Abstract: Background: Since survival in AL mainly depends on the extent of organ involvement of patients at presentation, early diagnosis and risk stratification are key to improve patients' outcome. Therefore, together with surrogates of organ involvement, biomarkers identifying patients with MGUS or MM at greater risk of developing AL would be highly valuable to prevent organ damage, to maximize therapeutic efficacy and to improve outcomes in AL. Aim: To investigate the value of multidimensional flow cytometry (MFC) for simultaneous fast diagnostic screening of plasma cell (PC) clonality and risk stratification, as well as to identify immunophenotypic markers useful for the selection of patients with monoclonal gammopathies candidates for monitoring of pre-symptomatic organ damage related to AL. Methods: We used MFC to characterize a large series of patients with newly-diagnosed (ND) AL (N=94) vs MGUS (N=20) and NDMM (N=52), as well as age-matched healthy adults (HA, N=30). For each patient with AL, automated risk stratification was performed using principal component analysis (PCA) based on the relative frequency of bone marrow (BM) PCs, plus the percentage of clonal and normal PCs within the whole BM PC compartment, vs a database containing information on the same three parameters from a total of 1,774 patients, including 497 MGUS and 1,227 NDMM. In parallel, immunophenotypic protein expression profiles (iPEP) of AL patients were clustered using t-SNE, and the comparison between the iPEP of clonal PCs from patients with AL vs MGUS and MM cases was performed using canonical-correlation analysis (CCA). To identify additional immunophenotypic hallmarks of AL, the BM cellular composition in HA, MGUS, AL and MM patients was compared using 2-dimensional minimum spanning tree (MST) force-directed classification to determine the distance among individual cases. Results: PC clonality was detected by MFC in 93/94 (99%) AL patients, whereas an M-component was detectable in 96% of cases by electrophoresis, immunofixation and sFLC. PCA as defined above, identified AL patients displaying an MM-like (n=6) and an MGUS-like (n=38) signature, as well as 49 cases with an intermediate signature between the MGUS and MM reference datasets. Multivariate analysis of baseline prognostic factors for survival, including patients' age, number of organs involved, Mayo staging, the percentage of BM PCs based on cytomorphology and eligibility for ASCT, showed that having an intermediate- or an MM-like profile had an independent adverse effect on patients' progression-free (PFS) and overall survival (OS) (HR:3.4; P≤.02). t-SNE based on the iPEP of clonal PCs revealed two major clusters of AL patients with significantly different PFS, defined by opposite patterns of expression for CD45, CD56 and CD138 (P≤.02). CCA of tumor iPEP showed partial overlap between AL vs MGUS and MM, with progressively higher percentages of cases with a CD38lo, CD45-ve, CD81-ve and CD138lo iPEP being observed from MGUS to AL and MM. In contrast, AL patients displayed significantly lower reactivity for CD56 (P≤ .03). Further characterization of the BM cellular composition allowed the systematic assessment of 16 cell populations and 18 phenotypic parameters that, by MST, mapped AL in between MGUS and MM. Of note, while AL patients displayed a predominantly-clonal PC compartment in the absence of an MM-like tumor PC expansion, the percentage of B-cell precursors was consistently lower in AL patients than in HA, MGUS and MM (P=.004). Thus, using optimal cut-off values to discriminate between AL vs MGUS and MM, we built a scoring model based on the presence of 〈 100% CD56+ve clonal PCs, 〈 0.1% B-cell precursors, 〉 80% clonal PCs within total BM PCs and 〈 2% BM PCs. Overall, a significant (P 〈 .001) association was found between a progressively higher score and the diagnosis of AL, with a 74% accurate classification based on ROC analysis (AUC of 0.74; 95% CI = 0.66 - 0.82; P 〈 .001) of the performance of the scoring model. Conclusions: We demonstrate the value of MFC for fast diagnostic screening of PC clonality in AL and simultaneous automated patient risk-stratification, based on the BM tumor burden and PC phenotype. In addition, our results also provide new immunophenotypic markers for the identification of patients with monoclonal gammopathies that are candidates for monitoring of pre-symptomatic organ damage related to AL. Disclosures Puig: Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Celgene: Honoraria, Research Funding. Ocio:Array Pharmaceuticals: Research Funding; Sanofi: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Pharmamar: Consultancy; BMS: Consultancy; AbbVie: Consultancy; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy; Mundipharma: Research Funding; Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Oriol:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. De La Rubia:Ablynx: Consultancy, Other: Member of Advisory Board. Martinez Lopez:Janssen: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau. Mateos:Amgen: 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; GSK: 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; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, 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. Lahuerta:Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria.

Abstract: Background: The transformation from a normal to a cancer cell is driven by the multistep acquisition of genetic alterations. Recently, shared mutations between clonal B cells in MBL/CLL and CD34+ hematopoietic progenitor cells (HPC) have been identified. Similarly, a HPC origin of BRAFV600E mutations in hairy cell leukemia (HCL) has been uncovered, strengthening the notion that at least a fraction of somatic mutations may occur in CD34+ HPC before the malignant transformation of some B cell neoplasms. Since almost all WM patients have mutated MYD88L265P, it is worthy to investigate if this disease follows a similar pathogenic process than that of MBL/CLL or HCL. Aim: Define the cellular origin of WM by comparing the genetic landscape of WM cells to that of CD34+ HPC, B cell precursors and residual normal B cells. Methods: We used FACSorting to isolate 57 cell subsets from bone marrow (BM) aspirates of 10 WM patients: CD34+ HPC, B cell precursors, residual normal B cells (if detectable), WM B cells, plasma cells (PCs) and T cells (germline control). Whole-exome sequencing (WES, mean depth 79x) was performed with 10XGenomics Exome Solution for low DNA-input due to limited numbers of some cell types. Single-cell RNA and B-cell receptor sequencing (scRNA/BCRseq) was performed in total BM B cells and PCs (n=32,720) from 3 IgM MGUS and 2 WM patients. Accordingly, the clonotypic BCR detected in WM cells was unbiasedly investigated in all B cell maturation stages defined according to their molecular phenotype. In parallel, MYD88p.L252P (orthologous position of the human L265P mutation) transgenic mice were crossed with conditional Sca1Cre, Mb1Cre, and Cγ1Cre mice to selectively induce in vivo expression of MYD88 mutation in CD34+ HPC, B cell precursors and germinal center B cells, respectively. Upon immunization, mice from each cohort were necropsied at 5, 10 and 15 months. Results: All 10 WM patients showed MYD88L265P and 3 had mutated CXCR4. Notably, we found MYD88L265P in B cell precursors from 1/10 cases and in residual normal B cells from 4/10 patients, which were confirmed by ASO-PCR and ddPCR. Indeed, these more sensitive methods detected MYD88L265P in B cell precursors from 6/10 cases and in residual normal B cells from 6/10 patients. CXCR4 was simultaneously mutated in B cell precursors and WM B cells from one patient. Overall, CD34+ HPC, B-cell precursors and residual normal B cells shared a median of 2 (range, 0-45; mean VAF, 0.13), 3 (range, 1-44; mean VAF, 0.168), and 6 (range, 1-56; mean VAF, 0.29) somatic mutations with WM B cells; some being found all the way from CD34+ HPC to WM B cells and PCs. Interestingly, concordance between the mutational landscape of WM B cells and PCs was & lt;100% (median of 79%, range: 55%-100%), suggesting that not all WM B cells differentiate into PCs. A median of 18 mutations (range, 3-26; median CCF and range, 0.72 [0.07 - 1]) were unique to WM cells. Importantly, clonal mutations in WM B cells were undetectable in normal cells. Thus, the few WM subclonal mutations observed in patients' lymphopoiesis could not result from contamination during FACSorting since in such cases, WM clonal mutations would become detectable in normal cells. Furthermore, copy number alterations (CNA) present in WM cells were undetectable in normal cells. scRNA/BCRseq unveiled that clonotypic cells were confined mostly within mature B cell and PC clusters in IgM MGUS, whereas a fraction of clonotypic cells from WM patients showed a transcriptional profile overlapping with that of B cell precursors. scRNA/BCRseq also uncovered transcriptional differences between clonal B cells from IgM MGUS vs WM patients (eg, proliferation, metabolism). In mice, induced expression of mutated MYD88 led to a moderate increase in the number of B220+CD138+ plasmablasts and B220-CD138+ PCs in lymphoid tissues and BM, but no signs of clonality or hematological disease. Interestingly, such increment was more evident in mice with activation of mutated MYD88 in CD34+ HPC and B-cell precursors vs mice with MYD88 L252P induced in germinal center B cells. Conclusions: We show for the first time that WM patients have somatic mutations, including MYD88L265P and CXCR4 at the B cell progenitor level. Taken together, this study suggests that in some patients, WM could develop from B cell clones carrying MYD88L265P rather than being the initiating event, and that other mutations or CNA are required for the expansion of B cells and PCs with the WM phenotype. Disclosures Motta: Roche: Honoraria; Janssen: Honoraria. Rossi:Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Advisory board; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Garcia-Sanz:Takeda: Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Gilead: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Amgen: Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Self: Patents & Royalties: BIOMED-2 PRIMERS FOR CLONALITY ASSESSMENT; IVS technologies: Consultancy, Patents & Royalties; Novartis: Research Funding. Roccaro:Transcan2-ERANET: Research Funding; European Hematology Association: Research Funding; Amgen: Other; AstraZeneca: Research Funding; Celgene: Other; Janssen: Other; Italian Association for Cancer Research (AIRC): Research Funding. 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. Paiva:Sanofi: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; SkylineDx: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Roche: Research Funding; Adaptive: Honoraria; Amgen: Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Kite: Consultancy.

Abstract: MYD88 L265P occurs in between a normal mutated lymphopoiesis and additional genetic alterations during lymphomagenesis.

Abstract: Background: SMM is a plasma cell (PC) disorder defined by the presence of at least 10% of PC and/or a serum M-component (MC) at least 3g/dl without end-organ damage. Due to the nature of the disease, SMM pts at high risk of progression to active MM ( 〉 50% at 2 yrs) have been identified using different criteria: bone marrow infiltration by 〉 10% of PCs & MC 〉 3g/dl (Kyle,NEJM 2007), or 〉 95% aberrant PC (aPC) by immunophenotyping plus immunoparesis (Pérez, Blood 2007), or abnormal FLCs (Dispenzieri,Blood 2008). The current standard of care for SMM is watchful waiting until disease progression. Although several small randomized studies have explored the value of early treatment with either conventional agents (melphalan/prednisone) or thalidomide, bisphosphonates, they showed no significant benefit. It should be noted that these trials did not focus on high-risk SMM. In 2007, the Spanish Myeloma Group initiated a randomized, phase III trial comparing lenalidomide (R) + low-dose dexamethasone (Rd) vs observation in high-risk SMM pts. After a median follow-up of 40 months, early treatment with Rd showed to be superior in terms of time to progression to active disease and overall survival (OS) (Mateos, NEJM 2014). Here we present an update after long-term median follow-up of 5 years. Pts and Methods: 119 pts with high-risk SMM were enrolled and randomized to Rd vs. observation. Pts in the treatment group received an induction regimen (lenalidomide 25 mg/day on days 1 to 21 plus dexamethasone 20 mg/day on days 1 to 4 and 12 to 15, at 4-week intervals for nine cycles) followed by a maintenance regimen (lenalidomide 10 mg/day on days 1 to 21 of each 28-day cycle). Maintenance therapy was initially given until disease progression, but an amendment limited the total treatment duration (induction plus maintenance) to 2 years, and the addition of dexamethasone (20 mg on days 1 to 4 of each cycle) for pts who developed asymptomatic biological progression during maintenance ( 〉 25% of increase in monoclonal component with no symptoms). The primary endpoint was time to progression to symptomatic disease (TTP), secondary endpoints included OS, response, and safety. Results: After a median follow-up of 64 months (range: 49-81), progression to symptomatic disease occurred in 23% of pts with Rd vs. 85% for the pts in the observation arm. Long term follow-up with early Rd treatment continues to show a significant benefit in TTP to active disease vs. watchful waiting (median TTP not reached vs. 21 months, HR= 6.21; 95% CI: 3.1-12.7, p 〈 0.0001). During maintenance therapy, 24 pts in the Rd arm experienced asymptomatic biological progression and low-dose dexamethasone was added in 18 pts. With a median follow-up of 50 months from biological progression, disease remains