further insight about long-term disease control ( 〉 5 years progression-free) in elderly MM we have analyzed a homogeneous population of 435 newly-diagnosed transplant-ineligible (TNE) patients enrolled in two consecutive Spanish clinical trials (GEM2005MAS65, GEM2010MAS65), that included both proteasome inhibitors and immunomodulatory drugs. Results: Amongst the 435 patients included in this post-hoc study, only 18.8% remained alive and progression-free after five years of initiating treatment. Noteworthy, in these patients the overall survival (OS) rate at 10-years was 69.4%, as compared to 11.4% for those patients progressing during the first five years (p 〈 0.001). Baseline variables significantly associated with long-term progression free survival in the univariate analysis were younger age, ISS 1, R-ISS 1, hemoglobin ≥ 12g/dl, normal LDH, and standard-risk cytogenetic abnormalities and the presence of a monoclonal gammopathy of unknown significance (MGUS)-like immunophenotypic profile in the bone marrow. Complete responses (CR) and minimal residual disease (MRD) negativity were also associated with long-term progression free survival. In the multivariate analysis, an hemoglobin level ≥12g/dl (OR 2.61; 95% CI 1.47 - 4.61, p=0.001) and a MGUS-like immunophenotypic profile in the bone marrow (OR 3.33; 95% CI 1.30 - 8.54, p=0.002) were the two baseline variables significantly and independently associated with a higher probability of long-term disease-free survival. When the depth of response (including MRD) was included in the logistic regression model, Hb level ≥12g/dl (OR 2.18; p=0.010) and the MGUS-like signature (OR 4.99, p 〈 0.001) retained their independent predictive value along with the achievement of MRD-negativity (OR 4.09, p 〈 0.001). Focusing on the 24 patients with an MGUS-like signature (based on the automated immunophenotyping analysis of the relative frequency of BM plasma cells (PCs) plus the percentage of clonal and normal PCs within the whole BM PC compartment), 50% percent of these patients displayed a long-term disease-free survival, as compared to only 17.5% of the remaining MM patients. The median OS for patients with MGUS-like signature was 90.2 months as compared to 62.6 for the MM-like patients. Most MGUS-like patients (90.5%) achieved a favorable response (10 complete response (CR) and 9 very good partial response (VGPR)). No differences in outcome were observed between VGPR and CR cases (p-value for OS 0.87) among MGUS-like patients. Conclusions: This study revealed that despite the usage of former novel agents, the probability of disease control at five years is still restricted to a small fraction (18.8%) of transplant-ineligible patients that achieve remarkable rates of long-term OS. Here, we identify that the combination of three biomarkers (normal Hb, MGUS-Like signature and MRD negativity) can help todefine elderly MM patients achieving long-term disease control. Our results highlight the presence of an MGUS-like signature in the bone marrow at diagnoses as the most powerful predictor for long-term disease free survival, and could be incorporated in clinical practice in order toimprove the prognostic information given to our patients. Disclosures Rodriguez Otero: Clínica Universidad de Navarra: Employment; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Bristol Myers Squibb: Research Funding. Mateos:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; 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; Amgen: 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. Martinez Lopez:Janssen: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau. Ocio:Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy; Pharmamar: Consultancy; Seattle Genetics: Consultancy; BMS: Consultancy; Novartis: Consultancy, Honoraria; Sanofi: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding. Puig:Takeda: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Oriol: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; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Bladé:Celgene: Honoraria; Amgen: Honoraria; Janssen: Honoraria. Lahuerta:Celgene: Honoraria; Amgen: Honoraria; Janssen: Honoraria. San-Miguel:Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria; Sanofi: Honoraria; Roche: Honoraria.

Abstract: 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. Although there are some reports indicating that 1q gains may be considered as a poor-risk feature, the information is not uniform. Furthermore, there are important controversies about whether or not novel agents-based combinations are able to overcome the poor prognosis of CA. Bortezomib-based combinations have shown to improve the outcome of patients with high-risk CA but they do not completely overcome their adverse prognosis. Here we report a preplanned analysis, in a series of elderly newly diagnosed myeloma patients included in the Spanish GEM2010 trial and receiving VMP and Rd, in a sequential or alternating approach, in order to evaluate the influence of CA by FISH on the response rate and outcome. Patients and methods: 242 pts were randomized to receive a sequential scheme consisting on 9 cycles of VMP followed by 9 cycles of Rd or the same regimens in an alternating approach (one cycle of VMP alternating with one Rd, up to 18 cycles. VMP included the iv administration of weekly bortezomib (except in the first cycle that was given twice weekly) at 1.3 mg/m2 in combination with oral melphalan 9 mg/m2 and prednisone 60 mg/m2 once daily on days 1-4. Rd treatment consisted on lenalidomide 25 mg daily on days 1-21 plus dexamethasone 40 mg weekly. FISH analysis for t(4;14), t(14;16), del(17p) and 1q gains was performed at diagnosis according to standard procedures using purified plasma cells. Results: In 174 out of the 233 patients evaluable for efficacy and safety, FISH analysis at diagnosis were available and two groups were identified: high-risk group (n= 32 patients with t(4;14) and/or t(14;16) and/or del(17p)) and standard-risk group (n=142 patients without high-risk CA). There weren't differences in the rates of CA according to the treatment arm. Response Rates (RR) were no different in the high-risk vs standard-risk groups, both in the sequential (74% vs 79% RR and 42% vs 39% CR) and alternating arms (69% vs 86% RR and 39% vs 38% CR). After a median follow-up of 37 months, high-risk patients showed shorter PFS as compared to standard risk in the alternating arm (24 versus 36 months, p=0.01, HR 2.2, 95% IC 1.1-4.2) and this also translated into a significantly shorter 4-years OS (27% vs 72%, p=0.006, HR 3.3, 95% IC 1.4-7.7). However, in the sequential arm, high-risk and standard-risk patients showed similar PFS (32 months vs 30 months) and 4-years OS (64% vs 60%). This effect was observed only in the sequential arm applied to either t(4;14) or del(17p). As far as 1q gains is concerned, 151 patients had 1q information and 76 of them had 1q gains (50.3%), defined as the presence of more than 3 copies in at least 10% of plasma cells. The rate of 1q gains was well balanced in both sequential and alternating arms. The ORR was similar in patients with or without 1q gains (83% vs 80%) as well as the CR rate (45% vs 31%), and no differences were observed between sequential and alternating arms. Patients with or without 1q gains had a similar PFS (33 months vs 30 months) and 4-years OS (58% vs 65%) in the whole series and no differences were observed in the sequential and alternating arms. This effect has been observed in patients with 1q gains as isolated CA and the outcome was slightly but not significantly worse when 1q gains were present plus either t(4;14) and/or del17p. Conclusions: The total therapy approach including VMP and Rd administered in a sequential approach is able to overcome the poor prognosis of the presence of high-risk CA in elderly patients with newly diagnosed MM. The presence of 1q gains has no impact in the PFS and OS of elderly patients treated with VMP and Rd. Disclosures Mateos: Celgene: Consultancy, Honoraria; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria; Takeda: Consultancy. Gironella:Celgene Corporation: Consultancy, Honoraria. Paiva:BD Bioscience: Consultancy; Binding Site: Consultancy; Sanofi: Consultancy; EngMab AG: Research Funding; Onyx: Consultancy; Millenium: Consultancy; Janssen: Consultancy; Celgene: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. San Miguel:Millennium: Honoraria; Janssen-Cilag: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Onyx: Honoraria; Sanofi-Aventis: Honoraria.

