Abstract: Background: The advent of immunotherapy renewed the interest in immune monitoring to identify determinants of treatment response. Flow cytometry is widely adopted in immunotherapy-based clinical trials, but manual analysis of multiparameter files poses a challenge to capture full cellular diversity and to provide unbiased reporting in large datasets. Methods: Here, we developed a semi-automated pipeline named "FlowCT" which, starting from compensated data obtained with standardized protocols, allows simultaneous analyses of multiple files and automated cell clustering. FlowCT starts with quality control and data normalization followed by an analytical stage with clustering algorithms, dimensional reduction techniques and cluster identification based on antigen expression. Statistical tools are included for immediate analysis of results. Results: As proof-of-concept, we used FlowCT in three different datasets. First, we applied FlowCT to bone marrow (BM) samples from three multiple myeloma (MM) patients stained with 17-color flow cytometry, to determine the increment in the complexity of analyzing 8 and 17 markers, chosen to characterize T cells. Of note, a single combination of CD3, CD4, CD8, CD45RA, CD56, CCR7, PD1 and TIGIT, allowed the identification of 31 lymphocyte subsets using FlowCT, which increased to 39 different clusters with 17 markers and unveiled a novel population of CD3- CD56- CD8+ CD16+ lymphoid cells in the MM immune microenvironment. Secondly, we applied FlowCT to matched peripheral blood (PB) and BM samples from 10 patients with smoldering MM, to objectively assess if PB represents a good surrogate of T-cell distribution in the BM. Using an 8-color combination to characterize CD4 T cells, up to 26 different subsets were identified, including several CD4 T helper (Th) type subsets. Of note, their distribution within PB CD4 T cells was similar to that found in BM, except for CD4 T CXCR3+CCR4+ effector memory and Th17 central memory subsets that decreased in the BM tumor immune microenvironment. Thirdly, we analyzed 30 BM samples from 10 MM patients studied every year during maintenance therapy, monitored with CD4, CD8, CD25, CD45RA, CD127, CCR7, PD1, and TCRγδ to characterize T cells. FlowCT identified 29 different T-cell populations, including 9 CD4 subsets, 14 CD8 subsets, 4 Tγδ cell subsets and 2 distinct Treg subsets. Longitudinal, semi-automated and unbiased analysis unveiled a significant fluctuation of CD4 naïve and transitional memory cells during maintenance, as well as a significant decrease of CD8 CD127- effector memory and transitional effectors cells after 2 years of maintenance. Conclusions: Here, we presented FlowCT, a pipeline optimized for the analysis of large flow cytometry datasets that could be easily implemented by research laboratories to unveil full cellular diversity, singular patterns of antigen expression, and to provide unbiased reporting in large studies, like clinical trials. Disclosures Puig: Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; The Binding Site: Honoraria; Takeda: Consultancy, Honoraria. Borrello:WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX; BMS: Consultancy; Celgene: Honoraria, Research Funding, Speakers Bureau. Rosinol Dachs:Janssen, Celgene, Amgen and Takeda: Honoraria. Mateos:Janssen, Celgene, Takeda, Amgen, GSK, Abbvie, EDO, Pharmar: Membership on an entity's Board of Directors or advisory committees; Janssen, Celgene, Takeda, Amgen, Adaptive: Honoraria; Amgen Inc, Janssen Biotech Inc: Other: Data and Monitoring Committee; Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Takeda Oncology.: Speakers Bureau; AbbVie Inc, Amgen Inc, Celgene Corporation, Genentech, GlaxoSmithKline, Janssen Biotech Inc, Mundipharma EDO, PharmaMar, Roche Laboratories Inc, Takeda Oncology: Other: Advisory Committee. Lahuerta:Takeda, Amgen, Celgene and Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bladé:Jansen, Celgene, Takeda, Amgen and Oncopeptides: Honoraria. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Paiva:Celgene, Janssen, Sanofi and Takeda: Consultancy; Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche and Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract: In MM patients relapsing after MRD-negativity, the disease could reemerge from immature cells or from undetectable MRD. However, it remains unknown if immature cells have the same genetic background as MM plasma cells (PCs), as well as the amount of MRD that persists below the limit of detection (LOD) of next-generation techniques. To obtain further insight, we compared the biological landscape of MM PCs at diagnosis to that of CD34 progenitors, B cells and normal PCs isolated from patients with negative MRD by next-generation flow (NGF) after treatment. We performed whole-exome sequencing (WES, mean depth: 90x) with the 10XGenomics Exome Solution for low DNA-input as well as deep NGS of B-cell receptor immunoglobulin (BcR IG) gene rearrangements (mean, 69,975 sequences), in a total of 68 cell-samples isolated from the bone marrow (BM) of 7 MM patients with MRD-negativity by EuroFlow NGF after induction with VRD and auto-transplant (GEM2012MENOS65 trial). Patients with negative MRD were intentionally selected to avoid contamination with MM PCs during sorting of CD34 progenitors, B-cell precursors, mature B cells and normal PCs after induction and transplant. We investigated in these populations the presence of somatic mutations and clonotypic BcR Ig rearrangements detectable in MM PCs sorted at diagnosis, using peripheral blood T cells as germline control. We also performed WES in matched diagnostic MM PCs and MRD cells persisting after VRD induction in 14 cases as control. In another 6 patients with untreated MM, we performed single-cell RNA and BcR IG sequencing (scRNA/BcRIGseq) of total BM B cells and PCs (n=16,380) to investigate before treatment, if the clonotypic BcR IG sequence of MM PCs was detectable in other B cell stages defined by their molecular phenotype. We used multidimensional flow cytometry (MFC) to investigate the frequency of B cell clonality in BM samples from a larger series of 195 newly-diagnosed MM patients, prospectively enrolled in the GEM-CLARIDEX trial. Somatic mutations present in diagnostic MM PCs were detectable in the lymphopoiesis of 5/7 patients achieving MRD-negativity after treatment. In one case, out of 55 mutations present in diagnostic MM PCs, a single mutation in PCSK1N (VAF: 0.30) was detectable in normal PCs. In the other four patients, a total of 85 mutations were present in MM PCs and up to 10 (median VAF, 0.16) were found all the way from CD34 progenitors into B-cell precursors, mature B cells and normal PCs, but not in T cells. Of note, most mutations were reproducibly detected in each cell type after induction and after transplant. All somatic mutations shared by MM PCs and normal cells were non-recurrent, and genes recurrently mutated in MM (eg. ACTG1, ATM, DIS3, FAM46C, KRAS, LTB, MAX, TRAF3) were found in MM PCs but never in normal cells. Copy number alterations (CNA) were found only in MM PCs. By contrast, up to 513/827 (62%) mutations and 48/67 (72%) CNA were detectable in matched diagnostic MM PCs and persistent MRD cells, indicating that the few somatic variants present in normal cells were unlikely related to contaminating MRD below NGF's LOD. Accordingly, MM clonotypic BcR IG rearrangements were detectable in normal PCs (4/7patients) and in immature B cells (5/7 patients) but at much lower frequencies (mean of 0.02% in both). Of note, 9 additional clonotypes (mean 8.4%) were found in MM PCs of 5/7 patients (range, 1-3). scRNR/BcRIGseq unveiled that clonotypic cells were confined mostly but not entirely within PC clusters, and that in 1 patient another clonotype was detectable in mature B cells. Accordingly, using MFC we found in a larger series that 25/195 (13%) of newly-diagnosed MM patients display B-cell clonality (median of 0.7% BM clonal B cells, range 0.02%-6.3%). In conclusion, we show for the first time that MM patients bear somatic mutations in CD34 progenitors that specifically differentiate into the B cell lineage, likely before the disease onset. Because diagnostic, MRD (and relapse) MM PCs display great genetic similarity, these results suggest that undetectable MRD & lt;10-6 rather than normal cells with a few non-recurrent mutations are responsible for relapses after MRD-negativity. This study also challenges