under control in 10 out of these 18 pts and they continue on therapy with lenalidomide plus dexamethasone at low doses. Pts who progressed to symptomatic disease and required to start systemic therapy were also followed for survival, and the percentage of pts who remain alive at 5 years after progression to active disease is 83% in the Rd vs. 58% in the observation arm. OS continues to be significantly longer with Rd vs. with the observation arm: 93% of the pts who received early treatment with Rd remain alive vs. 67% in the watchful waiting arm, (HR= 4.35, 95% CI 1.5-13.0, p=0.008). Only four pts died in the Rd arm, whilst 17 deaths occurred in the observation arm. In the Rd arm, only one patient’s death was considered treatment-related (pneumonia); disease progression was the cause of death in two pts and bronchoaspiration as consequence of surgery-related complication in the fourth pt. In the observation arm, disease progression was the most frequent cause of death, in 76% of the pts. As far as toxicity is concerned, no new second primary malignancy (SPM) occurred: four SPM were reported in 4 of the 62 pts with Rd (6%) and in 1 of the 63 pts in the observation group (2%). Conclusions: After long-term follow-up, Rd in high risk SMM pts as early treatment with Rd continued to show a significant reduction of not only in the risk of progression to active disease but also the risk of death. In addition, the long post relapse survival observed among pts who received early treatment with Rd and subsequently progressed to symptomatic disease indicate that this strategy does not induce more resistant clones. Disclosures Mateos: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Lenalidomide as first-line combination therapy for AL amyloidosis.. De La Rubia:Janssen: Honoraria; Celgene: Honoraria. Rosiñol:Janssen: Honoraria; Celgene: Honoraria. Oriol:Celgene Corporation: Consultancy. Blade:Janssen: Honoraria; Celgene: Honoraria. Lahuerta:Janssen: Honoraria; Celgene: Honoraria.

Abstract: Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation–related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies.

Abstract: MM patients are living longer with increasingly effective therapies, but long-term complications including second primary malignancies (SPMs) are becoming new challenges in designing optimal patient care. It has been demonstrated in large studies that amongst others, risk is particularly high for SPMs such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Importantly, such increased risk of MDS/AML has also been observed in MGUS patients, suggesting that increased risk for MDS/AML may not only be treatment related but inheritably high in MGUS/MM. Thus, there is need to investigate for biomarkers that uncover cellular alterations predisposing for higher risk of MDS/AML in MM. Here, we started by investigating in 312 newly diagnosed MM patients the presence of MDS-like phenotypic abnormalities (MDS-PA) in bone marrow (BM) neutrophil, monocytic, and erythroid lineages, using multidimensional flow cytometry 8 color combinations (CD138, CD27, CD38, CD56, CD45, CD19, CD117, CD81; and HLADR, CD45, CD36, CD13, CD34, CD117, CD11b, CD71). Up to 33/312 (11%) patients showed MDS-PA at diagnosis, which were more frequently observed in the neutrophil lineage (6%), followed by monocytic (4%) and erythroid (4%) lineages. Four cases had multilineage MDS-PA. Afterwards, we investigated if the presence of MDS-PA was associated with underlying somatic mutations by performing targeted sequencing of 54 MDS/AML related genes (depth ≥500x) in 44 patients from the previous series (10 with MDS-PA and 34 without). Next generation sequencing was performed, at diagnosis and after HDT/ASCT in FACS sorted CD34+ hematopoietic stem cells (HSCs) and dysplastic cell lineages from patients with MDS-PA, as well as in HSC from cases without MDS-PA. CD138+ BM plasma cells (PCs) from both cohorts were also sequenced using the same panel. Six out of the 10 cases with MDS-PA showed somatic mutations. Namely, HSCs from one patient had two mutations in TET2 [allele fraction (AF): 18%, ≥ 26017x] one in CALR (AF: 14%, 1158x) and another in ASXL1 (AF: 7%, 1339x). None of these mutations were present in myeloid/erythroid cells. A second patient had NPM1 mutated in HSCs (AF: 7%, 12825x), which was absent in neutrophils. A third case had TET2 mutated in HSCs (AF: 16%, 1233x) as well as in dysplastic monocytes (AF: 27%, 16647x) and neutrophils (AF: 23%, 21719x). In the fourth case, a mutation in BCORL1 was noted in dysplastic erythroid cells (AF: 10%, 796x). The fifth patient had TET2 mutated in both HSCs and dysplastic monocytes (AF: 45%-63%; ≥21799x). The sixth case had PHF6 mutated in HSCs (AF: 8%; 800x). In none of the patients were the mutations found in HSCs and/or dysplastic lineages, present in PCs. Within the control cohort of the 34 patients without MDS-PA, only two of them displayed somatic mutations in HSCs; one case had DNMT3A mutated (AF: 26%, 1900x) and the other TET2 (AF: 13%, 3400x). After demonstrating a correlation between MDS-PA and MDS/AML-related somatic mutations, we sought to analyze the prognostic significance of such alterations in MM. Since the follow-up of the present series of 312 cases is relatively short, we focused on a large series of 965 patients with longer follow up (median of 6.5 years) enrolled in GEM clinical trials, and for which the presence of CD56+ aberrant monocytes could be readily investigated. Noteworthy, this particular MDS-PA was again observed in a similar frequency as noted above (n=63; 6.5%) and as compared to the overall MM population, patients with MDS-PA showed significantly higher age, lower hemoglobin values and higher BMPC infiltration at diagnosis. Furthermore, they experienced more frequently hematological toxicity including anemia and neutropenia during treatment. Most interestingly, as compared to the overall MM population, patients with MDS-PA had significantly inferior progression-free (medians of 24 vs 37 months; P=.006) and overall survival (medians of 47 vs 73 months; P=.01). In conclusion, we showed for the first time that a fraction of newly diagnosed MM patients harbors MDS/AML-related somatic mutations in HSCs and myeloid/erythroid lineages, and that such patients could be predicted through flow-based screening for MDS-PA. The presence of MDS-PA identifies a subset of patients that experience more frequently hematological toxicity and display inferior survival; accordingly, screening for MDS-PA could become an important biomarker to tailor treatment in MM. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding. Oriol:Amgen: Honoraria, Other: Expert board committee; Janssen: Honoraria, Other: Expert board committee. Mateos:Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria.

Abstract: Early intervention in smoldering multiple myeloma (SMM) requires optimal risk stratification to avoid under- and overtreatment. We hypothesized that replacing bone marrow (BM) plasma cells (PC) for circulating tumor cells (CTC), and adding immune biomarkers in peripheral blood (PB) for the identification of patients at risk of progression due to lost immune surveillance, could improve the International Myeloma Working Group 20/2/20 model. Experimental Design: We report the outcomes of 150 patients with SMM enrolled in the iMMunocell study, in which serial assessment of tumor and immune cells in PB was performed every 6 months for a period of 3 years since enrollment. Results: Patients with & gt;0.015% versus ≤0.015% CTCs at baseline had a median time-to-progression of 17 months versus not reached (HR, 4.9; P  & lt; 0.001). Presence of & gt;20% BM PCs had no prognostic value in a multivariate analysis that included serum free light-chain ratio & gt;20, & gt;2 g/dL M-protein, and & gt;0.015% CTCs. The 20/2/20 and 20/2/0.015 models yielded similar risk stratification (C-index of 0.76 and 0.78). The combination of the 20/2/0.015 model with an immune risk score based on the percentages of SLAN+ and SLAN− nonclassical monocytes, CD69+HLADR+ cytotoxic NK cells, and CD4+CXCR3+ stem central memory T cells, allowed patient’ stratification into low, intermediate-low, intermediate-high, and high-risk disease with 0%, 20%, 39%, and 73% rates of progression at 2 years. Conclusions: This study showed that CTCs outperform BM PCs for assessing tumor burden. Additional analysis in larger series are needed to define a consensus cutoff of CTCs for minimally invasive stratification of SMM.