Abstract: Abstract 4077 Several murine models resembling multiple myeloma have been developed in the last years: the subcutaneous plasmocytoma model and the disseminated model, based on the iv or intracardiac injection of MM cells, have been widely used to test the efficacy of novel agents. Nevertheless, it is well known the crucial role that the interaction between MM plasma cells and the bone marrow microenvironment plays in MM pathogenesis as well as in the proliferation of MM cells and drug resistance; however, unfortunately, most murine models do not recapitulate these interactions. In order to overcome this caveat, other models such as the SCIDhu or the SCIDrab have been developed. However, in these models, the induced microenvironmental milleu is derived from a fetus and a rabbit respectively, which can be relatively different from that found in an adult patient with MM. We have developed a novel model of a human disseminated MM with the presence of human adult microenvironment. For this purpose, CB17-SCID mice, which lack B and T lymphocytes, were anesthetized and a human bone chip obtained from a healthy adult patient undergoing a hip replacement due to a non-malignant fracture, was subcutaneously implanted in the back of the mice. In the same day 3×106 MM1S-Luc cells were iv injected through the tail vein. Two groups were also included as controls in the study, one with MM1S-Luc cells without the bone chip, and the other with only the human bone chip (without MM cells). Luciferase activity was measured three times per week using a Xenogen-IVIS 50 system after ip injection of 0,15 mg/Kg luciferin. Mice were sacrificed when they developed signs associated with advanced disease, such as body weight loss or back limb paralysis. Although the group of mice receiving MM1S-Luc without bone, did develop a disseminated myeloma, this was much more precocious in the group of mice with a human bone subcutaneous implant. In fact, in mice with human bone and MM1S-Luc cells, the luciferase activity was detectable a median of 9 days after the injection of the cells whereas it was necessary 16 days for obtaining the same effect in mice without the bone chip. This is pointing out that the bone chip in these mice induces a favourable environment for myeloma growth and could be appropriate model to study the interactions of MM cells with human stroma. The increase of tumour growth rate also correlated with a decrease in survival. Median survival (CI 95%) of 64 (47-80) days for mice without bone vs 48 (45-50) days for mice with human bone (Log Rank=0.004). Regarding the sites of infiltration, luminescence images showed a disseminated involvement in all skeletal regions, and this was confirmed by histopathology, as there was a general infiltration of spine, femora, tibia and cranium of these mice. By contrast we did not find infiltration of extra-skeletal regions such as heart, lung or spleen in any mice and only unclear infiltrates were observed in the kidney and liver of some mice. In order to validate the suitability of this model for testing the in vivo antimyeloma activity of novel drugs in the presence of a human microenvironment, 6 mice bearing this novel model were randomized to receive: bortezomib (0.5 mg/Kg ip five days per week) + dexamethasone (0.5 mg/Kg 2 days per week) or vehicle control. A second group of the same number of mice with MM1S-Luc but without the bone chip were randomized in the same way and used as control. Bortezomib + dex significantly decreased tumour growth in both groups of mice without any clear differences in activity between both models, suggesting that this combination overcomes the influence of the microenvironment. In summary we have developed a murine model of MM that may allows us to 1) gain insights into the role of the BM microenvironment interactions in MM pathogenesis. 2) Evaluate the in vivo activity of different anti-MM agents in the presence of human microenvironment. 3) Potentially, this model could also contribute to analyze the effect of different agents in the myeloma bone disease. Disclosures: No relevant conflicts of interest to declare.

