ALBERT

Description: Patients with multiple myeloma (MM) carrying high-risk cytogenetic abnormalities (CA) have inferior outcome despite achieving similar complete response (CR) rates when compared to cases with standard-risk CA. This questions the legitimacy of CR as treatment endpoint for high-risk MM, and represents a biological conundrum regarding the nature of tumor reservoirs persisting after therapy in patients with standard- and high-risk CA. Here, we used next-generation flow (NGF) to evaluate measurable residual disease (MRD) in MM patients with standard- (N=300) vs high-risk CA (N=90) enrolled in the PETHEMA/GEM2012MENOS65 trial (NCT01916252), and to identify mechanisms determining MRD resistance in both patient subgroups (N=40). The 36-month progression-free and overall survival rates were higher than 90% in patients with undetectable MRD, with no significant differences (P≥0.202) between cases having standard- vs high-risk CA. Persistent MRD resulted in median progression-free survival of approximately three and two years in patients with standard- and high-risk CA, respectively (P

Description: 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

Description: Introduction Intensive consolidation chemotherapy in acute myeloid leukemia (AML) patients induces important hematologic toxicity with potential life-threatening infections that can lead to delays in further treatment or death in complete remission (CR). Recent single and multi-center studies (Guo et al. Blood 2011, JCO 2012, ASH 2015) have shown that the infusion of HLA-mismatched peripheral stem cell without immunosuppressive prophylaxis can accelerate hematologic recovery after chemotherapy, without developing graft versus host disease (GVHD). Objectives The primary objective of this phase I-II trial is to confirm the safety (absence of GVHD) and efficacy (reduction of neutropenia duration) of HLA-mismatched stem cells infusion after consolidation chemotherapy with idarubicine and cytarabine (3+7) in 50 younger patients with intermediate/high-risk AML. Herein we present the preliminary results of the phase I trial with 9 adult patients (safety cohort). Methods Patients younger than 65 years old with AML in CR after induction therapy that were assigned to receive consolidation course with idarubicin (12 mg/m2/day IV 1-3) and cytarabine (200 mg/m2/day IV 1-7) according to PETHEMA protocol were enrolled in this study in a single Spanish institution. To determine safety of HLA-mismatched stem cells infusion a standard 3+3 design was used in this preliminary study: cohorts of 3 patients were established with decreasing immunosuppressive prophylaxis, 3 additional patients would be enrolled with the same immunosuppression if limiting toxicity (LT) was observed in 1 out of 3 patients. LT was defined as global GVHD〉grade 2 or grade 4 infusion related reactions. The first cohort of 3 patients was assigned to receive immunosuppression with cyclosporine (1-1.5 mg/kg/bid) and prednisone (0.5 mg/kg/qid), the second cohort to receive only prednisone (0.5 mg/kg/qid), and the third would not receive immunosuppressive treatment. The immunosuppression resulting of this phase would be used in an expansion cohort. Stem cells were obtained after mobilization with G-CSF and apheresis from HLA-mismatched family-related donors and were infused the day 9 of the consolidation course. The donor and recipient HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 alleles were genotyped using a PCR-SSP method. Hematologic recovery was defined as days from start of chemotherapy to neutrophil count 〉0.5x109/L and to platelet count 〉50x109/L. G-CSF was administered only in case of severe infection. This study was approved by the Ethical Committee, and inform consent was obtained from all patients and donors. Results From March 2015 to June 2016, 9 patients were enrolled in this study. Median age was 46 (28-64) and 6 were male. All were in CR after induction therapy, 6 had intermediate risk cytogenetic, and 3 high risk cytogenetic/molecular AML. All the donors were family-related and HLA compatibility was 3/6 for 8 patients and 5/6 in one patient. HLA-mismatched stem cells infusion characteristics were: median number of mononuclear cells, CD34+ and CD3+ T cells infused per course was 4.5 (2.1-6.6)x108/kg, 3.3 (0.7-4.9)x106/kg and 1.7 (0.8-2.3)x108/kg, respectively. LT was not reached and no diagnosis of GVHD was made. Two patients developed cytarabine related rash and other 2 patients infectious diarrhea. No patient needed further immunosuppressive treatment. Median duration of neutropenia and thrombocytopenia was 30 (27-50) and 44 (22-51) days, respectively. 3 patients received G-CSF and 2 developed severe infections. Median blood cell unit and platelet units transfused was 4 (2-20) and 8 (2-32). These results are similar to those observed in a historical cohort (non-matched) of 59 patients with AML who received consolidation with 3+7 at the same institution between January 2010 to January 2015 (median duration of neutropenia and thrombocytopenia was 29 (17-57) and 36 (18-206) days, respectively). Conclusion The infusion of HLA-mismatched stem cell is safe after consolidation with idarubicin and cytarabine in younger patients. The methodology and in consequence the results of our safety cohort (with immunosuppressive prophylaxis) are not comparable to the previous experience reported by other groups. The reduction of hematologic recovery remains to be confirmed with this schedule in a larger cohort without immunosuppressive prophylaxis. Research granted by IIS La Fe (2014/0368) Disclosures Boluda: Instituto de Investigación Sanitaria La Fe: Employment.

Description: 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

Description: 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

Description: 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.

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

Description: 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

Description: Key Points In MM patients, stringent CR criteria, in particular the sFLC ratio, do not predict significantly better outcome among MM patients in conventional CR.

Description: 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