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: Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival (PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of ∼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species–mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252.

Abstract: Most multiple myeloma (MM) patients will experience relapse due to the persistence of residual tumor cells, or MRD. We compared the prognostic value of traditional response criteria with MRD measured by three different methods: a sequencing-based method, termed the LymphoSIGHT™ platform, multiparameter flow cytometry (MFC) and ASO-PCR of Ig genes in a cohort of 133 uniformly-treated MM patients from the Spanish Myeloma Group trials. Methods Bone marrow samples were obtained from 133 patients at diagnostic and post-treatment time points on GEM clinical trials (GEM00 and GEM05). All 133 patients were either in CR or VGPR at the post-treatment time point. Using the sequencing assay, we identified clonal rearrangements of immunoglobulin (IGH-VDJ, IGH-DJ, and IGK) genes in diagnostic samples. We then assessed MRD in follow-up samples, analyzing concordance between: sequencing, MFC and ASO-PCR of Ig genes MRD results, and comparing the prognostic value of each method with traditional response criteria. Results The sequencing assay detected the presence of a myeloma-specific gene rearrangement in diagnostic samples from 121 of 133 (91%) patients. We tested MRD in follow-up time points in 109 of the 121 patients. Of the 109 patients, 79 were positive by sequencing at MRD levels of 10-5 or higher and 30 were MRD negative. The Time to Tumor Progression (TTP) and Overall Survival (OS) were significantly longer in the MRD negative group compared with the MRD positive group by sequencing (TTP, median 80 vs. 31 months, p 〈 0.0001; and OS, median not reached vs. 80 months p=0.02) (Figures 1A and 1B). All patients in VGPR, with the exception of 4 cases, were MRD positive by sequencing (92%). Three of the 4 cases with MRD negative and positive immunofixation became immunofixation negative after maintenance treatment. When restricting the analysis to the 61 patients that were in conventional CR (negative immunofixation), 35 of 60 patients were positive by sequencing at MRD levels at 10-5 and higher and 26 were MRD negative. There was a significantly improved TTP in the MRD negative group compared with the MRD positive group (median 131 vs. 35 months, p=0.001) (Figure 1C). A high correlation between MFC and sequencing MRD results was observed (r2=0.87); as well as between ASO-PCR of Ig genes and sequencing (r2=0.89). Discordant results between FCM and sequencing (SEQ) included: 14 cases who were positive by SEQ and negative by FCM, and 5 cases negative by SEQ but positive by FCM; while the remaining cases were concordant: 63 cases were positive by both methods and 16 were negative by both methods. Correlation between ASO-PCR and SEQ was possible in 45 patients: 10 were discordant (9PCR-SEQ+ and 1 PCR+SEQ-) while 35 were concordant (20 PCR+SEQ+, 15 PCR-SEQ-). Conclusions The sequencing applicability for MRD studies in MM is 91%, which is essentially equivalent with that of MFC (96% Rawstron AC et al, JCO 2013). There is a high correlation between MRD levels by sequencing, ASO-PCR and MFC. Based on these results, MRD assessment by sequencing is a useful method for patient risk stratification and can be used to determine molecular CR in MM. Disclosures: Pepin: Sequenta, Inc.: Employment, Equity Ownership. Weng:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Abstract: Undetectable measurable residual disease (MRD) is a surrogate of prolonged survival in multiple myeloma. Thus, treatment individualization based on the probability of a patient achieving undetectable MRD with a singular regimen could represent a new concept toward personalized treatment, with fast assessment of its success. This has never been investigated; therefore, we sought to define a machine learning model to predict undetectable MRD at the onset of multiple myeloma. Experimental Design: This study included 487 newly diagnosed patients with multiple myeloma. The training (n = 152) and internal validation cohorts (n = 149) consisted of 301 transplant-eligible patients with active multiple myeloma enrolled in the GEM2012MENOS65 trial. Two external validation cohorts were defined by 76 high-risk transplant-eligible patients with smoldering multiple myeloma enrolled in the Grupo Español de Mieloma(GEM)-CESAR trial, and 110 transplant-ineligible elderly patients enrolled in the GEM-CLARIDEX trial. Results: The most effective model to predict MRD status resulted from integrating cytogenetic [t(4;14) and/or del(17p13)], tumor burden (bone marrow plasma cell clonality and circulating tumor cells), and immune-related biomarkers. Accurate predictions of MRD outcomes were achieved in 71% of cases in the GEM2012MENOS65 trial (n = 214/301) and 72% in the external validation cohorts (n = 134/186). The model also predicted sustained MRD negativity from consolidation onto 2 years maintenance (GEM2014MAIN). High-confidence prediction of undetectable MRD at diagnosis identified a subgroup of patients with active multiple myeloma with 80% and 93% progression-free and overall survival rates at 5 years. Conclusions: It is possible to accurately predict MRD outcomes using an integrative, weighted model defined by machine learning algorithms. This is a new concept toward individualized treatment in multiple myeloma. See related commentary by Pawlyn and Davies, p. 2482

Abstract: Response criteria for multiple myeloma (MM) require monoclonal protein (M-protein)–negative status on both serum immunofixation electrophoresis (sIFE) and urine (uIFE) immunofixation electrophoresis for classification of complete response (CR). However, uIFE is not always performed for sIFE-negative patients. We analyzed M-protein evaluations from 384 MM patients (excluding those with light-chain-only disease) treated in the GEM2012MENOS65 (NCT01916252) trial to determine the uIFE-positive rate in patients who became sIFE-negative posttreatment and evaluate rates of minimal residual disease (MRD)–negative status and progression-free survival (PFS) among patients achieving CR, CR but without uIFE available (uncertain CR; uCR), or very good partial response (VGPR). Among 107 patients with M-protein exclusively in serum at diagnosis who became sIFE-negative posttreatment and who had uIFE available, the uIFE-positive rate was 0%. Among 161 patients with M-protein in both serum and urine at diagnosis who became sIFE-negative posttreatment, 3 (1.8%) were uIFE positive. Among patients achieving CR vs uCR, there were no significant differences in postconsolidation MRD-negative ( & lt;10−6; 76% vs 75%; P = .9) and 2-year PFS (85% vs 88%; P = .4) rates; rates were significantly lower among patients achieving VGPR. Our results suggest that uIFE is not necessary for defining CR in MM patients other than those with light-chain-only disease.

Abstract: Introduction: Although multiple myeloma (MM) is typically described as an incurable disease, it has been shown in recent years that a small fraction of patients may reach more than 10-years progression-free survival (PFS), which is considered as the minimum threshold to identify patients in "operational cure". However, because of the scarcity of available data there is significant lack of knowledge in MM regarding the frequency of cases attaining operational cure, nor the existence of biomarkers that could prospectively predict such curability. Methods: Herein, we sought to define the frequency as well as the biomarkers predictive of operational cure in a large series of uniformly-treated transplant-eligible patients enrolled in the PETHEMA/GEM2000 protocol (VBMCP/VBAD followed by HDT/ASCT and 2 years of maintenance with interferon and prednisone). Patients' follow-up was updated at the time of abstract submission, and the median follow-up of the series is now of 12-years. We used an automated multiparameter flow cytometric (MFC) classification model focused on the analysis of the bone marrow plasma-cell compartment to identify among newly diagnosed symptomatic MM those with MGUS-like vs. MM-like phenotypic signatures. Minimal residual disease (MRD) was monitored using a first-generation 4-color MFC assay (CD38-FITC / CD56-PE / CD19-PerCPCy5.5 / CD45-APC) with a limit of detection of 10-4. PFS and overall survival (OS) were measured from the time of diagnosis. Results: From a total of 1075 patients enrolled in the GEM2000 protocol, 763 were eligible for this analysis because they either relapsed or died during the first 10-years from diagnosis (n=666; 87%), or remained progression-free and alive for more than 10-yers (n=97; 13%); accordingly, all patients remaining progression-free and alive but for which the follow-up was inferior to 10-years were excluded from the analysis. We then investigated the biomarkers that could help to identify patients reaching operational cure after HDT/ASCT. As compared to the vast majority of cases, patients reaching 〉 10-years PFS (13%) had significantly less frequent anemia (76% vs. 60%, respectively; P=.002), as well as more frequent Durie-Salmon stage IA (14% vs. 6%; P=.004), MGUS-like signature as determined by the automated MFC algorithm (28% vs. 6%; P 〈 .001), complete response (CR) after HDT/ASCT (51% vs. 35%; P=.003), and MRD-negativity by MFC (72% vs. 31%; P 〈 .001). Other biomarkers such as ISS, LDH, ploidy and proliferation were not significantly different among patients reaching 〉 10-years PFS vs. those who relapsed earlier. On multivariate analysis, only the presence of an MGUS-like signature at baseline (P=.04; HR: 3.9) and MRD-negativity at day+100 after HDT/ASCT (P=.006; HR: 6.3) emerged as independent predictive markers for 〉 10-years PFS; anemia, Durie-Salmon and CR status were not retained in the logistic regression model. Accordingly, patients with an MFC-defined baseline MGUS-like signature reaching MRD-negativity after HDT/ASCT (n=14) had a median PFS of 10-years and a 10-year OS rate of 79%, which were significantly superior to those observed among cases with MM-like signatures being MRD-negative (n=54) or positive (n=99) after therapy [median PFS of 6 and 3 years (P 〈 .001); 10-year overall survival rates of 55% and 19% (P 〈 .001)]. Conclusions: We demonstrated that operational cure (i.e.: 〉 10-years PFS) was possible for 13% of transplant-eligible MM patients before the era of novel agents. Curability rates were particularly frequent among patients with a benign phenotypic signature at diagnosis and MRD negativity after HDT/ASCT, suggesting a remarkable clinical benefit of attaining deep remissions after intensive treatment for patients with early MM. Disclosures Paiva: Sanofi: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Onyx: Consultancy; BD Bioscience: Consultancy; EngMab AG: Research Funding; Binding Site: Consultancy; Celgene: Consultancy. Puig:The Binding Site: Consultancy; Janssen: Consultancy. Mateos:Takeda: Consultancy; Celgene: Consultancy, Honoraria; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria. San Miguel:Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Onyx: Honoraria; Sanofi-Aventis: Honoraria; Novartis: Honoraria; Millennium: Honoraria; Janssen-Cilag: 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: 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: 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.