Abstract: Abstract 3792 Poster Board III-728 Background p53 mutation is the most frequent single genetic abnormality found in therapy related AML and is also quite frequent in de novo AML with an incidence between 10% and 25%; Moreover patients with p53 mutations characteristically present complex karyotypes and complicated chromosome rearrangements, leading to an adverse prognosis and resistance to conventional chemotherapeutic agents. Consequently, there is a need for novel antileukemic drugs that could work on both p53-wild type and p53-mutated AML patients. In this study we have evaluated the activity of Zalypsis, a novel alkaloid from marine origin, in several AML cell lines and patients samples with different p53 status. Methods The efficacy of Zalypsis was analyzed in four AML cell lines (HL60, HEL, MV4.11 and KG1) and in cells from fresh bone marrow samples from ten newly diagnosed AML patients. The cytotoxicity was analyzed by means of MTT assay and with a multiparametric flow cytometry (MFC) technique that allowed us to study the efficacy of the drug in both mature and immature blast cell compartments. Proteomic and genomic changes induced after treatment with Zalypsis were analyzed in two AML cell lines with different p53 status (HEL and HL60) by Western-Blot and gene expression profile studies. Results Zalypsis showed a very potent antileukemic activity in the four AML cell lines tested, with IC50s at 48 hours below 1 nM. When compared with the in vitro activity of conventional antileukemic agents, such as cytarabine, doxorubicine or fludarabine, Zalypsis turned out to be 10-100 times more potent. It also showed remarkable ex vivo potency in freshly isolated blasts from ten AML patients. In these patients samples, we had the opportunity to separately analyze by multiparametric flow cytometry, the activity of Zalypsis in the different blasts populations, and we constantly observed similar activity in the most mature blast population and in the most immature one (CD34+, CD38- Lineage-), which is thought to include the leukemic stem cells and which is usually more resistant to conventional chemotherapy. Regarding toxicity, Zalypsis preserved the CD34+ non tumoral hematopoietic progenitor cells. The combination of this novel drug with conventional antileukemic agents indicated that Zalypsis is a good partner for combination with all of them, being the combination with cytarabine and daunorubicin particularly attractive for the clinic. Interestingly, as already said, all cell lines were extremely sensitive to Zalypsis, independently of the p53 status, as some of them displayed high basal levels of this protein by Western-Blot and had mutated p53 (HEL and MV 4.11), whereas its protein expression in the remaining cell lines was low, with KG1 bearing a mutation of the gene and HL60 a p53 deletion. Nevertheless, these last cells with low levels of the protein were a bit more sensitive to the drug, pointing out to a role of p53 in Zalypsis induced cell death. This was in agreement with a clear induction of DNA double strand breaks after treatment with Zalypsis, which was evidenced by an increase in phospho Histone H2AX, phospho CHK1 and phospho CHK2 after treatment of both HEL and HL60 with this compound. This was followed by the overexpression of p53 in the AML cell line bearing low basal levels of this protein (HL60). These results were further confirmed in the gene expression profile studies, in which treatment of AML cells with Zalypsis resulted in an important deregulation of genes involved in DNA damage response, including genes implicated in the ATM repair pathway and other mRNAs related to DNA repair, such as TLK2, ATR, ATMIN, CHEK2, RAD5, RAD52, RAD54L, BRCA1, BRCA2 and GADD45B among others. This response ended up in a clear induction of apoptosis as assessed by annexin V studies, caspase and PARP cleavage with partial rescue of cell death with the caspase inhibitor ZVAD-FMK, DNA laddering and loss of mitochondrial membrane potential analyzed by DiOC6 with the subsequent release of cytochrome C and AIF into the cytosol. Conclusion The potent and selective antileukemic effect observed with Zalypsis in AML cell lines and in patient's samples through a DNA damage response, together with its activity in both p53 mutated- and deleted-cells, provides the rationale for the investigation of Zalypsis in clinical trials for patients with AML. Disclosures: Galmarini: Pharmamar: Employment. Avilés:Pharmamar: Employment. Cuevas:Pharmamar: Employment. Pandiella:Pharmamar: Research Funding. San-Miguel:Pharmamar: Research Funding.

Abstract: Background: Since survival in AL mainly depends on the extent of organ involvement of patients at presentation, early diagnosis and risk stratification are key to improve patients' outcome. Therefore, together with surrogates of organ involvement, biomarkers identifying patients with MGUS or MM at greater risk of developing AL would be highly valuable to prevent organ damage, to maximize therapeutic efficacy and to improve outcomes in AL. Aim: To investigate the value of multidimensional flow cytometry (MFC) for simultaneous fast diagnostic screening of plasma cell (PC) clonality and risk stratification, as well as to identify immunophenotypic markers useful for the selection of patients with monoclonal gammopathies candidates for monitoring of pre-symptomatic organ damage related to AL. Methods: We used MFC to characterize a large series of patients with newly-diagnosed (ND) AL (N=94) vs MGUS (N=20) and NDMM (N=52), as well as age-matched healthy adults (HA, N=30). For each patient with AL, automated risk stratification was performed using principal component analysis (PCA) based on the relative frequency of bone marrow (BM) PCs, plus the percentage of clonal and normal PCs within the whole BM PC compartment, vs a database containing information on the same three parameters from a total of 1,774 patients, including 497 MGUS and 1,227 NDMM. In parallel, immunophenotypic protein expression profiles (iPEP) of AL patients were clustered using t-SNE, and the comparison between the iPEP of clonal PCs from patients with AL vs MGUS and MM cases was performed using canonical-correlation analysis (CCA). To identify additional immunophenotypic hallmarks of AL, the BM cellular composition in HA, MGUS, AL and MM patients was compared using 2-dimensional minimum spanning tree (MST) force-directed classification to determine the distance among individual cases. Results: PC clonality was detected by MFC in 93/94 (99%) AL patients, whereas an M-component was detectable in 96% of cases by electrophoresis, immunofixation and sFLC. PCA as defined above, identified AL patients displaying an MM-like (n=6) and an MGUS-like (n=38) signature, as well as 49 cases with an intermediate signature between the MGUS and MM reference datasets. Multivariate analysis of baseline prognostic factors for survival, including patients' age, number of organs involved, Mayo staging, the percentage of BM PCs based on cytomorphology and eligibility for ASCT, showed that having an intermediate- or an MM-like profile had an independent adverse effect on patients' progression-free (PFS) and overall survival (OS) (HR:3.4; P≤.02). t-SNE based on the iPEP of clonal PCs revealed two major clusters of AL patients with significantly different PFS, defined by opposite patterns of expression for CD45, CD56 and CD138 (P≤.02). CCA of tumor iPEP showed partial overlap between AL vs MGUS and MM, with progressively higher percentages of cases with a CD38lo, CD45-ve, CD81-ve and CD138lo iPEP being observed from MGUS to AL and MM. In contrast, AL patients displayed significantly lower reactivity for CD56 (P≤ .03). Further characterization of the BM cellular composition allowed the systematic assessment of 16 cell populations and 18 phenotypic parameters that, by MST, mapped AL in between MGUS and MM. Of note, while AL patients displayed a predominantly-clonal PC compartment in the absence of an MM-like tumor PC expansion, the percentage of B-cell precursors was consistently lower in AL patients than in HA, MGUS and MM (P=.004). Thus, using optimal cut-off values to discriminate between AL vs MGUS and MM, we built a scoring model based on the presence of 〈 100% CD56+ve clonal PCs, 〈 0.1% B-cell precursors, 〉 80% clonal PCs within total BM PCs and 〈 2% BM PCs. Overall, a significant (P 〈 .001) association was found between a progressively higher score and the diagnosis of AL, with a 74% accurate classification based on ROC analysis (AUC of 0.74; 95% CI = 0.66 - 0.82; P 〈 .001) of the performance of the scoring model. Conclusions: We demonstrate the value of MFC for fast diagnostic screening of PC clonality in AL and simultaneous automated patient risk-stratification, based on the BM tumor burden and PC phenotype. In addition, our results also provide new immunophenotypic markers for the identification of patients with monoclonal gammopathies that are candidates for monitoring of pre-symptomatic organ damage related to AL. Disclosures Puig: Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Celgene: Honoraria, Research Funding. Ocio:Array Pharmaceuticals: Research Funding; Sanofi: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Pharmamar: Consultancy; BMS: Consultancy; AbbVie: Consultancy; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy; Mundipharma: Research Funding; Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Oriol:Takeda: 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; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. De La Rubia:Ablynx: Consultancy, Other: Member of Advisory Board. Martinez Lopez:Janssen: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau. Mateos:Amgen: 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; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: 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. Lahuerta:Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria.