Abstract: Background: CTCs may be responsible for MM spreading and accordingly, their numbers in peripheral blood (PB) could be a potential surrogate marker for the rate of dissemination and overall tumor burden in bone marrow (BM). In such case, CTCs may be a powerful biomarker of malignant transformation and disease aggressiveness. Aim: To investigate the clinical significance of CTCs in patients with smoldering (SMM), newly diagnosed (NDMM) and relapsed/refractory MM (RRMM), and to compare the transcriptional profile of CTCs across the disease spectrum. Methods: Next-generation flow (NGF) cytometry was used to assess the percentage of CTCs in PB of 1,157 patients: 316 with SMM, 650 with NDMM and 191 with RRMM. In each disease setting, patients were sub-classified into three groups with undetectable, low and high percentage of CTCs. Cutoffs were defined using maximally selected rank statistics adjusted for time to progression (TTP) in SMM and progression free survival (PFS) in NDMM/RRMM. A subset of SMM patients (n=86) was enrolled in GEM-CESAR. Transplant eligible (n=374) and ineligible (n=276) NDMM, as well as RRMM patients, were homogenously treated according to the GEM2012MENOS65, GEM-CLARIDEX and GEM-KYCYDEX clinical trials, respectively. In 40 patients (2 SMM, 33 NDMM and 5 RRMM) paired CTCs and BM tumor cells were FACSorted and their transcriptional profile was analyzed using RNAseq. Differentially expressed genes were investigated using DESeq2. Results: CTCs were detected in 248/316 (78%), 597/650 (92%) and 170/192 (89%) of SMM, NDMM and RRMM patients. Median CTC frequencies were: 0.001% (0.05 CTCs/µL), 0.01% (0.64 CTCs/µL) and 0.005% (0.22 CTCs/µL), respectively. There were 79 genes differentially expressed between patient-matched CTCs and BM tumor cells (e.g., FLNA, EMP3, LGALS9, MUC1). These were functionally related with TNFα signaling and inflammatory response (enriched in CTCs), as well as to cell cycle and MYC targets (enriched in BM tumor cells). Interestingly, the enrichment of these signatures in CTCs and BM tumor cells was progressively more pronounced from SMM to NDMM and RRMM. Altogether, these data suggest that the CTC-based dissemination potential peaks at the stage of NDMM, which could be related to greater inflammation in BM and cell cycle arrest driving tumor cell egression into PB. There were significant associations between the percentage of CTCs and the 2/20/20 IMWG risk model in SMM, the ISS in NDMM, and high-risk cytogenetics in all three-disease settings. Untreated SMM patients (n=230) with high CTC levels (≥0.02%) showed ultra-high risk of transformation vs those with low and undetectable CTCs (median TTP of 11 months vs not reached [NR] in both; P & lt; .0001). Notably, SMM patients with ≥0.02% CTCs enrolled in GEM-CESAR have not reached a median TTP; thus, early intervention abrogated the poor prognosis of high CTC levels. Transplant-eligible NDMM patients stratified by undetectable, low and high (≥0.2%) CTC levels showed median PFS of NR, 78 and 47 months, respectively (P & lt; .0001). Significant risk stratification was further observed in transplant ineligible NDMM (median PFS: NR, 31 and 14 months, respectively, P = .002) and RRMM (median PFS: NR, 24 and 7 months, respectively, P = .004). In untreated SMM, multivariate analysis of TTP including CTCs, serum M-component ( & gt;2 g/dL), sFLC ratio ( & gt;20) and BM plasma cells ( & gt;20%) selected CTCs as an independent prognostic factor (hazard ratio [HR]: 1.61, P = .015) together with the M-component and sFLC ratio. In NDMM, multivariate analysis of PFS including CTCs, BM plasma cells counts by morphology and flow cytometry, ISS, LDH, cytogenetics and transplant eligibility showed that high CTC levels had independent prognostic value (HR: 1.43, P = .003). Only the achievement of undetectable measurable residual disease (MRD) abrogated the poor prognosis of high CTC levels. Conclusions: This is the largest study investigating the role of CTCs in smoldering and active MM. Our results show that tumor cells are continuously trafficking in PB, possibly through a dynamic mechanism of egression that peaks in NDMM. Evaluation of CTCs in PB outperformed quantification of BM tumor burden in SMM and NDMM, and showed prognostic value in all three-disease stages. Thus, CTC assessment should be part of the diagnostic workup of MM. Early intervention in high risk SMM and undetectable MRD in NDMM may abrogate dismal outcomes associated with high CTC levels. Disclosures Puig: Amgen, Celgene, Janssen, Takeda: Consultancy; Celgene: Speakers Bureau; Celgene, Janssen, Amgen, Takeda: Research Funding; Amgen, Celgene, Janssen, Takeda and The Binding Site: Honoraria. Cedena: Janssen, Celgene and Abbvie: Honoraria. Oriol: GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Sureda: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite, a Gilead Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau; Roche: Other: Support for attending meetings and/or travel; Bluebird: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Consultancy; MSD: Consultancy, Honoraria, Speakers Bureau; BMS/Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending meetings and/or travel, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending meetings and/or travel, Research Funding, Speakers Bureau. De Arriba: Amgen: Consultancy, Honoraria; Glaxo Smith Kline: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; BMS-Celgene: Consultancy, Honoraria, Speakers Bureau. Moraleda: Pfizer: Other: Educational Grants, Research Funding; Sanofi: Other: Educational Grants, Research Funding; MSD: Other: Educational Grants, Research Funding; ROCHE: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Takeda: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Sandoz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Gilead: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Educational Grants, Research Funding; NovoNordisk: Other: Educational Grants, Research Funding; Janssen: Other: Educational Grants, Research Funding; Celgene: Other: Educational Grants, Research Funding; Amgen: Other: Educational Grants, Research Funding. Terpos: GSK: Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Goldschmidt: Incyte: Research Funding; Adaptive Biotechnology: Consultancy; BMS: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Celgene: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Chugai: Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; GSK: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Johns Hopkins University: Other: Grant; Molecular Partners: Research Funding; MSD: Research Funding; Mundipharma: Research Funding; Dietmar-Hopp-Foundation: Other: Grant; Sanofi: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding. Avet-Loiseau: Adaptive Biotechnologies: Honoraria, 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; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: 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; Bristol Myers Squibb: 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. Roccaro: AstraZeneca,: Research Funding; Amgen, Celgene, Janssen, Takeda: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per la Ricerca sul Cancro (AIRC): Research Funding; European Hematology Association: Research Funding; Fondazione Regionale per la Ricerca Biomedica (FRRB), Transcan-2 ERA-NET: Research Funding. Martinez-Lopez: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: 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; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: 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. Lahuerta: Celgene: Other: Travel accomodations and expenses; Celgene, Takeda, Amgen, Janssen and Sanofi: Consultancy. Ocio: Amgen: Consultancy, Honoraria; Bristol-Myers Squibb/Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria; Karyopharm: Consultancy; MSD: Honoraria; Oncopeptides: Consultancy, Honoraria; Pfizer: Consultancy; Secura-Bio: Consultancy. Rosinol: Janssen, Celgene, Amgen and Takeda: Honoraria. Bladé Creixenti: Janssen, Celgene, Takeda, Amgen and Oncopeptides: Honoraria. Mateos: AbbVie: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Honoraria; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird bio: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Regeneron: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: 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; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene - Bristol Myers Squibb: 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; Oncopeptides: Honoraria; Sea-Gen: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel: AbbVie, Amgen, Bristol-Myers Squibb, Celgene, GlaxoSmithKline, Janssen, Karyopharm, Merck Sharpe & Dohme, Novartis, Regeneron, Roche, Sanofi, SecuraBio, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Paiva: Adaptive, Amgen, Bristol-Myers Squibb-Celgene, Janssen, Kite Pharma, Sanofi and Takeda: Honoraria; Bristol-Myers Squibb-Celgene, Janssen, and Sanofi: Consultancy; Celgene, EngMab, Roche, Sanofi, Takeda: Research Funding.

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: Patients with multiple myeloma (MM) may show patchy bone marrow (BM) infiltration and extramedullary disease. Notwithstanding, quantification of plasma cells (PCs) continues to be performed in BM since the clinical translation of circulating tumor cells (CTCs) remains undefined. PATIENTS AND METHODS CTCs were measured in peripheral blood (PB) of 374 patients with newly diagnosed MM enrolled in the GEM2012MENOS65 and GEM2014MAIN trials. Treatment included bortezomib, lenalidomide, and dexamethasone induction followed by autologous transplant, consolidation, and maintenance. Next-generation flow cytometry was used to evaluate CTCs in PB at diagnosis and measurable residual disease (MRD) in BM throughout treatment. RESULTS CTCs were detected in 92% (344 of 374) of patients with newly diagnosed MM. The correlation between the percentages of CTCs and BM PCs was modest. Increasing logarithmic percentages of CTCs were associated with inferior progression-free survival (PFS). A cutoff of 0.01% CTCs showed an independent prognostic value (hazard ratio: 2.02; 95% CI, 1.3 to 3.1; P = .001) in multivariable PFS analysis including the International Staging System, lactate dehydrogenase levels, and cytogenetics. The combination of the four prognostic factors significantly improved risk stratification. Outcomes according to the percentage of CTCs and depth of response to treatment showed that patients with undetectable CTCs had exceptional PFS regardless of complete remission and MRD status. In all other cases with detectable CTCs, only achieving MRD negativity (and not complete remission) demonstrated a statistically significant increase in PFS. CONCLUSION Evaluation of CTCs in PB outperformed quantification of BM PCs. The detection of ≥ 0.01% CTCs could be a new risk factor in novel staging systems for patients with transplant-eligible MM.

Abstract: Introduction: Carfilzomib dosed at 56 mg/m2 twice a week in combination with dexamethasone (Kd) is a standard of care for RRMM after 1-3 prior lines (PL) based on the ENDEAVOR study. Later, the ARROW study showed Kd dosed at 70 mg/m2 weekly to be superior to Kd dosed at 27 mg/m2 twice a week on RRMM patients (pts) after 2-3 PL. On the other side, Cyclophosphamide is an alkylating agent that has been widely combined with proteasome inhibitors and immunomodulatory drugs in MM, improving their efficacy with a good safety profile. In this phase 2 randomized study, we have compared Kd plus cyclophosphamide (KCyd) with Kd in RRMM after 1-3PL, both with K dosed weekly at 70 mg/m2. Patients and methods: RRMM after 1-3 PL of therapy were included in the trial. Consistently with the ENDEAVOR population, previous therapy with proteasome inhibitors was allowed but refractory patients were excluded. Pts were randomized 1:1 to receive K at a dose of 70 mg/m2 iv on days 1, 8 and 15 plus dexamethasone at a dose of 20 mg PO the day on and the day after K plus/minus KCyd at a dose of 300 mg/m2 IV on days 1, 8 and 15 of each 28 days-cycle, as continuous treatment until progressive disease or unacceptable toxicity. The primary endpoint was PFS and key secondary endpoints included response rates, safety profile, and OS. Results: Between January 2018 and February 2020, 198 RRMM pts were included. 97 pts were randomized to KCyd and 101 to Kd. The baseline characteristics of the patients were well balanced between both groups. The median age was 70 years, and 70% and 28% of pts were older than 65 and 75. The median number of PL was one; 61% of pts had received 1 prior line. 94% and 92% of patients had been exposed to bortezomib in the KCyd and Kd and all of them were sensitive. 72% and 67% of patients had been exposed to IMiD's and 51% and 55% of them were IMiD's-refractory in the KCyd and Kd. Only 4 and 6 patients in KCyd and Kd, had received anti-CD38 antibodies being all refractory. After a median f/u of 15.6 months, median PFS was 20.7 m and 15.2 m in KCyd and Kd (p=0.2). In pts after 1PL, median PFS has not been reached in any arm (p=0.4) and in patients after 2-3PL, KCyd resulted in a median PFS of 20.7 vs 11m for Kd (p=0.4). Of note, in the IMiD-refractory population, the addition of Cy to Kd resulted in a significant benefit in terms of PFS: 26.2 months vs 7.7 months in the Kd arm (p=0.01). OS is immature with 23 and 25 events so far in KCyd and Kd, respectively. The ORR was 78% for KCyd and 73% for Kd: 20% of patients in both arms achieved at least complete response, 33% and 28% very good partial response, respectively, and 25% partial response in both arms. The MRD-ve rate was 4% and 5%. As far as toxicity is concerned, neutropenia was the only hematological adverse event more frequently reported in KCyd compared with Kd, of any grade (24% vs 11%) and grade 3-4 (13% vs 7%). This did not translate into more infections and the rate was comparable in both arms (5% G3-4 in both arms). Thrombocytopenia of any grade and grade 3-4 occurred in 14%/1% and 18%/10% in KCyd/Kd. Cardiovascular events of any grade occurred in 22% and 30% of patients in KCyd and Kd. Nine pts in KCyd developed G3-4 cardiovascular events, these included atrial fibrillation (1pt), cardiac failure (2 pts), myocardial infarct (2 pts), and hypertension (4 pts). In the Kd arm, 11 patients developed G3-4 cardiovascular events and consisted of hypertension in most of them (9 pts). Conclusion: Cyclophosphamide added to Kd 70 mg/m2 weekly in RRMM pts after 1-3 PL prolonged the PFS as compared to Kd particularly in the lenalidomide-refractory population. The administration of K at a dose of 70 mg/m2 weekly was safe and more convenient and overall, the toxicity profile was manageable in both arms. Disclosures Mateos: Abbvie/Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Regeneron: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Honoraria; PharmaMar-Zeltia: Consultancy; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ocio:Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria; Asofarma: Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Honoraria; GSK: Consultancy; MDS: Honoraria; Secura-Bio: Consultancy; Oncopeptides: Consultancy. Sureda Balari:Novartis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria; Merck Sharpe and Dohme: Consultancy, Honoraria, Speakers Bureau; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; BMS: Speakers Bureau; Incyte: Consultancy; Celgene: Consultancy, Honoraria; Gilead/Kite: Consultancy, Honoraria. Oriol:Celgene/Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees. Rosinol Dachs:Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Sanofi: Honoraria. Blade Creixenti:Takeda: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: 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; Amgen: Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, BMS, Celgene, Janssen, MSD, Novartis, Takeda, Sanofi, Roche, Abbvie, GlaxoSmithKline and Karyopharm: Consultancy, 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: INTRODUCTION: There is expectation of using biomarkers to personalize treatment in MM. Yet, a successful treatment selection cannot be confirmed before 5 or 10 years of progression-free survival (PFS). Treatment individualization based on the probability of an individual patient to achieve undetectable MRD with a singular regimen, could represent a new model towards personalized treatment with fast assessment of its success. This idea has not been investigated previously. AIM: Develop a machine learning model to predict undetectable MRD in newly-diagnosed transplant-eligible MM patients, treated with a standard of care. METHODS: This study included a total of 278 newly-diagnosed and transplant-eligible MM patients treated with proteasome inhibitors, immunomodulatory drugs and corticosteroids. The training (n=152) and internal validation cohort (n=60) consisted of 212 active MM patients enrolled in the GEM2012MENOS65 trial. The external validation cohort was defined by 66 high-risk smoldering MM patients enrolled in the GEM-CESAR trial, which treatment differed only by the substitution of bortezomib by carfilzomib during induction and consolidation. RESULTS: We started by investigating patients' MRD status after VRD induction, HDT/ASCT and VRD consolidation according to their ISS and R-ISS, LDH levels, and cytogenetic alterations. Surprisingly, neither the ISS nor the R-ISS predicted significantly different MRD outcomes. Indeed, high LDH levels and del(17p13) were the only parameters associated with lower rates of undetectable MRD. Because these two features are relatively infrequent at diagnosis, we next aimed to evaluate other disease features and develop integrative, weighted and more effective models based on machine learning algorithms. Of 37 clinical and biological parameters evaluated, 17 were associated with MRD outcomes. These were subsequently modeled using logistic regression for machine learning classification, where the sum of the weighted coefficients multiplied by its input variable, is transformed into a probability outcome that ranges from 0 to 1 using a logit sigmoid function. The most effective model resulted from integrating cytogenetic [t(4;14) and/or del(17p13)], tumor burden (plasma cell [PC] clonality in bone marrow and CTCs in blood) and immune related (myeloid precursors, mature B cells, intermediate neutrophils, eosinophils, CD27 negCD38 pos T cells and CD56 brightCD27 neg NK cells) biomarkers. Of note, immune biomarkers displayed the highest coefficient weights and were determinant to predict patients' MRD status in this model. Data obtained for an individual patient can be substituted into our formula, which results in a numerical probability of achieving undetectable ( & gt;0.5) vs persistent ( & lt;0.5) MRD after treatment. If probability outcomes are & gt;0.685 or & lt;0.365 (observed in 102/212 patients), MRD outcomes are respectively predicted with higher confidence. Standard-confidence, high-confidence, and external validation predictions were accurate in 152/212 (71.7%), 85/102 (83.3%), and 48/66 (72.7%) patients respectively. Similarly, the external validation set exhibited a similar receiver operating characteristic (ROC) curve as the internal test set (AUC of 0.73 and 0.77 respectively). Patients predicted to achieve undetectable MRD using standard and high-confidence values showed longer PFS and overall survival (OS) than those with probability of persistent MRD. In fact, patients with & gt;0.687 probability of achieving undetectable MRD showed 86% PFS and 94% OS at five years, whereas those in whom persistent MRD was predicted ( & lt;0.365), median PFS was 44 months, and 69% OS was observed at five years. These data indicate that the combination of cytogenetics, tumor burden in bone marrow plus peripheral blood, and immune profiling, may also be explored to identify a subset of patients that have a singular disease biology and long-term survival. CONCLUSION: We demonstrated that it is possible to predict patients' MRD status with significant accuracy, using an integrative, weighted model based on machine learning algorithms. Although immune biomarkers are not commonly used, the raw data from which these can be developed is generally obtained in diagnostic laboratories using flow cytometry to screen for PC clonality. Furthermore, we made the model available to facilitate its use in clinical practice at www.MRDpredictor.com. Disclosures Puig: Celgene, Janssen, Amgen, Takeda: Research Funding; Celgene: Speakers Bureau; Amgen, Celgene, Janssen, Takeda: Consultancy; Amgen, Celgene, Janssen, Takeda and The Binding Site: Honoraria. Cedena: Janssen, Celgene and Abbvie: Honoraria. Oriol: Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. De Arriba: Amgen: Consultancy, Honoraria; Glaxo Smith Kline: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; BMS-Celgene: Consultancy, Honoraria, Speakers Bureau. Martínez-López: Janssen, BMS, Novartis, Incyte, Roche, GSK, Pfizer: Consultancy; Roche, Novartis, Incyte, Astellas, BMS: Research Funding. Lahuerta: Celgene: Other: Travel accomodations and expenses; Celgene, Takeda, Amgen, Janssen and Sanofi: Consultancy. Rosinol: Janssen, Celgene, Amgen and Takeda: Honoraria. Bladé Creixenti: Janssen, Celgene, Takeda, Amgen and Oncopeptides: Honoraria. Mateos: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria; Bluebird bio: Honoraria; GSK: Honoraria; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sea-Gen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene - Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Regeneron: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel: AbbVie, Amgen, Bristol-Myers Squibb, Celgene, GlaxoSmithKline, Janssen, Karyopharm, Merck Sharpe & Dohme, Novartis, Regeneron, Roche, Sanofi, SecuraBio, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Paiva: Bristol-Myers Squibb-Celgene, Janssen, and Sanofi: Consultancy; Adaptive, Amgen, Bristol-Myers Squibb-Celgene, Janssen, Kite Pharma, Sanofi and Takeda: Honoraria; Celgene, EngMab, Roche, Sanofi, Takeda: Research Funding.

Abstract: Background: Novel insights into the biology of myeloma cells have led to the identification of relevant prognosis factors. CA has become one of the most important prognostic factors, and the presence of t(4;14), t(14;16) or del(17p) are associated with poor prognosis. Although there are some reports indicating that 1q gains may be considered as a poor-risk feature, the information is not uniform. Furthermore, there are important controversies about whether or not novel agents-based combinations are able to overcome the poor prognosis of CA. Bortezomib-based combinations have shown to improve the outcome of patients with high-risk CA but they do not completely overcome their adverse prognosis. Here we report a preplanned analysis, in a series of elderly newly diagnosed myeloma patients included in the Spanish GEM2010 trial and receiving VMP and Rd, in a sequential or alternating approach, in order to evaluate the influence of CA by FISH on the response rate and outcome. Patients and methods: 242 pts were randomized to receive a sequential scheme consisting on 9 cycles of VMP followed by 9 cycles of Rd or the same regimens in an alternating approach (one cycle of VMP alternating with one Rd, up to 18 cycles. VMP included the iv administration of weekly bortezomib (except in the first cycle that was given twice weekly) at 1.3 mg/m2 in combination with oral melphalan 9 mg/m2 and prednisone 60 mg/m2 once daily on days 1-4. Rd treatment consisted on lenalidomide 25 mg daily on days 1-21 plus dexamethasone 40 mg weekly. FISH analysis for t(4;14), t(14;16), del(17p) and 1q gains was performed at diagnosis according to standard procedures using purified plasma cells. Results: In 174 out of the 233 patients evaluable for efficacy and safety, FISH analysis at diagnosis were available and two groups were identified: high-risk group (n= 32 patients with t(4;14) and/or t(14;16) and/or del(17p)) and standard-risk group (n=142 patients without high-risk CA). There weren't differences in the rates of CA according to the treatment arm. Response Rates (RR) were no different in the high-risk vs standard-risk groups, both in the sequential (74% vs 79% RR and 42% vs 39% CR) and alternating arms (69% vs 86% RR and 39% vs 38% CR). After a median follow-up of 37 months, high-risk patients showed shorter PFS as compared to standard risk in the alternating arm (24 versus 36 months, p=0.01, HR 2.2, 95% IC 1.1-4.2) and this also translated into a significantly shorter 4-years OS (27% vs 72%, p=0.006, HR 3.3, 95% IC 1.4-7.7). However, in the sequential arm, high-risk and standard-risk patients showed similar PFS (32 months vs 30 months) and 4-years OS (64% vs 60%). This effect was observed only in the sequential arm applied to either t(4;14) or del(17p). As far as 1q gains is concerned, 151 patients had 1q information and 76 of them had 1q gains (50.3%), defined as the presence of more than 3 copies in at least 10% of plasma cells. The rate of 1q gains was well balanced in both sequential and alternating arms. The ORR was similar in patients with or without 1q gains (83% vs 80%) as well as the CR rate (45% vs 31%), and no differences were observed between sequential and alternating arms. Patients with or without 1q gains had a similar PFS (33 months vs 30 months) and 4-years OS (58% vs 65%) in the whole series and no differences were observed in the sequential and alternating arms. This effect has been observed in patients with 1q gains as isolated CA and the outcome was slightly but not significantly worse when 1q gains were present plus either t(4;14) and/or del17p. Conclusions: The total therapy approach including VMP and Rd administered in a sequential approach is able to overcome the poor prognosis of the presence of high-risk CA in elderly patients with newly diagnosed MM. The presence of 1q gains has no impact in the PFS and OS of elderly patients treated with VMP and Rd. Disclosures Mateos: Celgene: Consultancy, Honoraria; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria; Takeda: Consultancy. Gironella:Celgene Corporation: Consultancy, Honoraria. Paiva:BD Bioscience: Consultancy; Binding Site: Consultancy; Sanofi: Consultancy; EngMab AG: Research Funding; Onyx: Consultancy; Millenium: Consultancy; Janssen: Consultancy; Celgene: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. San Miguel:Millennium: Honoraria; Janssen-Cilag: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Onyx: Honoraria; Sanofi-Aventis: Honoraria.

Abstract: Background: MRD is an established biomarker to evaluate treatment efficacy, define patients at risk based on persistent MRD, and eventually, act as surrogate for prolonged survival based on sensitive MRD-negative definitions. Accordingly, the IMWG has developed criteria for MRD-negativity defined by next-generation sequencing, NGF or PET/CT, and has recommended their inclusion in clinical trials. Notwithstanding, most flow cytometry results have been obtained using less sensitive methods and in fact, there is no data about the impact of NGF-based MRD assessment in clinical trials. Aim: To define the feasibility, sensitivity and clinical impact of NGF-based MRD assessment in the phase III PETHEMA/GEM2012 trial. Methods: A total of 458 patients were enrolled into the PETHEMA/GEM2012 trial. MRD was predefined to be prospectively assessed at three time-points: after six induction cycles with bortezomib, lenalidomide, and dexamethasone (VRD), after HDT/ASCT, and after two courses of consolidation with VRD. MRD monitoring was performed blinded for clinical outcomes in four PETHEMA/GEM laboratory cores, and data was centralized for MRD analyses. MRD assessment was performed following EuroFlow SOPs in a total of 1,134 bone marrow (BM) samples from 419 patients. The 39 cases without MRD assessment had suboptimal response to induction and were thus considered as MRD+ for intention-to-treat analyses. Noteworthy, in 14 BM samples with undetectable MRD, B-cell precursors, erythroblasts and mast cells represented & lt;0.01% of BM cells, and these samples were thus considered as hemodiluted and inadequate for MRD assessment. The limit of detection (LOD) was determined for each of the 1,117 BM samples representative for MRD assessment, according to the formula: (20/nucleated viable cells) x 100; the median LOD achieved by NGF in the PETHEMA/GEM2012 trial was of 3x10-6. Results: Overall, 225/458 (49%) patients had undetectable MRD at the latest time-point in which MRD was assessed and were thus classified as MRD-. Conversely, 233/458 (51%) cases remained MRD+: 28% with ≥10-4 MRD, 12% with 10-5 MRD, and 11% with 10-6 MRD. Detailed analyses of MRD kinetics in 320 patients with available MRD results at all three time-points, showed that the percentage of MRD- patients increased from 35% into 54% and 58% after induction, HDT/ASCT and consolidation, respectively. Furthermore, a restricted analysis among MRD+ patients showed that whereas after induction only 8% of them had MRD levels as low as 10-6, subsequent intensification with HDT/ASCT and consolidation could reduce MRD levels down to 10-6 in 32% of MRD+ cases. Progression-free survival (PFS) rates at 3-years were of 92%, 70%, 54% and 44% for patients being MRD-negative, MRD+ 10-6, 10-5 and ≥10-4, respectively (P & lt;.001; Figure). Thus far, only 6/225 (3%) MRD- patients have relapsed; strikingly, all 6 cases had extramedullary plasmacytomas at diagnosis, all relapsed with extramedullary plasmacytomas, and only 2 had concomitant serological relapse. The favorable outcome of MRD- patients encouraged us to investigate the impact of MRD negativity in both standard- and high-risk patients defined by FISH [i.e.: t(4;14), t(14;16), and/or del(17p)]. Even though MRD- rates were significantly inferior in patients with high- vs standard-risk FISH (37% vs 50%, respectively; P=.03), 3-year PFS rates were similar between patients with high- and standard-risk FISH reaching MRD-negativity (94% and 91%, respectively; P=.56); by contrast, MRD+ cases with high- and standard- risk FISH had median PFS of 27 and 35 months, respectively (P=.025). Conclusions: This is the largest study of MRD monitoring in MM based on the total number of samples analyzed (n=1,134). Our results show that NGF-based MRD assessment is feasible in large multicenter clinical trials, is highly-sensitive, and allows the identification of hemodiluted BM samples inadequate for MRD assessment. Risk of relapse among MRD-negative patients was remarkably reduced (3%), and was particularly related to the reappearance of extramedullary plasmacytomas, which urges the need for combined cellular and imaging MRD monitoring in these patients; by contrast, even MRD levels as low as 10-5 and 10-6 conferred significantly inferior PFS. Overall, this study defines MRD-negativity as the most relevant clinical endpoint for both standard- and high-risk transplant-eligible MM patients. Figure Figure. Disclosures Paiva: Sanofi: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria; Merck: Honoraria; Novartis: Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; EngMab: Research Funding. Oriol: Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored symposia, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored symposia, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored symposia; Celgene: Speakers Bureau. de la Rubia: Janssen: Other: Honoraria; Amgen: Other: Honoraria; Celgene: Other: Honoraria. Rosinol: Celgene: Honoraria; Janssen: Honoraria. Mateos: Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Lahuerta: Amgen: Honoraria; Celgene: Honoraria; Janssen: Honoraria. San Miguel: Roche: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; MSD: 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; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees.