Abstract: The International Staging System (ISS) and the Revised International Staging System (R-ISS) are commonly used prognostic scores in multiple myeloma (MM). These methods have significant gaps, particularly among intermediate-risk groups. The aim of this study was to improve risk stratification in newly diagnosed MM patients using data from three different trials developed by the Spanish Myeloma Group. For this, we applied an unsupervised machine learning clusterization technique on a set of clinical, biochemical and cytogenetic variables, and we identified two novel clusters of patients with significantly different survival. The prognostic precision of this clusterization was superior to those of ISS and R-ISS scores, and appeared to be particularly useful to improve risk stratification among R-ISS 2 patients. Additionally, patients assigned to the low-risk cluster in the GEM05 over 65 years trial had a significant survival benefit when treated with VMP as compared with VTD. In conclusion, we describe a simple prognostic model for newly diagnosed MM whose predictions are independent of the ISS and R-ISS scores. Notably, the model is particularly useful in order to re-classify R-ISS score 2 patients in 2 different prognostic subgroups. The combination of ISS, R-ISS and unsupervised machine learning clusterization brings a promising approximation to improve MM risk stratification.

Abstract: Assessing measurable residual disease (MRD) has become standard with many tumors, but the clinical meaning of MRD in multiple myeloma (MM) remains uncertain, particularly when assessed by next-generation flow (NGF) cytometry. Thus, we aimed to determine the applicability and sensitivity of the flow MRD-negative criterion defined by the International Myeloma Working Group (IMWG). PATIENTS AND METHODS In the PETHEMA/GEM2012MENOS65 trial, 458 patients with newly diagnosed MM had longitudinal assessment of MRD after six induction cycles with bortezomib, lenalidomide, and dexamethasone (VRD), autologous transplantation, and two consolidation courses with VRD. MRD was assessed in 1,100 bone marrow samples from 397 patients; the 61 patients without MRD data discontinued treatment during induction and were considered MRD positive for intent-to-treat analysis. The median limit of detection achieved by NGF was 2.9 × 10 −6 . Patients received maintenance (lenalidomide ± ixazomib) according to the companion PETHEMA/GEM2014MAIN trial. RESULTS Overall, 205 (45%) of 458 patients had undetectable MRD after consolidation, and only 14 of them (7%) have experienced progression thus far; seven of these 14 displayed extraosseous plasmacytomas at diagnosis and/or relapse. Using time-dependent analysis, patients with undetectable MRD had an 82% reduction in the risk of progression or death (hazard ratio, 0.18; 95% CI, 0.11 to 0.30; P 〈 .001) and an 88% reduction in the risk of death (hazard ratio, 0.12; 95% CI, 0.05 to 0.29; P 〈 .001). Timing of undetectable MRD (after induction v intensification) had no impact on patient survival. Attaining undetectable MRD overcame poor prognostic features at diagnosis, including high-risk cytogenetics. By contrast, patients with Revised International Staging System III status and positive MRD had dismal progression-free and overall survivals (median, 14 and 17 months, respectively). Maintenance increased the rate of undetectable MRD by 17%. CONCLUSION The IMWG flow MRD-negative response criterion is highly applicable and sensitive to evaluate treatment efficacy in MM.

Abstract: Monitoring of the monoclonal protein (M-protein) by electrophoresis and/or immunofixation (IFE) has long been used to assess treatment response in multiple myeloma (MM). However, with the use of highly effective therapies, the M-protein becomes frequently undetectable, and more sensitive methods had to be explored. We applied IFE and mass spectrometry (EXENT & FLC-MS) in serum samples from newly diagnosed MM patients enrolled in the PETHEMA/GEM2012MENOS65 obtained at baseline (n = 223), and after induction (n = 183), autologous stem cell transplantation (n = 173), and consolidation (n = 173). At baseline, the isotypes identified with both methods fully matched in 82.1% of samples; in the rest but 2 cases, EXENT & FLC-MS provided additional information to IFE with regards to the M-protein(s). Overall, the results of EXENT & FLC-MS and IFE were concordant in & gt;80% of cases, being most discordances due to EXENT & FLC-MS+ but IFE− cases. After consolidation, IFE was not able to discriminate 2 cohorts with different median progression-free survival (PFS), but EXENT & FLC-MS did so; furthermore, among IFE− patients, EXENT & FLC-MS identified 2 groups with significantly different median PFS (P = .0008). In conclusion, compared with IFE, EXENT & FLC-MS is more sensitive to detect the M-protein of patients with MM, both at baseline and during treatment, and provides a more accurate prediction of patients’ outcome. This trial was registered at www.clinicaltrials.gov as #NCT01916252.

Abstract: Introduction : In patients (pts) with multiple myeloma (MM), next generation flow cytometry (NGF) and next generation sequencing have shown an increased capacity to identify the presence of disease and to anticipate patient's prognosis as compared to serum protein immunofixation (IFE). However, both methods rely on bone marrow (BM) samples and it is important to explore alternative techniques applicable in more accessible samples such as peripheral blood. Patients and Methods: Newly diagnosed MM pts enrolled in the PETHEMA/GEM2012MENOS65 trial received six cycles of induction with bortezomib, lenalidomide and dexamethasone (VRD), intensification with high-dose therapy (melphalan or busulfan and melphalan) followed by autologous stem cell transplantation and consolidation with two more cycles of VRD. At the end of the treatment (post-consolidation), the M-protein (MP) was analyzed in serum by conventional IFE and by EXENT Quantitative Immunoprecipitation Mass Spectrometry using IgG/A/M, κ, λ, free κ and free λ isotypic beads; negative samples by EXENT were re-analyzed by liquid chromatography mass spectrometry (LC-MS). The presence of clonal plasma cells in BM samples was investigated by NGF following the Euroflow guidelines (sensitivity≥10 -5). Samples from the first 164 pts enrolled in the trial were analyzed in this study. Results : After consolidation, persistent disease was detected in 42 (26%) pts by IFE, in 56 (34%) by EXENT and in 72 (44%) by NGF. Surprisingly, the presence or absence of disease by IFE did not discriminate pts with different progression-free survival (PFS), but both EXENT and NGF segregated two cohorts with a significantly different PFS (p=0.0016 and p & lt;0.0001, respectively; fig1A and 1C). Importantly, we observed that among pts in complete response (IFE negative), EXENT and NGF also distinguished two groups with different PFS (p=0.002 and p=0.0001, respectively.) Analyzing the results obtained with EXENT and NGF, we found that 76% were concordant (44 EXENT +/NGF +, 80 EXENT -/NGF -) and 24% discordant (28 EXENT -/NGF + and 12 EXENT +/NGF -). When we compared pts´ PFS according the combined results of both methods (fig 1D), we learnt that the 3 comparisons reaching statistical significance were EXENT +/NGF + vs EXENT -/NGF - (p & lt;0.0001), EXENT +/NGF + vs EXENT +/NGF - (p=0.0394) and EXENT -/NGF - vs EXENT -/NGF + (p=0.0363). Considering NGF as a reference, the negative predictive value (NPV) of EXENT was 74% overall (96% in MRD levels ≥10 -4, p & lt;0.0001; 89% in & lt;10 -4 - ≥10 -5 p & lt;0.0001; and 84% in ≥10 -6 cases, p=0.0524). Samples deemed negative by EXENT were re-analyzed with LC-MS. In 63 of them (58%) the MP was identified using LC-MS and therefore a total of 119 samples (73%) were considered positive with EXENT & LC-MS. We first confirmed that EXENT & LC-MS segregated two cohorts with different PFS (fig 1B, p=0.0193) Then, as earlier, we analyzed the results obtained with EXENT & LC-MS and NGF, finding that 65% were concordant (67 EXENT & LC-MS +/NGF +, 40 EXENT & LC-MS -/NGF -) and 35% discordant (5 EXENT & LC-MS -/NGF + and 52 EXENT & LC +/NGF -). Again, we compared pts´ PFS according the combined results of both methods (fig 1E) learning now that only the comparisons EXENT & LC-MS +/NGF + vs EXENT & LC-MS -/NGF - (p=0.0006) and EXENT & LC-MS +/NGF + vsEXENT & LC-MS +/NGF - (p=0.0006) reached statistical significance. With these results, the NPV of EXENT & LC-MS was 89% overall (100% in MRD levels ≥10 -4 p & lt;0.0001; 100% in & lt;10 -4 - ≥10 -5 p & lt;0.0001; and 89% in ≥10 -6 cases p=0.0914). Finally, we observed that whereas among pts deemed EXENT +, NGF identified 2 cohorts with different PFS (a NGF -group of 12 pts displaying a longer PFS as compared with those NGF +; median PFS: 4 years vs not reached, p=0.039) among EXENT & LC-MS - cases (n=45) NGF was not able to show any added clinical value. Conclusions : The use of IFE post-consolidation in pts with MM, as opposed to EXENT and NGF, did not identify pts with different PFS. When referred to NGF, EXENT provided a similar clinical value and displayed a very high NPV, increased further with the addition of LC-MS; this could be utilised to avoid the performance of a BM aspiration after treatment in pts with undetectable disease. Regarding EXENT + and/or LC-MS + cases future quantitative and sequential studies could reveal whether are due to the long half-lives of the MP or represent quiescent low-level disease. Figure 1 Figure 1. Disclosures Puig: Amgen, Celgene, Janssen, Takeda and The Binding Site: Honoraria; Amgen, Celgene, Janssen, Takeda: Consultancy; Celgene: Speakers Bureau; Celgene, Janssen, Amgen, Takeda: Research Funding. 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. Cedena: Janssen, Celgene and Abbvie: Honoraria. Rosinol: Janssen, Celgene, Amgen and Takeda: Honoraria. Martínez-López: Roche, Novartis, Incyte, Astellas, BMS: Research Funding; Janssen, BMS, Novartis, Incyte, Roche, GSK, Pfizer: Consultancy. Oriol: 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; Sanofi: Consultancy, Honoraria, 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. Sureda: Bluebird: Membership on an entity's Board of Directors or advisory committees; Kite, a Gilead Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, 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; GSK: Consultancy, Honoraria, Speakers Bureau; Roche: Other: Support for attending meetings and/or travel; 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; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Mundipharma: Consultancy; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Moraleda: Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Gilead: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Novartis: Consultancy, Honoraria, Other: Educational Grants, Research Funding; Sandoz: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Other: Educational Grants, Research Funding; ROCHE: Consultancy, Honoraria, Other: Educational Grants, Research Funding; MSD: Other: Educational Grants, Research Funding; Sanofi: Other: Educational Grants, Research Funding; Pfizer: 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. Bladé Creixenti: Janssen, Celgene, Takeda, Amgen and Oncopeptides: Honoraria. 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. Mateos: Regeneron: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria; 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; Oncopeptides: 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; Janssen: 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; 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; 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; Bluebird bio: Honoraria; GSK: Honoraria; Oncopeptides: Honoraria.

Abstract: Background: Transplant-eligible MM patients are achieving unprecedented CR rates with frontline therapy. This urges the question about what other tests are informative upon a negative immunofixation (IFx-), as well as if patients with short duration CR continue having dismal survival with modern frontline plus salvage therapies and if so, how to predict risk of unsustained CR. Aim: To provide an optimal definition of unsustained CR and biomarkers to predict it in transplant-eligible MM patients treated with optimal therapy. Methods: A total of 262 patients enrolled in the PETHEMA/GEM2012MENOS65 trial and who were in CR after receiving six induction cycles of bortezomib, lenalidomide and dexamethasone (VRD), autologous transplant and two consolidation cycles of VRD, were included in this study. Afterwards, patients were enrolled in the PETHEMA/GEM2014MAIN trial. Median follow-up of the series was 38 months after consolidation (53 months since diagnosis). Serum free light-chains (sFLC) were measured in 252 cases. MRD was assessment with next-generation flow (NGF) in 257 patients (median limit of detection of 2.8x10-6). FISH was performed in CD138-enriched plasma cells (PCs) from 223 patients at diagnosis [high-risk was defined by the presence of t(4;14), t(14;16) and/or del(17p)]. To understand the relationship between duration of CR and outcome, patients were segmented into 6-monht increments (range, 0 - 48 months) in time since response assessment after consolidation and loss of CR. Results: We first investigated what other tests commonly performed upon the achievement of IFx- were informative, particularly those employed to define stringent CR. The median percentage of PCs by morphology, at the time of IFx-, was 1.7% (range 0-5). Only 4 patients out of 266 (1.5%) with a negative immunofixation had & gt;5% BM PCs and therefore, were not classified as in CR. Almost one-fourth of patients in CR display an abnormal sFLC ratio (56/248, 23%), but their PFS was identical to that of cases with normal sFLC ratio (3 years-PFS rates of 70% vs 72%; P=.6). BM biopsies were not performed in this study to evaluate PC clonality by immunohistochemistry but we noted that in CR patients with persistent MRD, the median percentage of clonal and normal PCs among total PCs identified by NGF was of 3% and 97%, respectively. Thus, the median percentage of normal PCs is 24-fold greater than clonal PCs within the PC compartment, and therefore simple κ/λ ratios measured in ≥100 PCs are unable to detect such low-levels of residual disease. Indeed, persistent MRD was detectable by NGF in 73/252 (29%) CR patients at a median level of 0.03% (range 0.0002% - 0.59%), and resulted in significantly inferior PFS (3-year rates of 49% vs 83% in cases with persistent vs undetectable MRD, P & lt; .00001) and OS (3 years-PFS rates of 84% vs 95%; P=.001). Afterwards, we sought to identify the optimal landmark to define unsustained CR and to identify which biomarkers could identify patients at risk. A duration of CR of & lt;24 months emerged as optimal criteria to identify a numerically relevant subset of patients (39/262, 15%) with dismal OS (median of 48 months vs not reached in patients with duration of CR ≥24 months, P & lt; .00001). We then performed a logistic regression to identify biomarkers with independent value to predict unsustained CR. Elevated LDH levels (HR: 2.9 [1.1 - 8.0], P =.038) and & gt;20% PCs (HR: 2.8 [1.2 - 6.7], P =.020) at diagnosis together with persistent MRD (HR: 4.3 [1.9 - 9.6] , P & lt;.001) had independent value to predict unsustained CR in a multivariable analysis. Thus, a scoring system with the three parameters yielded significant stratification of patients in CR into favorable (no risk factors), intermediate (any risk factor) and dismal (all risk factors) PFS (3-year rates of 92%, 67% and 0%, respectively; P & lt;.0001; Figure 1) and OS (3-year rates of 99%, 90% and 83%, respectively; P=.001). Conclusions: This is the first study evaluating which baseline and response assessments are useful to stratify transplant-eligible patients with unsustained CR in the context of modern therapy; a simple model based on LDH levels and PC counts at diagnosis plus MRD status was identified and can be used broadly. These findings are clinically meaningful because patients with unsustained CR remain a high-risk population despite optimal therapy, and those at risk should be offered alternative treatment strategies before insurmountable disease progression occurs. Figure 1 Disclosures Paiva: SkylineDx: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Roche: Research Funding; Adaptive: Honoraria; Amgen: Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Kite: Consultancy; Sanofi: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau. Oriol:Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy; Celgene: Consultancy, Speakers Bureau. Sureda Balari:Roche: Honoraria; Celgene: Consultancy, Honoraria; BMS: Speakers Bureau; Incyte: Consultancy; Janssen: Consultancy, Honoraria; Gilead/Kite: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Merck Sharpe and Dohme: Consultancy, Honoraria, Speakers Bureau; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau. de la Rubia:Janssen: Consultancy, Other: Expert Testimony; Amgen: Consultancy, Other: Expert Testimony; Celgene: Consultancy, Other: Expert Testimony; Ablynx/Sanofi: Consultancy, Other: Expert Testimony. Moraleda:Takeda: Consultancy, Other: Travel Expenses; Sandoz: Consultancy, Other: Travel Expenses; Novartis: Consultancy, Other: Travel Expenses; Gilead: Consultancy, Other: Travel Expenses; Jazz Pharmaceuticals: Consultancy, Research Funding. Mateos:Pfizer: 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; GlaxoSmithKline: Honoraria; Seattle Genetics: 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; Amgen: 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; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: 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; Adaptive: 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. San-Miguel:GlaxoSmithKline: 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; Roche: 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; MSD: 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; AbbVie: 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; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); 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.

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