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We investigated genomic and transcriptomic changes in paired tumor samples of 29 in-house multiple myeloma (MM) patients and 28 patients from the MMRF CoMMpass study before and after treatment. A change in clonal composition was found in 46/57 (82%) of patients, and single-nucleotide variants (SNVs) increased from median 67 to 86. The highest increase in prevalence of genetic aberrations was found in RAS genes (60% to 72%), amp1q21 (18% to 35%), and TP53 (9% to 18%). The SBS-MM1 mutation signature was detected both in patients receiving high and low dose melphalan. A total of 2589 genes were differentially expressed between early and late samples (FDR XPO1 and ABC transporters. Our study demonstrates a transcriptomic convergence of pathways supporting increased proliferation and metabolism during disease progression in MM.

Progression and persistence of malignancies are influenced by the local tumor microenvironment, and future eradication of currently incurable tumors will, in part, hinge on our understanding of malignant cell biology in the context of their nourishing surroundings. Here, we generated paired single-cell transcriptomic datasets of tumor cells and the bone marrow immune and stromal microenvironment in multiple myeloma. These analyses identified myeloma-specific inflammatory mesenchymal stromal cells, which spatially colocalized with tumor cells and immune cells and transcribed genes involved in tumor survival and immune modulation. Inflammatory stromal cell signatures were driven by stimulation with proinflammatory cytokines, and analyses of immune cell subsets suggested interferon-responsive effector T cell and CD8+ stem cell memory T cell populations as potential sources of stromal cell–activating cytokines. Tracking stromal inflammation in individuals over time revealed that successful antitumor induction therapy is unable to revert bone marrow inflammation, predicting a role for mesenchymal stromal cells in disease persistence. Multiple myeloma disease progression and therapy response are influenced by the bone marrow niche in which the tumor cells reside. To characterize this supportive niche, Cupedo and colleagues use single-cell transcriptomic analysis of bone marrow stromal cell populations from individuals with multiple myeloma. They identify a myeloma-specific inflammatory mesenchymal stromal cell (iMSC) population that spatially colocalizes with tumor cells. Anti-myeloma induction therapy does not influence iMSC presence, suggesting a role for bone marrow inflammation in myeloma persistence or relapse.

Minimal residual disease (MRD) tracking, by next generation sequencing of immunoglobulin sequences, is moving towards clinical implementation in multiple myeloma. However, there is only sparse information available to address whether clonal sequences remain stable for tracking over time, and to what extent light chain sequences are sufficiently unique for tracking. Here, we analyzed immunoglobulin repertoires from 905 plasma cell myeloma and healthy control samples, focusing on the third complementarity determining region (CDR3). Clonal heavy and/or light chain expression was identified in all patients at baseline, with one or more subclones related to the main clone in 3.2%. In 45 patients with 101 sequential samples, the dominant clonal CDR3 sequences remained identical over time, despite differential clonal evolution by whole exome sequencing in 49% of patients. The low frequency of subclonal CDR3 variants, and absence of evolution over time in active multiple myeloma, indicates that tumor cells at this stage are not under selective pressure to undergo antibody affinity maturation. Next, we establish somatic hypermutation and non-templated insertions as the most important determinants of light chain clonal uniqueness, identifying a potentially trackable sequence in the majority of patients. Taken together, we show that dominant clonal sequences identified at baseline are reliable biomarkers for long-term tracking of the malignant clone, including both IGH and the majority of light chain clones.

Minimal residual disease (MRD) by multiparameter flow cytometry (MFC) is the most effective tool to define a deep response in multiple myeloma (MM). We conducted an MRD correlative study of the EMN02/HO95 MM phase III trial in newly diagnosed MM patients achieving a suspected complete response before maintenance and every 6 months during maintenance. Patients received high-dose melphalan (HDM) versus bortezomib-melphalan-prednisone (VMP) intensification, followed by bortezomib-lenalidomide-dexamethasone (VRd) versus no consolidation, and lenalidomide maintenance. Bone marrow (BM) samples were processed in three European laboratories, applying EuroFlow-based MFC protocols (eight colors, two tubes) with 10−4−10−5 sensitivity. At enrollment in the MRD correlative study, 76% (244/321) of patients were MRD-negative. In the intention-to-treat analysis, after a median follow-up of 75 months, 5-year progression-free survival was 66% in MRD-negative versus 31% in MRD-positive patients (HR 0.39; p p

Background Primary plasma cell leukemia (pPCL) is an aggressive subtype of multiple myeloma that is characterized by ≥20% circulating tumor cells (CTCs), whereas CTC levels in newly diagnosed multiple myeloma (NDMM) are Methods Transcriptomic data were generated of CD138-enriched bone marrow (BM) plasma cells from both NDMM patients enrolled in the Cassiopeia (NCT02541383) and HO143 (EudraCT 2016-002600-90) trials and pPCL patients from the EMN12/HO129 (EudraCT 2013-005157-75) trial. NDMM CTC levels were determined with the EuroFlow NGF protocol. HR FISH was defined as the presence of either t(4;14), t(14;16) or del17p.154 NDMM and 29 pPCL patients were divided into a discovery and validation cohort to construct and test a classifier for PCL-like disease. Subsequently, data from 8 additional NDMM cohorts were used to assess the association of PCL-like status with progression-free survival (PFS) and overall survival (OS) in Cox proportional hazards (PH) models including conventional HR markers. Results Baseline CTC levels were determined in 297 NDMM and 51 pPCL patients. CTCs could be detected in 87% of NDMM patients, with a limit of detection Conclusions (1) pPCL cannot only be identified clinically, but also molecularly. (2) PCL-like status is a novel marker for HR disease in NDMM that identifies patients with a tumor transcriptome similar to pPCL and has independent prognostic value in the context of conventional HR markers. (3) PCL-like status could help detect NDMM patients with early stage or borderline pPCL.

Introduction Multiple myeloma (MM) is a malignant plasma cell disorder of which the prognosis has significantly improved since the introduction of proteasome inhibitors (PIs) and immune modulatory drugs (IMiDs) in standard-of-care treatment protocols. Despite this progress, many patients still suffer from progressive disease, of which the underlying biological mechanisms are mainly unknown. To elucidate these, we have studied the genomic evolution of MM tumor samples in 29 patients before and after receiving a PI-based and/or IMiD-based treatment, using whole exome sequencing (WES) and mRNA sequencing (RNA-Seq). Methods Patient samples were selected from the Norwegian Biobank for Multiple Myeloma and the HOVON-87/NMSG-18 MM and EMN-02/HOVON-95 MM studies. Tumor DNA was isolated from CD138-positive MM cells with control DNA from peripheral blood samples. WES libraries were prepared using the SureSelectXTsample prep kit and the SureSelectXT Human All Exon V5 target 50 Mb kit (Agilent), followed by paired-end sequencing on a HiSeq2500 instrument (Illumina). Somatic, non-synonymous variants (SNSVs) were called using MuTect and Strelka. Copy number aberrations (CNAs) were estimated using Sequenza. RNA-Seq libraries were generated using the TruSeq® Stranded mRNA kit (Illumina).DESeq2 paired analysis was performed in R Studio to find differentially expressed genes. RNA-Seq expression data from diagnosis/relapse pairs from patients treated with a PI and/or IMiD were selected from the CoMMpass IA10 release database and used as validation cohort. Results We obtained 21 diagnosis/relapse pairs, as well as 8 relapse/relapse pairs, with a median time between samples of 21 months, respectively 16 months. In addition, for 3 of the patients, we had a 3rd sample included. We found a median of 54 SNSVs in our cohort, with an increase in mutational load from the first to the second sample (median 44 to 61). Three different patterns of tumor evolution were observed, confirming previous reports:stable evolution (29% of patients), where the tumor had no or minor changes;linear evolution (29% of patients), where there were acquired mutations and/or CNAs at relapse;differential clonal response (42% of patients), where there was a disappearance of certain mutations/CNAs at relapse and appearance of new ones, suggesting a shift in dominance of different clones. Of interest, of the 7 relapse-relapse tumor pairs that were included in the analysis, 5 were defined to have a differential clonal response, while the remaining 2 had linear evolution, suggesting that the tumors are more prone to genomic change later in the disease course.Tumors shifting their clonal dominance tended to select for a clone with more high-risk features later in the disease course, as illustrated by the appearance of a deletion of chromosome 17p, TP53 mutations and a gain of chromosome 1q. There was a shorter average time interval between sample 1 and 2 for those having a stable evolution pattern (average 12 months, range [5-16]), than those having a differential clonal response (19 months [5-70]), and the highest interval was in patients with linear evolution patters (29 months [15-50]). Tumors having a differential clonal response and linear evolution, showed more often a deeper treatment response (43% CR/nCR, 40% VGPR and 22% CR/nCR, 56% VGPR, respectively) compared to those with stable evolution patterns (14% VGPR), suggesting that tumors that respond well to treatment are more prone to evolve. RNA-Seq data showed a higher expression of genes involved in G2M checkpoint, E2F targets and mitotic spindle at relapse. In line with this, relapsed tumors were found to have an increased proliferation index (GEP; Zhan et al, Blood, 2006). This was confirmed in RNA-Seq data from 27 diagnosis-relapse samples from the CoMMpass dataset. Conclusion This study has performed a comprehensive genomic and transcriptomic analysis of the clonal evolution of MM tumors after treatment with PIs and IMIDs. Our data suggest that clones with high-risk factors and increased expression of genes involved in proliferation and cell cycle regulation are selected for under the pressure of these treatment regimens. The data also suggest that there is a relation between the pattern of genomic evolution and the obtained depth of response, which deserves further investigation. Disclosures Sonneveld: Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

8011 Background: MRD detection is a sensitive tool to measure response in MM. We assessed MRD by MFC in newly diagnosed MM patients (pts) enrolled in the EMN02/HO95 phase 3 trial. Methods: Pts were ≤65 years old and received Bortezomib-Cyclophosphamide-Dexamethasone (VCD) induction, intensification with Bortezomib-Melphalan-Prednisone (VMP) vs High-Dose-Melphalan (HDM) followed by stem cell transplant, consolidation with Bortezomib-Lenalidomide-Dexamethasone (VRD) vs no consolidation, and Lenalidomide maintenance. MRD analysis was performed in pts achieving at least a very good partial response (VGPR) before starting maintenance (after HDM, VMP or VRD) and during maintenance every 6-12 months; samples were centralized to 3 European labs. MFC was performed on bone marrow according to Euroflow-based methods (8 colors, 2 tubes) with a sensitivity of 10-5. Quality checks were performed to compare sensitivity and to show correlation between protocols (Hofste op Bruinink D ASH 2016 abstract 2072). Results: 316 pts were evaluable before maintenance: median age was 57 years, 18% (57/316) pts had ISS III and 22% (70/316) had high risk cytogenetic (HR-C) defined as having at least one among del17, t(4;14) or t(14;16); 63% (199/316) had received HDM and 37% (117/316) VMP; thereafter 51% (160/316) had received VRD. 76% (239/316) were MRD negative (MRD-) of whom 64% (153/239) received HDM vs 36% (86/239) VMP, with a median follow-up time of 30 months from MRD enrolment. 3-year PFS was 50% in MRD positive (MRD+) vs 77% in MRD- pts (HR: 2.87, p < 0.001). Subgroup analyses were performed to evaluate the risk factors for MRD+ according to baseline characteristics and therapies: HR-C was the most important risk factor (HR 9.87, interaction-p = 0.001). Finally, 48% of MRD+ pts at pre-maintenance who had a second MRD evaluation after at least 1 year of lenalidomide became MRD-. Conclusions: MRD by MFC is a strong prognostic factor in MM pts receiving intensification with novel agents or transplant; lenalidomide maintenance further improved depth of response; HR-C is the most important prognostic factor in MRD+ pts. Clinical trial information: NCT01208766.

Background Deletion of chromosome 17p (del17p) is detected in 10% of multiple myeloma (MM) patients at diagnosis and is associated with both a dismal prognosis and increased prevalence after treatment. Even though this suggests that it might be a driver of disease progression, relatively little is known about the genomic landscape of these tumors. With this study, we aimed to identify recurrent aberrations in a large cohort of del17p MM patients and to assess their prognostic value within this patient group. Methods The study design consisted of a discovery phase and a validation phase. For the discovery phase, 44 newly diagnosed MM (NDMM) patients were included with a del17p in at least 50% of plasma cells, as detected with fluorescent in situ hybridization (FISH). From 12 del17p patients, 2 or 3 tumor samples were available to analyze clonal evolution during disease progression. DNA was isolated from peripheral blood and CD138+ enriched bone marrow mononuclear cells, followed by a custom capture of the whole exome, Chr17p and the IgH, Igk, IgL and MYC regions (SeqCap EZ Exome Plus, Nimblegen) and paired-end sequencing. Significantly mutated genes were determined with MutSigCV and significantly deleted or amplified genomic regions with GISTIC2. Single nucleotide variants (SNVs) in TP53, FAM46C, KRAS, NRAS, DIS3 and BRAF were validated using a custom amplicon panel (TruSeq Custom Amplicon Assay v1.5, Illumina), followed by deep sequencing on a MiSeq System. Findings were validated in a cohort of 463 NDMM patients of the UK Myeloma XI trial, from which whole exome sequencing data were available for paired tumor and germline DNA (Walker et al. - J Clin Oncol 2015). All samples in both the discovery and the validation cohort were reanalyzed with one bioinformatic pipeline, from alignment to variant calling. The copy number status of TP53 was determined with Sequenza, followed by selection of del17p samples after manual inspection of the results. From a third cohort of 233 MM patients with progressive disease (PDMM) treated at the University of Arkansas for Medical Sciences (UAMS), 406 cancer-related genes were paired-end sequenced from tumor DNA with the FoundationOne Heme assay (He et al. - Blood 2016). Results In the discovery cohort, we identified a commonly deleted region (CDR) on Chr17p of 235 kb, in which one or more somatic, nonsilent aberrations (SNSA) in TP53 were detected in 25/44 (57%) patients (adj. p In the UK Myeloma XI cohort, we detected a del17p in 32/463 (7%) of patients. Of these, 15/32 (47%) had an SNSA in the remaining allele. Follow-up data were available of 73/76 del17p NDMM patients from both cohorts (n=46 transplant-eligible (TE), n=27 nontransplant-eligible (NTE)), with a median follow-up time of 17 months. TP53 mutated patients had a worse overall survival (OS) than TP53 wildtype patients (p=0.02), of which the strongest effect on OS was seen of TP53 missense mutations (p We went on to look in the FoundationOne cohort of PDMM patients and detected a del17p in 37/233 (16%) of patients and an SNSA in TP53 in 25/37 (68%) of del17p PDMM patients, which is a higher rate than in the 2 NDMM cohorts. Conclusions (1) TP53 is somatically mutated in half of del17p MM patients at diagnosis, validated in 2 independent cohorts, and this percentage is higher in del17p MM patients at progression. (2) Particularly TP53 missense mutations have a significant, negative impact on both PFS and OS in del17p MM patients. (3) The rest of the genomic landscape of del17p MM is characterized by significant mutations in FAM46C and KRAS, as well as deletions of RB1, TRAF3 and FAM46C. Disclosures Ashby: University of Arkansas for Medical Sciences: Employment. He:Foundation Medicine, Inc: Employment, Equity Ownership. Bailey:Foundation Medicine, Inc: Employment, Equity Ownership. Mughal:Foundation Medicine: Employment, Equity Ownership. Waage:Novartis, Amgen, Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Celgene: Consultancy, Honoraria. Avet-Loiseau:celgene: Consultancy; sanofi: Consultancy; janssen: Consultancy; amgen: Consultancy. Jackson:Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau. Morgan:Univ of AR for Medical Sciences: Employment; Takeda: Consultancy, Honoraria; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol Meyers: Consultancy, Honoraria. Sonneveld:Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding.

Background Multiple myeloma (MM) is a malignant plasma cell disorder, in which at diagnosis circa 10% of tumors are characterized by a deletion of chromosome 17p (del17p). Previously, others and we reported that this aberration is associated with a significantly inferior prognosis, classifying it as high-risk disease. As TP53 is located on chromosome 17p (Chr17p), most research has thus far been focused on this gene. However, only in a minority of del17p MM patients a mutation could be detected (Lodé et al. - Haematologica 2010, Kortüm et al. - Br J Haematol 2015). We therefore hypothesized that other relevant mutations might be present in TP53, its surrounding genes, or disease relevant pathways. Aim With this study, we sought to identify commonly mutated genes in del17p MM, which could explain its aggressive clinical behavior. Material and Methods We obtained high molecular DNA from CD138+ purified MM cells and matched peripheral blood from patients with a del17p in ≥50% of plasma cells, as detected with fluorescent in situ hybridization (FISH). Libraries were generated using a custom capture of 111 Mb (SeqCap EZ Exome Plus, Nimblegen), comprising the whole exome, Chr17p and the IgH, Igk, IgL and MYC regions. Paired-end sequencing was performed on a HiSeq2500 platform, followed by data-analysis using an in-house bioinformatics pipeline at Erasmus MC. Results Matched tumor and germline DNA was sequenced from 54 patients, with sequential tumor samples available from 12 patients. This resulted in an average coverage of 88x and overall coverage of 94%. Of 44 del17p patients, tumor DNA was obtained before treatment start. Clinical data were collected from 40/44 patients, with a median follow-up time of 18 months. We found TP53 to be mutated most frequently. 31/54 (57%) patients had a somatic nonsynonymous mutation, insertion or deletion (71% missense, 19% stopgain/frameshift, 10% splicing). Although no hotspot mutations were detected, most TP53 mutations resided in the DNA binding domain. Of note, most TP53wt/- patients had no clonal somatic nonsynonymous mutations in other p53 pathway genes, or on Chr17p. To assess the prognostic impact of TP53 mutations in del17p MM, we focused on the 40 chemotherapy-naive patients and stratified for age ≤65 years (n = 25) and >65 years (n = 15). TP53mut/- patients had a significantly worse overall survival (OS) than TP53wt/- patients (p = 0.002). Strikingly, TP53 missense mutations showed the worst OS (p Conclusions (1) In contrast with previous reports, we find TP53 to be somatically mutated in the majority of MM patients with a clonal del17p aberration. (2) Particularly TP53 missense mutations have a significantly negative impact on both PFS and OS in del17p MM patients. This study was supported by the Plaisier Foundation, Erasmus MC Grant and the Dutch CTMM Project. Citation Format: Davine Hofste op Bruinink, Remco Hoogenboezem, Eric Bindels, Mathijs Sanders, Claudia Erpelinck - Verschueren, Paulina van Strien, Jasper Koenders, Kristine Misund, Berna Beverloo, Bronno van der Holt, Ivo Touw, Anders Waage, Hervé Avet-Loiseau, Pieter Sonneveld. Multiple myeloma with a clonal del17p aberration is characterized by somatic TP53 mutations, which negatively affect prognosis in this cytogenetic subgroup. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5023.

Introduction: Customized gene panel sequencing is an attractive approach to genomic tumor characterization in clinical care. Based on published MM exome data we developed a MM Mutation Panel (M3 P) that includes the most commonly mutated genes, actionable drug targets, genes targeted by current standard of care (SOC) therapies and which allows tracking of clonal evolution, copy number and sample purity. Methods and Material: M3 P (v3.0) covers 88 genes (1327 amplicons, 181kb). MM samples from 504 patients (pts) have been analyzed (corresponding germline in 81%) through collaborations between Mayo Clinic and Hospital-12-de-Octubre (Madrid, Spain), the DSMM and GMMG (Würzburg, Ulm, Freiburg and Heidelberg, Germany) and the HOVON trial groups (Rotterdam, The Netherlands). The investigated cohort includes 410 untreated pts (81%), which includes a high risk cohort of 72 pts with del17p, 25 paired samples with later follow up from the same cohort, and 94 relapsed patients, of which 50 were relapsed and refractory. Results: Overall coverage per mutation averaged >500x depth. We identified 945 variants (1.9 per pt) and in 83% of the pts a mutation was found. Clonal heterogeneity was assessed with mutations ranging from 3%-100% variant reads suggesting the presence of a significant number of subclones (e.g. 21% of mutations were in < 10% of reads). The mutation incidence was compared with and closely resembles the most recent MM comprehensive genomic data from the MMRF CoMMpass study: We compare here all pts sequenced by M3 P, untreated pts sequenced by M3 P and CoMMpass: KRAS (24%/23%/24%), NRAS (20%/20%/18%), DIS3 (13%/14%/10%), BRAF (9%/7%/6%), FAM46C (6%/6%/8%) and TRAF3 (6%/6%/7%). TP53 mutation incidence, however, was significantly increased in our cohort (14%/12%/4.2%), a difference explained by the inclusion of del17p (untreated) and relapsed refractory MM in panel sequenced pts, cohorts with elevated incidences of TP53 mutations (32% / 26% respectively). Potentially actionable targets include BRAF mutationsin 43 patients (9%), with druggable p.V600E in 19 or 5%, 8 pts (2%) with FGFR3 (p.R248C and p.G375C one patient each), p.R132 mutation in 4 out of 5 IDH1 (1%) and p.R172K IDH2 mutation in 1 of 3 (1%) pts. Mutations in the MAPK pathway (NRAS, KRAS, BRAF) were detected in 59% of pts, ranging from 36% untreated MM to 72% in refractory MM. Similarly, the CRBN/CUL4B/IKZF1/IKZF3/IRF4 pathway, important for IMiD function, harbored a significant enrichment of mutations in advanced disease (6% untreated vs 17% relapsed), including CRBN mutations (0.5% vs 7%). Nine of 17 IRF4 mutations were located at the p.K123R hotspot, with minor difference between early or late disease (1% vs 3%). Notably, in 8 of 9 pts with CRBN mutation and clinical information, all were unresponsive to IMiD therapy, supporting association of these mutations with resistance to IMiDs. Conversely, M3 P genes related to other SOC therapies, including NR3C1 (targeted by steroids) and 5 proteasome subunit genes (proteasome inhibitors), were rarely mutated across the cohorts not exceeding 1% mutation incidence for each gene. Significant differences in DIS3 and FAM46C mutation incidences were observed across cohorts: DIS3 mutations are more common in untreated pts with a 1.7 fold increased predominance (14% untreated and 8% treated). FAM46C has an expected incidence of 8% but was rarely mutated in untreated del17p high risk disease with only one of 100 patients harboring both mutations. The significance of this finding needs to be determined but implies a possible overlap in function. Finally we assessed impact on survival of the mutation variants identified in 142 untreated Mayo patients and found STAT3 mutations negatively impacting PFS (p=0.034) and OS (p=0.001). This gene is rarely mutated in MM (2% of the total cohort) thus the sample size was small and this finding needs further validation. Conclusion: We here describe 504 MM patients sequenced using the M3 P gene panel, which identified mutations in >80% of investigated patients, overlaps well with published whole exome sequence data and provides clinically relevant information. New findings were the high frequency of minor clones, the relative lack of overlap of del17 and FAM46C mutation, a higher frequency of DIS3 mutation at diagnosis compared to relapse, the prognostic significance of STAT3 mutation and the frequent presence of CRBN pathway mutation in drug resistant relapsed patients. Disclosures Sonneveld: Janssen-Cilag, Celgene, Onyx, Karyopharm: Honoraria, Research Funding; novartis: Honoraria. Mai:Janssen-Cilag: Other: Travel Grant; Onyx: Other: Travel Grant; Mundipharma: Other: Travel Grant; Celgene: Other: Travel Grant. Goldschmidt:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millenium: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Knop:Celgene Corporation: Consultancy. Kull:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Martinez-Lopez:Novartis: Honoraria, Research Funding; Bristol-Meyer Squibb: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Einsele:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Speakers Bureau. Raab:Novartis: Research Funding. Stewart:Oncospire Inc.: Equity Ownership; Celgene: Consultancy; Novartis: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees.

Background The introduction of novel treatment strategies against multiple myeloma (MM) has resulted in a major improvement in the overall outcome, which has led to an increased need for highly sensitive methods to detect minimal residual disease (MRD) in each patient. MRD assessment by multicolor flowcytometry (MFC) has been shown to be of prognostic value in many treatment protocols over the last decade, making it an attractive method to assess response in clinical trials. However, it is currently not known (1) what the best timing is to perform MFC MRD analysis in the context of a treatment protocol including induction, intensification, consolidation and maintenance treatment, (2) which patients should be selected for this analysis, and (3) what its feasibility is in a large international trial. The ongoing EMN-02 MRD Study aims to answer these questions within the framework of the EMN-02/HOVON-95 MM trial. Here, we describe our methods and the results of our first quality assessment round to compare the sensitivity of the used protocols. Methods The EMN-02/HOVON-95 MM trial is a randomized, multicenter, phase 3 trial in which newly diagnosed MM patients 18-65 years received 4 cycles of bortezomib, cyclophosphamide and dexamethasone (VCD) as induction treatment, followed by a first randomization between either 4 cycles of bortezomib, melphalan and prednisone (VMP), or high dose melphalan (HDM) and 1 or 2 ASCT as intensification treatment. Subsequently, patients were randomized between 2 cycles of bortezomib, lenalidomide and dexamethasone (VRD) or no consolidation treatment, followed by lenalidomide maintenance treatment for all until progression or toxicity occurred. Patients undergoing a bone marrow (BM) aspiration for complete response (CR) confirmation according to the International Myeloma Working Group (IMWG) criteria (Rajkumar et al. - Blood 2011) anytime during the trial were eligible for the EMN-02 MRD Study. BM samples from patients from 13 European countries were sent to 4 central MFC MRD laboratories in the Netherlands (A), Czech Republic (B), Denmark (C) and Italy (D), either using the strict Euroflow protocol (A) (Van Dongen et al. - Leukemia 2012) or Euroflow-based methods (B, C & D). In order to check compatibility between protocols, 5 bone marrow samples from MM patients with a clinical response ranging from progressive disease (PD) to CR were each divided in equal volumes and sent to the respective laboratories on 3 different days. MFC MRD analysis was performed on a FACS Canto II (BD) (A-C) or Coulter Navios flowcytometer (D). Protocols A, B & C used the Euroflow Plasma Cell Disorder (PCD) tube 1 and 2 combination of antibodies, containing the backbone markers CD138-PO, CD38-FITC, CD45-PB and CD19-PE-Cy7, with CD56-PE, B2micro-PerCP-Cy5.5, cyIgK-APC and cyIgL-APC-C750 in tube 1, and CD28-PE, CD27-PerCP-Cy5.5, CD117-APC, CD81-APC-H7 in tube 2. Protocol D had the same backbone markers (CD138-PerCP-Cy5.5, CD38-PB, CD45-KO and CD19-PE-Cy7), together with CD27-PE, CD81-FITC and CD20-APC in tube 1 and cyIgK-FITC, cyIgL-PE, CD56-APC and CD117-APC-AF 750 in tube 2. Bulk lysis was performed in protocols A, B and D. Every laboratory acquired at least 2x10e6 leukocytes (or at least 1x10e4 plasma cells) and performed data-analysis in Infinicyt version 1.6 or higher (A, B & C) or Navios Kaluza (D), using a threshold ranging from 10-25 aberrant plasma cell events as cutoff for MFC MRD positivity. Results Acquisition of events occurred the day after BM aspiration for all samples. The total number of acquired events per sample was dependent on the level of MRD, ranging from 3x10e5 to 2x10e7 leukocytes. MFC MRD results were very comparable between labs with a 1:1 correlation between results at every level of residual disease, being 1x10e-2, 1x10e-4, 1x10-4, 1x10e-5 and 1 MRD negative sample at the level of Conclusions This is the first time that a European framework has been set up between laboratories to test MFC MRD analysis in the context of an international trial. The sensitivity of the protocols has been compared in a quality assessment round, which showed a high correlation of results. Disclosures Oliva: Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria. Boccadoro:CELGENE: Honoraria, Research Funding; Mundipharma: Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Abbivie: Honoraria; SANOFI: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Hajek:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sonneveld:Celgene: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria, Research Funding. Palumbo:Takeda: Employment, Honoraria; Janssen Cilag: Honoraria.