Abstract: The genetic heterogeneity of multiple myeloma (MM) makes it unlikely that established or novel chemotherapy could be equally effective in all genetic subgroups. Therefore, genetics alone is insufficient to fully capture different disease outcomes, and there is growing body of evidence showing that detection of minimal residual disease (MRD), using immunophenotypic or molecular-based approaches, also provides powerful independent prognostic information particularly among transplant-eligible patients. However, it is perhaps in elderly MM, the major patient subgroup and in which optimal balance between efficacy and toxicity is critical, that sensitive response assessment could help to tailor patients’ treatment. Here, we used for the first time sensitive 8-color multidimensional flow cytometry (cut-off of 10-5) to monitor MRD among elderly MM patients included in the PETHEMA/GEM2010MAS65 trial (sequential chemotherapy with 9 cycles of bortezomib-melphalan-prednisone (VMP) followed by 9 cycles of lenalidomide-low dose dexamethasone (Rd), or alternating cycles of VMP and Rd up to 18 cycles). A single 8-color antibody combination (CD45-PacB/CD138-OC515/CD38-FITC/CD56-PE/CD27-PerCPCy5.5/CD19-PECy7/CD117-APC/CD81-APCH7) was used to detect phenotypically aberrant clonal plasma cells (PCs), and MRD-negativity was defined when & lt;20 clonal PCs were detected among ≥2.000.000 leukocytes ( & lt;0.001%). MRD assessment was centralized in three PETHEMA/GEM laboratory-cores, cytometrists were blinded to all clinical data, and results were prospectively uploaded into a locked intranet dataset. Median follow-up of the series was 27 months, and time-to-progression (TTP) / overall survival (OS) was measured from the moment of MRD assessment. First, we evaluated the MRD status at cycle 9 of chemotherapy (n=117), and no significant differences were observed for MRD-negative rates between the sequential vs alternating regimens (23% vs 25%; P = .86). However, when we focused on patients in complete response (CR; n=41) and compared the quality of CR achieved in each arm according to patients’ MRD status, we found significantly higher frequencies of MRD-negative rates after the sequential vs alternating schema (75% vs 40%; P = .03). Patients in CR attaining MRD-negativity at cycle 9 showed a significantly prolonged TTP (100% vs 41% at 2-years; P = .001) as well as OS (100% vs 71% at 2-years; P= .03) as compared to patients in CR but with persistent MRD cells. To understand the kinetics of MDR response with sequential vs alternating 18 cycles of chemotherapy, we focused on 72 patients with paired Flow-MRD assessments at cycles 9 and 18. No MRD-negative patients at cycle 9 turned into MRD-positive at cycle 18; however, 21% of MRD-positive patients at cycle 9 became MRD-negative at cycle 18, with no significant differences between rates of transformation after sequential vs alternating regimens (P = .23). At the end of cycle 18, MRD-negative rates among patients randomized to the sequential vs alternating schema were of 48% vs 31% (P = .08), and the quality of CR (according to patients’ MRD status) was slightly but not significantly superior in the sequential vs alternating arm (66% vs 48%; P = .16). Again, patients in CR at cycle 18 attaining MRD-negativity showed superior TTP as compared to those in CR with persistent MRD: TTP at 2-years of 83% vs 56% (P= .06). We also compared the impact of Flow-MRD among cytogenetically defined standard- and high-risk [+1q, t(4;14), t(14;16), and/or del(17p)] patient subgroups (n=125). As expected, standard-risk patients attaining MRD-negativity had significantly prolonged TTP as compared to MRD-positive patients (94% vs 58% at 2-years; P = .035); however, also high-risk cytogenetic patients achieving Flow-CR showed significantly superior TTP (median not reached vs 10 months; P= .001). In summary, we unravel the clinical impact of sensitive Flow-MRD monitoring (10-5) among elderly MM patients in which attaining MRD-negativity, particularly early in therapy, translated into virtually relapse-free intervals at 2-years. In parallel, we also show the value of sensitive MRD kinetics to understand the benefit of additional (sequential or alternating) chemotherapy to further reduce MRD levels, as well as the significance of Flow-MRD among cytogenetically defined standard- and high-risk patents. Disclosures Paiva: Millenium: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Ocio:Array Biopharma: Honoraria, Research Funding. Rosiñol:Janssen: Honoraria; Celgene: Honoraria. Oriol:Celgene Corporation: Consultancy. Gutierrez:Celgene: Honoraria; Janssen: Honoraria. Blade:Janssen: Honoraria; Celgene: Honoraria. Lahuerta:Janssen: Honoraria; Celgene: Honoraria. Mateos:Celgene: Honoraria; Janssen: Honoraria. San Miguel:Janssen: Honoraria; Celgene: Honoraria.

Abstract: Background: MM usually affects elderly patients and although novel agents-based combinations have substantially improved MM outcome, it is possible that this benefit would be particularly relevant for the "youngest-elderly" patients (65-80y). According to the frailty score published by IMWG, the chronological age ≥80 years identifies itself a frail patient population with poor outcome and we have here evaluated the efficacy, safety and outcome of patients included in the GEM2010 trial according to the age to identify the group of patients who benefit most of this total therapy approach. Patients and methods: 242 patients 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. Results: 233 pts were evaluable for safety and efficacy (118 in the sequential and 115 in the alternating arm). One hundred and fifteen patients (49%) were between 65-75 years, 69 patients (30%) were aged between 75-80 years and 49 patients (21%) were older than 80 years. The allocation in both sequential and alternating arms was well balanced. There were not significant differences in the baseline characteristics of the three subgroups of patients. The ORR was similar in both patients aged 65-75 and 75-80 (80% and 83%), but significantly lower in patients older than 80 years (68%) (p=0.007). The CR rate was also almost identical in patients between 65-75 (45%) and 75-80 (49%), but significantly lower in those aged over 80 years (10%) (p 〈 0.0001). The median PFS was 35 m and 32 m in the group of patients aged between 65-75 and 75-80, respectively (p=NS), but PFS was only 25 months in patients older than 80 (p=0.02). In terms of OS, 75% of the patients between 65-75 years remained alive at 4 years, 60% of patients between 75-80 years (p=0.05), as compared to only 30% of patients older than 80 years (p=0.003). There were no significant differences between the sequential and alternating arms. Hematological and non-hematological toxicity was not different in the different groups according to the age, but it is important to note that 63% of patients older than 80 years early discontinued the trial due to toxicity or informed consent withdrawal, while this occurred in only 30% of patients aged 65-75 and 75-80. Sixty-eight percent and 59% of the patients aged 65-75 and 75-80, respectively, completed the 18 planned cycles, as compared to only 36% of patients aged over 80 years. The median cumulative dose of bortezomib and lenalidomide was significantly higher in the group of patients aged 65-75 (44 mg/m2 for bz and 4410 mg for len) and 75-80 (41 mg/m2 for bz and 4078 mg for len) compared to patients over 80 years (33 mg/m2 for bz and 1783 mg for len). These differences were maintained in both sequential and alternating arms. Conclusions: The present therapeutic approach, based on VMP and Rd for newly diagnosed elderly MM patients probably represents the optimal therapeutic option for elderly patients between 65 and 80 years in a sequential or alternating approach. By contrast, further optimization for the patient population over 80 years is still required. Disclosures Mateos: Janssen: 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; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. 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. Oriol:Celgene, Janssen, Amgen: Consultancy, Speakers Bureau. Gironella:Celgene Corporation: Consultancy, Honoraria. Paiva:Celgene: Consultancy; Binding Site: Consultancy; Millenium: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; EngMab AG: Research Funding; BD Bioscience: Consultancy; Onyx: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. San Miguel:Celgene: Honoraria; Janssen-Cilag: Honoraria; Novartis: Honoraria; Sanofi-Aventis: Honoraria; Bristol-Myers Squibb: Honoraria; Millennium: Honoraria; Onyx: Honoraria.

Abstract: Introduction: Although in multiple myeloma (MM) failure to attain deep remissions after therapy typically limits the chances of long-term survival, such paradigm does not apply to all patients since a fraction of patients with persistent disease may be progression-free for more than 10-years even without continuous treatment. Accordingly, it could be hypothesized that prolonged survival in such patients is related to their immune surveillance in controlling detectable (MRD-positive) or undetectable (MRD-negative) residual disease. Unfortunately, while the immune impairment in newly-diagnosed MM as well as the presence of unique immune profiles among patients attaining long-term disease control have been described, no studies have been performed after therapy, during MRD monitoring, to develop an immune signature capable to predict patients' outcome. Methods: We have investigated the immune signature of 146 elderly patients enrolled in the GEM2010MAS65 clinical trial after therapy, during MRD monitoring. Briefly, patients were treated with sequential chemotherapy with 9 cycles of bortezomib-melphalan-prednisone (VMP) followed by 9 cycles of lenalidomide-low dose dexamethasone (Rd) (n=72), or alternating cycles of VMP and Rd up to 18 cycles (n=74). A single 8-color antibody combination (CD45-PacB/CD138-OC515/CD38-FITC/CD56-PE/CD27-PerCPCy5.5/CD19-PECy7/CD117-APC/CD81-APCH7) was used to monitor MRD, and allowed for the enumeration of not only normal and clonal plasma cells, but also erythroid and myeloid hematopoietic progenitors, erythroblasts, mast cells, eosinophils, basophils, monocytes, neutrophils, B-cells and their respective precursor, naïve and memory subsets, as well as T-cells plus TNK- and NK-cells. Median follow-up of the series was 3-years; time-to-progression (TTP) and overall survival (OS) were measured from diagnosis. Results: Principal component analysis (PCA) based on the bone marrow distribution of the 13 immune cell populations revealed the presence of 3 clusters (Panel 1): A (n=16), B (n=117) and C (n=13). When comparing cluster A with clusters B and C, there was a decrease in mean values of erythroblasts (25%, 15% and 13%; P=.03) combined with a trend for increased neutrophils (52%, 59% and 60%; P=.07). The distribution of different maturation subsets within the B-cell compartment was also significantly altered between clusters C and B vs. A, with decreased numbers of B-cell precursors (4%, 0.6% and 1%; P 〈 .001) but increased frequencies of naïve (0.1%, 0.09% and 0.5%; P 〈 .001) and antigen-experienced memory (0.05%, 0.03% and 0.3%; P=.006) B-cells (Panel 2). There were no significant differences in cluster frequency according to treatment schema, nor according to baseline ISS or FISH risk-stratification. Most interestingly though, particularly when compared to cluster C, patients clustering in group A had a trend toward superior TTP (median of 44 vs 35 months, respectively; P=.08) and significantly superior OS (3-year rates of 77 vs 100%, respectively; P=.02); patients belonging to cluster B had intermediate outcome (median TTP of 37 months and 82% 3-year OS rate) (Panels 3 and 4). Noteworthy, there were no significant differences according to patients' MRD status across the different clusters; accordingly, even among MRD-positive patients immune profiling continued to impact patients survival with 3-year OS rates of 62%, 77% and 100% for clusters C, B and A, respectively (P=.02). Conclusions: We showed for the first time that immune profiling in MM after therapy during MRD monitoring is prognostically relevant and allows the identification of patients with either poor survival or sustained disease control. Accordingly, flow-based MRD monitoring offers complementary information to quantification of MRD levels, and may contribute to identify patients that albeit being MRD-positive can still experience prolonged survival due to a unique immune signature particularly characterized by increased peripheral B-cell maturation. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Paiva: BD Bioscience: Consultancy; Binding Site: Consultancy; Onyx: Consultancy; Millenium: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Sanofi: Consultancy; EngMab AG: Research Funding. Puig:Janssen: Consultancy; The Binding Site: Consultancy. Gironella:Celgene Corporation: Consultancy, Honoraria. Mateos:Janssen-Cilag: Consultancy, Honoraria; Takeda: Consultancy; Celgene: Consultancy, Honoraria; Onyx: Consultancy. San Miguel:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Janssen-Cilag: Honoraria; Millennium: Honoraria; Novartis: Honoraria; Sanofi-Aventis: Honoraria; Onyx: Honoraria.

Abstract: Multiparameter flow cytometry (MFC) is commonly used to monitor minimal residual disease (MRD) in MM due to its widespread availability, fast turnaround, and the amount of information obtained upon enumeration of different cell populations and their corresponding antigen expression levels. Thus, MFC could potentially be used not only to monitor MRD, but also to offer additional prognostic information based on MM plasma cell (PC) phenotypes. However, in MM there is lack of consensus about which markers are prognostically relevant because of technical variability between centers and the paucity of studies in large series of patients. Before investigating the prognostic value of those antigens evaluated in MRD studies, we first demonstrated their stability over time by comparing the phenotypic profile of MM-PCs from diagnosis to the MRD stage using principal component analysis (PCA). Accordingly, PCA of merged diagnostic and MRD profiles showed phenotypic overlap between both (MM-PC references in red and blue, respectively; Panel A), that was also demonstrable at the individual patient level [Panel B, in which diagnostic and MRD phenotypic profiles from individual patients (n=14) are represented with the same color]. After demonstrating antigen stability from diagnosis to the MRD stage, we then investigated their prognostic value in a large series of 1265 newly-diagnosed patients enrolled in four consecutive GEM/PETHEMA clinical trials (GEM2000 and GEM2005MENOS65 for transplant-eligible, GEM2005MAS65 and GEM2010 for elderly patients). As compared to cases with bright CD38 expression, patients with aberrantly low CD38 had inferior PFS (medians of 38 vs 30 months; P 〈 .001) and OS (medians of 92 vs 55 months; P 〈 .001). Similar results were observed while comparing the outcome of patients with bright vs. low CD138 expression (median PFS of 34 vs 29 months; P=.003 / median OS of 67 vs 55 months; P=.05). CD81-positive patients had inferior survival than CD81-negative cases (median PFS of 25 vs 42 months; P 〈 .001 / median OS of 55 vs 92 months; P 〈 .001). Interestingly, CD117-negativity conferred inferior PFS and OS in transplant-eligible patients treated without novel agents (ie. GEM2000) but not in the era of novel agents (ie. GEM2005MENOS65). On the other hand, CD45-positive vs. negative expression resulted in inferior PFS (medians of 23 vs 31 months; P=.03) and OS (medians of 43 vs 61 months; P=.03) only among elderly (ie. GEM2005MAS65 and GEM2010) but not in transplant-eligible patients. Importantly, the prognostic value of CD38, CD138 and CD81 continued to be noted in patients with persistent MRD; furthermore, in a multivariate analysis with other prognostic factors such as ISS, FISH cytogenetics, CR and MRD status after therapy, CD38 and CD81 retained independent prognostic value for PFS and OS. Subsequently, we determined the extent of antigenic heterogeneity in MM by enumerating how many different phenotypic profiles could be observed for the combination of markers consensually used in MRD studies: CD38, CD138, CD19, CD27, CD45, CD56, CD81 and CD117. Detailed analysis of 222 newly-diagnosed patients enrolled in the GEM2010MAS65 study and for which all the above markers were simultaneously evaluated, revealed that a total of 94 different phenotypes were identified (Panel D); the most common antigenic profile was CD38+d CD138+ CD19- CD27+ CD45- CD56+ CD81- CD117+ in 13 of the 222 patients (6%). Since aberrant protein expression could result from specific genetic abnormalities, we subsequently investigated for a relationship between both. Interestingly, we found a phenotypic profile determined by combined CD45-CD56+ CD117- expression that was present in 39% of the patients and that in comparison against other profiles was particularly enriched in high-risk cytogenetic abnormalities such as t(4;14) (19% vs 8%; P 〈 .001), and del(17p) (13% vs 7%, P=.01). In summary, we unravel the heterogeneous phenotypic landscape of MM and shed light in the relationship between patients' phenotype and genotype. In addition, CD81, CD38 and CD138 were prognostically relevant in one of the largest series of MM patients ever studied; because their expression is stable over time, these results indicate that MRD monitoring by MFC can offer additional prognostic information based on the phenotypic profile of the chemoresistant PC clone. Figure 1. Figure 1. Disclosures Paiva: Onyx: Consultancy; Celgene: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Binding Site: Consultancy; EngMab AG: Research Funding; BD Bioscience: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. Gironella:Celgene Corporation: Consultancy, Honoraria. Mateos:Takeda: Consultancy; Janssen-Cilag: Consultancy, Honoraria; Onyx: Consultancy; Celgene: Consultancy, Honoraria. San Miguel:Novartis: Honoraria; Onyx: Honoraria; Janssen-Cilag: Honoraria; Millennium: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Sanofi-Aventis: Honoraria.

Abstract: Introduction: Several cooperative MM groups have shown that MRD monitoring may be relevant as biomarker to evaluate the efficacy of different treatment strategies, to support treatment decisions, and to act as surrogate for overall survival (OS) in MM. Because of its wider applicability, a significant fraction of available MRD data has been obtained using MFC that originally, was limited to 4- or 6-colors and measured a limited number of cells. It is assumed that the sensitivity of MFC can increase by usage of ≥8 markers and acquisition of greater cell numbers, but the degree of improved specificity and sensitivity remains unknown. Methods: We aimed at determining the increment in specificity and sensitivity upon transition from first-generation 4-color into a second-generation 8-color MFC assay, by applying new computational tools developed by the EuroFlow consortium in elderly MM patients, enrolled in the GEM2010MAS65 study, for which MRD monitoring was performed with an 8-color monoclonal antibody combination - CD45-PacB/CD138-OC515/CD38-FITC/CD56-PE/CD27-PerCPCy5.5/CD19-PECy7/CD117-APC/CD81-APCH7 - and acquisition of ≥2x106 leukocytes (detection limit: 10-5). Time-to-progression (TTP) and OS were measured from diagnosis. Results: First, we created a reference data file of normal (n=17) and clonal (n=71) plasma cells (PCs) derived from bone marrow samples of healthy individuals and MM patients (Figure 1A) in order to determine the individual contribution of each marker to the discrimination between normal vs. clonal PCs. Principal component analysis (PCA) showed that CD19 ranked as the most significant marker followed by CD56, CD81, CD27, CD117, CD45, forward scatter (FSC), CD38, CD138 and sideward scatter (SSC). Accordingly, the 8-color combination resulted in improved discrimination between normal vs. clonal PCs as compared to the former 4-color approach based only on CD38/CD56/CD19/CD45 (Figure 1B); in fact, CD81, CD27 and CD117 had higher independent value than CD45 in the PCA. Afterwards, we focused on 50 randomly selected MRD-positive patients enrolled in the GEM2010MAS65 study, to compare the performance of an 8- vs. 4-color software-guided classification of MRD cells. PCA based on 8-colors showed that all but two patients were accurately located in the clonal PC reference and outside 1 or 2 standard deviation (SD) curves of the normal PC reference (96% accuracy; Figure 1C); by contrast, using 4-color software-guided classification up to 9 patients became located in the overlapping area between 1 and 2 SD of the normal and clonal PCs references (82% accuracy; Figure 1D). Afterward, we investigated the increment in sensitivity due to the evaluation of 2x106 leukocytes with the second-generation 8-color flow assay instead of the standard 2x105 cells with the first-generation 4-color approach, by determining how many of the 50 MRD-positive patients would turn into MRD-negative if only 2x105 leukocytes had been analyzed (detection limit: 10-4). Interestingly, by reducing the number of visible events to 2x105, our results showed that up to 15 out of the 50 cases (30%) would become wrongly classified as MRD-negative. Then, we investigated the impact in TTP and OS of having MRD levels of 10-5 within a series of 163 patients enrolled in the GEM2010MAS65 and with MRD assessment. Accordingly, 88 cases had detectable MRD levels ≥10-4, 21 patients had persistent MRD at 10-5, and the remaining 54 cases were MRD-negative. Importantly, MRD-positive patients at 10-5 had similar outcome as compared to cases with MRD levels ≥10-4 (both had median TTP of 31 months; 3-year OS rates were 80% and 74%, respectively) and significantly inferior to that of MRD-negative patients [median TTP not reached (P 〈 .001); 3-year OS rate of 93% (P =.05)]. Conclusions: We showed that the transition from a first-generation 4-color into a second-generation 8-color MFC assay that measured ten-times more cells resulted in increased specificity and sensitivity. MRD detection at the 10-5 level is clinically relevant, since it identifies a subset of patients with inferior survival than MRD-negative cases, similar to that of the overall MRD-positive patient population. Figure 1. Figure 1. Disclosures Paiva: Millenium: Consultancy; BD Bioscience: Consultancy; Celgene: Consultancy; Janssen: Consultancy; EngMab AG: Research Funding; Binding Site: Consultancy; Onyx: Consultancy; Sanofi: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. Gironella:Celgene Corporation: Consultancy, Honoraria. van Dongen: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 ; 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: 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.. Mateos:Takeda: Consultancy; Onyx: Consultancy; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees.

Abstract: Introduction: In MM, there is growing body of evidence showing the importance of MRD monitoring particularly among transplant-eligible patients. However, it is perhaps in elderly MM, the major patient subgroup and in which optimal balance between efficacy and toxicity is critical, that sensitive response assessment could help to tailor patients' treatment. Methods: We used an 8-color second-generation flow assay to monitor MRD among elderly MM patients (n=163) included in the PETHEMA/GEM2010MAS65 trial (sequential chemotherapy with 9 cycles of bortezomib-melphalan-prednisone (VMP) followed by 9 cycles of lenalidomide-low dose dexamethasone (Rd), or alternating cycles of VMP and Rd up to 18 cycles). A single 8-color antibody combination (CD45-PacB/CD138-OC515/CD38-FITC/CD56-PE/CD27-PerCPCy5.5/CD19-PECy7/CD117-APC/CD81-APCH7) was used to detect phenotypically aberrant clonal plasma cells (PCs), and MRD-negativity was defined when 〈 20 clonal PCs were detected among ≥2.000.000 leukocytes ( 〈 0.001%; limit of detection: 10-5). MRD assessment was centralized in three PETHEMA/GEM laboratory-cores, cytometrists were blinded to all clinical data, and results were prospectively uploaded into a locked intranet dataset. Median follow-up was 3-years; time-to-progression (TTP) and overall survival (OS) were measured from diagnosis. Results: MRD-negative rates at cycle 9 of chemotherapy (n=128) were similar between the sequential vs alternating regimens (20% vs 24%; P=.37). Patients attaining MRD-negativity at cycle 9 showed a significantly prolonged TTP (median not reached -NR- vs 35 months; P =.001) as well as OS (100% vs 72% at 3-years; P=.02) as compared to patients with persistent MRD. Even among patients in complete response at cycle 9 (n=43), MRD-persistence continued to result in significantly inferior OS (100% vs 74% OS at 3-years; P=.02). To understand the kinetics of MRD response with sequential vs alternating 18 cycles of chemotherapy, we focused on patients with paired MRD assessments at cycles 9 and 18. Up to 19% of MRD-positive patients at cycle 9 became MRD-negative at cycle 18, with no significant differences between rates of transformation after sequential vs alternating regimens (P=.28); by contrast, no MRD-negative patients at cycle 9 turned into MRD-positive at cycle 18. At the end of cycle 18 (n=119), MRD-negative rates were slightly higher among patients randomized to the sequential vs alternating schema: 46% vs 33% (P=.19). In contrast to previous studies in which MRD assessment has been performed at intermediate stages of patients' treatment (eg: before maintenance), the design of the GEM2010MAS65 trial allowed to investigate the immediate impact in patients' outcome according to their depth of response without additional (maintenance) therapy. Thus, whereas the median TTP was not reached for patients in CR plus MRD-negativity, it became significantly shorter for cases in CR and less than CR but remaining MRD-positive (42 vs. 30 months, respectively; P 〈 .001). Afterwards, we also compared the impact of MRD-negativity among cytogenetically defined standard- and high-risk t(4;14), t(14;16), and del(17p)] patient subgroups (total of 132). As expected, standard-risk patients attaining MRD-negativity had significantly prolonged TTP as compared to MRD-positive patients (median 40 vs 31 months; P =.002); interestingly, also high-risk cytogenetic patients reaching MRD-negativity showed significantly superior time-to progression (median NR vs 26 months; P=.007). Lastly, we assessed if the impact of attaining MRD-negativity was equally beneficial according to patients' age. Interestingly, while median TTP was not reached for patients with 65-75 and 75-80 years reaching MRD-negativity, it became of 32, 28 and 22 months for MRD-positive patients with 65-75 vs 75-80 vs 〉 80 years, respectively (P 〈 .001). Only 2 cases older than 80 years old reached MRD-negativity (both relapse-free). Conclusions: Here, we show the clinical impact of depth of response including MRD-negativity in elderly MM patients, which translated into significantly improved survival irrespectively of patients' age and cytogenetic risk. Conversely, patients with standard-risk MM as well as those in CR but remaining MRD-positive experience poor outcomes, and warrant potential treatment individualization to improve their survival. Disclosures Paiva: Celgene: Consultancy; Onyx: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; BD Bioscience: Consultancy; Millenium: Consultancy; Binding Site: Consultancy; EngMab AG: Research Funding. Puig:Janssen: Consultancy; The Binding Site: Consultancy. Gironella:Celgene Corporation: Consultancy, Honoraria. Mateos:Takeda: Consultancy; Celgene: Consultancy, Honoraria; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria. San Miguel:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Millennium: Honoraria; Novartis: Honoraria; Sanofi-Aventis: Honoraria; Onyx: Honoraria; Janssen-Cilag: Honoraria.

Abstract: B-cell lymphopoiesis ends in terminally differentiated bone marrow (BM) plasma cells (PCs). While initially PCs have been viewed mainly as short-lived end-stage B-cells, there is now evidence that this is a heterogeneous cellular compartment in which new-born plasmablasts and long-lived PCs also coexist. The transition between both is characterized by phenotypic modifications, which include higher CD38/CD138 expression alongside HLADR and CD19 down-regulation. CD19 function and expression are regulated by CD81 and accordingly, three normal BM PC subsets can be delineated according to the expression levels of both markers: CD19+/CD81+, CD19-/CD81+, and CD19-/CD81-. However, no additional phenotypic information exists on such normal subsets, nor about how myeloma PC differentiation relates to its normal counterpart. Here, we used 23-color multidimensional flow cytometry (MFC) and principal component analysis (PCA) to phenotypically characterize normal BM PC differentiation in 10 healthy donors and its malignant counterpart in 115 elderly newly-diagnosed multiple myeloma (MM) patients included in the PETHEMA/GEM2010MAS65 study. First, we adopted novel MFC software technology to, after merging and calculating phenotypic data obtained from four 8-color combinations, define the immunophenotypic expression profile (iPEP) of normal BM PCs and characterize their differentiation pathway through PCA. Accordingly, we observed that from the CD19+/CD81+ subset through the CD19-/CD81+ and CD19-/CD81- stages (corresponding to 64%, 32% and 4% of total PCs, respectively) there is a continuous down-regulation on the amount of expression of CD54 (P=.008), CD44 (P=.03) and CD27 (P=.07). By contrast, a trend towards increased levels of CD28 (P=.06), CD38 (P=.05) and CD56 (P=.09) was also observed, suggesting an accumulation of potentially less active and more differentiated PCs from the CD19+/CD81+ to the CD19-/CD81- stages. Using the same approach as described above to determine the iPEP of myeloma PC clones from each individual patient, we then integrated such iPEPs into the normal PC differentiation pathway to investigate, through PCA, the stage and corresponding normal counterpart of each patient myeloma PC clone. From the 115 patients included in this analysis, 3 (3%) had myeloma PCs fitting within the CD19+/CD81+ differentiation stage and 21 (18%) within the CD19-/CD81+ subset, whereas the remaining 91 cases (79%) fitted within potentially more differentiated CD19-/CD81- stages. Virtually no cases with myeloma PC clones corresponding to the CD19+/CD81+ and CD19-/CD81+ differentiation stages had ISS stage I (8%), as compared to 30% in patients with a more differentiated PC CD19-/CD81- signature (P=.03). Furthermore, patients with myeloma PC clones matching the CD19+/CD81+ and CD19-/CD81+ differentiation stages showed a trend for higher incidence of extramedullary plasmacytomas (22% vs 9%; P=.09). Interestingly, almost half of patients with myeloma PC clones in potentially less differentiated stages had no cytogenetic abnormalities [t(IGH), +1q, del(13q), and/or del(17p)] as compared to cases with myeloma PCs matching with the CD19-/CD81- normal PC counterpart (44% vs. 16%, respectively; P=.01). However, whenever present, the type (standard- vs high-risk) of such cytogenetic abnormalities did not differ between both subgroups. Finally, we investigated if the differentiation stage of myeloma PC clones influenced patients’ prognosis, and noted that progression-free survival (PFS) of cases in less and intermediate differentiation stages was significantly inferior PFS as compared to patients with a mature CD19-/CD81- myeloma PC clone (median of 24 months vs not reached, respectively; P=.02). Noteworthy, identical patient prognostication for PFS (P=.02) was observed when the analysis was restricted to cytogenetically-defined standard-risk cases, thus identifying a subgroup of patients with more aggressive MM despite favorable cytogenetic profiles. In summary, we showed that in the vast majority (~80%) of MM patients the PC clone phenotypically matches more differentiated normal PC counterpart subsets. Patients harboring less differentiated clones show a higher incidence of ISS stage II/III and extramedullary disease despite fewer cytogenetic abnormalities, as well as significantly inferior PFS as compared to cases with more differentiated myeloma PC clones. Disclosures Ocio: Array Biopharma: Honoraria, Research Funding.

Abstract: The existence of patients with multiple myeloma (MM) and light-chain (AL) amyloidosis who present with a monoclonal gammopathy of undetermined significance (MGUS)–like phenotype has been hypothesized, but methods to identify this subgroup are not standardized and its clinical significance is not properly validated. PATIENTS AND METHODS An algorithm to identify patients having MGUS-like phenotype was developed on the basis of the percentages of total bone marrow (BM) plasma cells (PC) and of clonal PC within the BM PC compartment, determined at diagnosis using flow cytometry in 548 patients with MGUS and 2,011 patients with active MM. The clinical significance of the algorithm was tested and validated in 488 patients with smoldering MM, 3,870 patients with active MM and 211 patients with AL amyloidosis. RESULTS Patients with smoldering MM with MGUS-like phenotype showed significantly lower rates of disease progression (4.5% and 0% at 2 years in two independent series). There were no statistically significant differences in time to progression between treatment versus observation in these patients. In active newly diagnosed MM, MGUS-like phenotype retained independent prognostic value in multivariate analyses of progression-free survival (PFS; hazard ratio [HR], 0.49; P = .001) and overall survival (OS; HR, 0.56; P = .039), together with International Staging System, lactate dehydrogenase, cytogenetic risk, transplant eligibility, and complete remission status. Transplant-eligible patients with active MM with MGUS-like phenotype showed PFS and OS rates at 5 years of 79% and 96%, respectively. In this subgroup, there were no differences in PFS and OS according to complete remission and measurable residual disease status. Application of the algorithm in two independent series of patients with AL predicted for different survival. CONCLUSION We developed an open-access algorithm for the identification of MGUS-like patients with distinct clinical outcomes. This phenotypic classification could become part of the diagnostic workup of MM and AL amyloidosis.

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: We report for the first time the biological features of MRD cells in MM and unravel that clonal selection is already present at the MRD stage. MRD cells show a singular phenotypic signature that may result from persisting clones with different genetic and gene expression profiles.