our understanding of myelomagenesis and clonal heterogeneity, and proposes that mutated lymphopoiesis may increase risk of developing B cell and PC oligoclonality, which precedes secondary driver mutations or CNA leading to the expansion of MM PCs. Disclosures Puig: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda, Amgen: Consultancy, Honoraria; The Binding Site: Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Martinez-Lopez:BMS: Honoraria, Other: Advisory boards; Janssen: Honoraria, Other: Advisory boards and Non-Financial Support ; Amgen: Honoraria, Other: Non-Financial Support ; Celgene: Honoraria, Other: Advisory boards and Non-Financial Support ; Incyte: Honoraria, Other: Advisory boards; Novartis: Honoraria, Other: Advisory boards; VIVIA Biotech: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria. Lahuerta:Takeda, Amgen, Celgene and Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosinol Dachs:Janssen, Celgene, Amgen and Takeda: Honoraria. Bladé:Jansen, Celgene, Takeda, Amgen and Oncopeptides: Honoraria. Mateos:EDO: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract: Continuous treatment with lenalidomide (R) and dexamethasone (d) is a standard of care for multiple myeloma (MM) patients (pts) not candidates for autologous stem cell transplantation (ASCT). As previously reported, the addition of Clarithromycin (C) to Rd has proven to be safe and effective, and case-control analyses suggested a significant additive value with the combination. C optimizes the therapeutic effect of glucocorticoids by increasing the area under the curve, has immunomodulatory effects and may have direct antineoplastic properties. However, there are not randomized phase III trials confirming these results. GEM-Claridex in an open, randomized, phase III trial for untreated newly diagnosed MM pts ineligible for ASCT. Enrolled pts were randomly assigned 1:1 to receive 28-day cycles of R (25mg po qd days 1-21), d (40mg po [20mg in pts & gt;75 years], days 1, 8, 15 and 22) plus or minus C (500mg po bid) until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall response rate (ORR), overall survival (OS) and minimal residual disease (MRD) negativity rate and safety. MRD was evaluated in 99 pts using Euroflow NGF (limit of detection, 2x10-6). As expected, most pts in CR were tested for MRD whereas the majority of pts with missing MRD data achieved VGPR or less and were thus considered as MRD-positive for intent to treat analyses. Two hundred and eighty-eight pts were included (144 to C-Rd and 144 to Rd). Median age was 76 (range: 65-93), 36.8% of pts had ISS 3 and 15.6% presented with high-risk cytogenetic abnormalities. Key baseline characteristics were well balanced between the two arms. The addition of C to Rd resulted in deeper responses with a ≥ complete response (CR) rate of 20.1% in the C-Rd arm compared to 11.2% in the Rd arm (p = 0.037). Also, the ≥ very good partial response (VGPR) rate was 52.8% in the C-Rd arm as compared to the 37.1% in the Rd arm (p = 0.007). MRD analysis was performed at suspected CR and yearly afterwards. On intent-to-treat, 5/144 (3,5%) and 9/143 (6,2%) of pts achieved undetectable MRD with C-Rd and Rd, respectively (p = 0,7). With a median follow-up of 16 months (range, 1-47), no significant differences were observed in PFS: in the C-Rd arm the median was 23 months and has not been reached in the Rd arm (p = 0.09); furthermore, although disease progression and/or death rate was comparable in both arms (C-Rd: 57/144 [39.6%] vs Rd: 45/144 [31.2%] ), a trend towards shorter PFS was observed in the C-Rd group (Figure 1). This effect was less evident in younger ( & lt;75) pts (median PFS, C-Rd: 24 months vs Rd NR, p = 0,588) but, in older pts (≥ 75), the addition of C to Rd resulted into a significant deleterious effect on PFS (median PFS, C-Rd: 19 vs Rd 28 months, p = 0.03) (Figure 2a and 2b). Irrespectively of treatment arm, pts with MRD negative had significantly longer PFS (NR vs 26 months, p = 0,03). Concerning OS, no differences have been identified (p = 0.41), although median has not been reached yet in any arm. Out of the 33 and 28 deaths documented in the C-Rd and Rd arms respectively, the percentage of pts dying w/o documented PD was significantly higher in the C-Rd group (27/33 [82%] vs 13/27 [48%] , p = 0.004). Furthermore, in the C-Rd arm, the most frequent causes of death were severe infections (14/27 [52%] and cardiovascular events 6/27 [22%] ) the majority of them occurring in older (≥75) pts (20/27, 74%). The most common G3-4 adverse events (AE) in the C-Rd and Rd arms were hematologic (neutropenia: 10,4% vs 16,7% [p = ns] and anemia: 2,1% vs 6,9% [p = 0,04] , respectively). G3-4 infections occurred in 16% of cases in both arms and were the most frequent non-hematological AE. 7% of pts in both arms developed G3-4 GI toxicity and there were no differences between the two arms in G3-4 skin-related AEs (2,8% vs 3,5%). Only one case of invasive SPM (colon cancer) in the C-Rd arm was reported. In conclusion, the addition of C to Rd in transplant ineligible newly diagnosed MM pts significantly increases the rate and depth of responses but it is not associated with an improved PFS and OS due to a higher proportion of deaths in the C-Rd arm, mostly infectious, in pts & gt; 75 years and being early deaths. Overexposure to steroids due to the delayed clearance induced by C in this elderly population could explain our results. Figure Disclosures Puig: The Binding Site: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Rosinol Dachs:Janssen, Celgene, Amgen and Takeda: Honoraria. De Arriba:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Honoraria. Oriol:Celgene Corporation: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy; Amgen: Consultancy, Speakers Bureau. De La Rubia:AbbVie: Consultancy; AMGEN: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Janssen: Consultancy. Amor:Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Martín Sánchez:GILEAD SCIENCES: Research Funding. Rossi:BMS: Research Funding; Janssen, Celgene, Amgen: Consultancy. Coleman:Merck: Research Funding; Pharmacyclics: Speakers Bureau; Kite Pharmaceuticals: Equity Ownership; Gilead, Bayer, Celgene: Consultancy, Research Funding, Speakers Bureau. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Bladé:Jansen, Celgene, Takeda, Amgen and Oncopeptides: Honoraria. Niesvizky:Takeda, Amgen, BMS, Janssen, Celgene: Consultancy, Research Funding. Mateos:EDO: Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria.

Abstract: Background: The broad use of immunomodulatory drugs (IMiDs) and the breakthrough of novel immunotherapies in MM, urge the optimization of immune monitoring to help tailoring treatment based on better prediction of patients' response according to their immune status. For example, current T cells immune monitoring is of limited value because the phenotype of tumor-reactive T cells is uncertain. Aims: To characterize the MM immune microenvironment at the single-cell level and to identify clinically relevant subsets for effective immune monitoring. Methods: We used a semi-automated pipeline to unveil full cellular diversity based on unbiased clustering, in a large flow cytometry dataset of 86 newly-diagnosed MM patients enrolled in the PETHEMA/GEM2012MENOS65 clinical trial, including immune monitoring at diagnosis, after induction with bortezomib, lenalidomide, dexamethasone (VRD), autologous transplant and VRD consolidation. Immunophenotyping was performed using the first 8-color combination (CD19, CD27, CD38, CD45, CD56, CD81, CD117, CD138) of the next-generation flow (NGF) panel for MRD assessment. Results were then validated in additional 145 patients enrolled in the same trial. Deep characterization of T cells was performed using 17-color multidimensional flow cytometry (TIM3, CD160, TIGIT, CD57, CD8, PD1, CD45RA, CD56, BTLA, CD4, CD3, CD39, CD137, CTLA4, CCR7, CD16, CD27) and combined single-cell (sc) RNA/TCR sequencing (10xGenomics). Results: Simultaneous analysis of the entire dataset (n=333 files) unbiasedly identified 25 cell clusters (including 9 myeloid and 13 lymphocytes subsets) in the MM immune microenvironment. Afterwards, we correlated a total of 120 immune parameters derived from the cellular abundance of each cluster and specific cell ratios determined at all time points, with a total of 20 clinical parameters including the International Staging System (ISS) and FISH cytogenetics. Twelve variables had significant impact in progression-free survival (PFS) and the ratio between CD27- vs CD27+ T cells emerged as an independent prognostic factor (HR:0.09, p=0.04) together with the ISS in a Cox regression model. The 3-year PFS rates of patients with high vs low CD27-/CD27+ ratios were 94% vs 71% (p=0.02), respectively; these findings being confirmed in the validation dataset. Thus, we observed in the entire cohort (n=231) that a prognostic score including the CD27-/CD27+ T cell ratio (HR:0.21, p=0.013) and ISS (HR:1.41, p=0.015) outperformed each parameter alone (HR:0.06, p=0.007). To gain further insight into the biological significance of the CD27-/CD27+ T cell ratio, we performed scRNA/TCRseq in 44,969 lymphocytes from 9 MM patients. Downstream analysis unveiled that CD27- T cells were mostly CD8 and included senescent, effector and exhausted clusters. By contrast, CD27+ T cells were mainly CD4 and the remaining CD8 T cells had a predominant immune suppressive phenotype (ie. high GZMK, TIGIT, LAG3 and PD1 expression levels). Such T cell clustering was validated by 17-color multidimensional flow cytometry that confirmed the cellular distribution identified by scRNAseq, as well as higher reactivity for PD1, TIGIT, BTLA and TIM3 in CD27+ vs CD27- T cells. Simultaneous scTCRseq revealed a total of 90 different clonotypes (median of 12 per patient). Interestingly, most clonotypes where found in CD27- (74/90) as opposed to CD27+ T cells and, using the VDJB database, the CDR3 sequences of clonotypic effector/exhausted CD27- T cells were predicted to recognize MM-related epitopes such as MLANA, HM1.24 (CD319), TKT, or IMP2. In selected patients, we performed exome- and RNA-sequencing of tumor cells and analyzed their HLA profile. Using the T Cell Epitopes - MHC Binding Prediction tool from the IEDB Analysis Resource, we found expression of mutated genes (e.g. UBXN1, UPF2, GNB1L) predicted to bind MHC class I molecules on tumor cells and potentially recognized by autologous clonotypic CD27- T cells. Conclusion: We show for the first time that potential MM-reactive T cells are CD27-negative and that their abundance in the immune microenvironment of newly-diagnosed MM patients is prognostic, possibly due to their reactivation after treatment with IMiDs and autologous transplant. Because NGF is broadly used, these results are readily applicable for effective T cell immune monitoring. Disclosures Puig: Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria; The Binding Site: Honoraria; Takeda: Consultancy, Honoraria. Rosinol Dachs:Janssen, Celgene, Amgen and Takeda: Honoraria. Oriol:Janssen: Consultancy; Takeda: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Speakers Bureau. Rios:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sureda:Takeda: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria; Gilead: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Roche: Honoraria; Sanofi: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support; Amgen: Membership on an entity's Board of Directors or advisory committees. De La Rubia:Takeda: Consultancy; Janssen: Consultancy; Celgene Corporation: Consultancy; AMGEN: Consultancy; AbbVie: Consultancy. Mateos:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria; EDO: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lahuerta:Takeda, Amgen, Celgene and Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bladé:Irctures: Honoraria; Janssen, Celgene, Amgen, Takeda: Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract: Introduction: SMM is an asymptomatic and heterogeneous plasma cell disorder. Both Spanish Myeloma and ECOG Groups have demonstrated that pts at high risk of progression to active MM benefit from early treatment with R-based regimens. Our next step was to design this phase 2, single arm trial, focusing on the same population, but with the potential goal of cure, defined by sustained minimal residual disease negativity (MRD-ve) at 5 years after HDT-ASCT. Patients and methods: Ninety SMM pts at high-risk of progression ( & gt;50% at 2 yrs), younger than 70 years and transplant candidates were included. The high risk was defined by the presence of both ≥PC 10% and MC ≥3g/dL (Mayo) or ifonly one criterion was present, pts must & gt;95%of aberrant PCs within the total PCsBM compartment by immunophenotyping plus immunoparesis (Spanish). Induction therapy consisted on six 4-weeks cycles of KRd in which K was given at dose of 36 mg/m2 twice per week plus R at dose of 25 mg on days 1-21 and dexamethasone at dose of 40 mg weekly. Melphalan at dose of 200 mg/m2 followed by ASCT was given as intensification therapy followed by two KRd consolidation cycles and maintenance with R at dose of 10 mg plus dexamethasone at dose of 20 mg weekly for up to 2 yrs. The primary end-point was to evaluate the MRD-ve rate by next generation flow (NGF) after induction and ASCT and our aim was to increase the MRD -ve rate from 34% (reported in NDMM pts after VTD and ASCT) to at least 50%. Results: Between June 2015 and June 2017, 90 high-risk SMM pts were recruited. Twenty-eight pts (32%) shared at least one of the new biomarkers predicting imminent risk of progression to MM. On February 4th, 2019, 71 pts were already receiving maintenance treatment; 7 pts had finalized the treatment and there were 11 early discontinuations (4 biochemical relapses during maintenance, 2 Informed Consent refusal, 3 adverse events and two deaths). After a median follow-up of 32 months (8-128), 93% of pts remain alive and free of progression and 98% of them alive. In the intent-to-treat pts' population, after induction, the ≥CR rate was 41% and increased to 59% after HDT-ASCT and to 70% after consolidation. In the same analysis, MRD-ve rate was observed in 30% of pts after induction, 52% after HDT-ASCT and 57% after consolidation. If we focus on the 83 pts who completed induction, HDT-ASCT and consolidation, the ≥CR/undetectable MRD rates were 42%/31%, 64%/56% and 76%/63% after each step, respectively. Concerning toxicity, during induction, G3-4 neutropenia and thrombocytopenia were reported in 5 (6%) and 10 pts (11%), respectively. G3-4 infections were reported in 16 pts (18%), followed by skin rash in 8 pts (9%). One patient reported G1 atrial fibrillation and another cardiac failure secondary to respiratory infection. Three pts reported hypertension (G2 in two and G3 in one). In all but two of the pts, PBSC collection was successful with a median of 4.10 x 106/Kg CD34 cells collected. All pts engrafted but one patient developed late graft failure. During consolidation, 2 pts developed G3-4 neutropenia, 3 pts G3-4 infections and 1 pt skin rash. Maintenance treatment is ongoing and one patient had to discontinue due to a second primary malignancy (lung cancer) and other due to sustained thrombocytopenia. Conclusions: The primary end point of the trial was met, and 56% of the pts who completed induction and HDT-ASCT achieved MRD-ve. This "curative strategy for high risk SMM" continues being encouraging and 93% of pts remain alive and progression-free at 30 months and 98% of pts alive. Disclosures Mateos: GSK: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees; EDO: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rodriguez Otero:Celgene Corporation: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria; Takeda: Consultancy; BMS: Honoraria; Kite Pharma: Consultancy. Oriol:Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy; Takeda: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Speakers Bureau. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. Rosinol Dachs:Janssen, Celgene, Amgen and Takeda: Honoraria. Amor:Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Puig:Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria; The Binding Site: Honoraria. De La Rubia:AMGEN: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Janssen: Consultancy; AbbVie: Consultancy. De Arriba:Takeda: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Lopez Jimenez:GILEAD SCIENCES: Honoraria, Other: Education funding. Ocio:Celgene: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Mundipharma: Research Funding; AbbVie: Consultancy; Sanofi: Research Funding; Seattle Genetics: Consultancy; Array Pharmaceuticals: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Pharmamar: Consultancy. Bladé:Jansen, Celgene, Takeda, Amgen and Oncopeptides: Honoraria. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria.