Abstract: Purpose: Immune checkpoint inhibitors have recently revolutionized cancer immunotherapy. On the basis of data showing KIR-ligand mismatched natural killer (NK) cells reduce the risk of leukemia and multiple myeloma relapse following allogeneic hematopoietic stem cell transplantation, investigators have developed a checkpoint inhibition antibody that blocks KIR on NK cells. Although in vitro studies suggest the KIR2D-specific antibody IPH2101 induces KIR-ligand mismatched tumor killing by NK cells, our single-arm phase II clinical trial in patients with smoldering multiple myeloma was prematurely terminated due to lack of clinical efficacy. This study aimed at unveiling the underlying mechanisms behind the lack of clinical efficacy. Experimental Design: Treatment-naïve patients received an intravenous infusion of 1 mg/kg IPH2101 every other month for up to a year. Peripheral blood was collected at baseline and 24 hours after first infusion, followed by weekly samples for the first month and monthly samples thereafter. NK cell phenotype and function was analyzed using high-resolution flow cytometry. Results: Unexpectedly, infusion of IPH2101 resulted in rapid reduction in both NK cell responsiveness and KIR2D expression on the NK cell surface. In vitro assays revealed KIR2D molecules are removed from the surface of IPH2101-treated NK cells by trogocytosis, with reductions in NK cell function directly correlating with loss of free KIR2D surface molecules. Although IPH2101 marginally augmented the antimyeloma cytotoxicity of remaining KIR2Ddull patient NK cells, the overall response was diminished by significant contraction and reduced function of KIR2D-expressing NK cells. Conclusions: These data raise concerns that the unexpected biological events reported in this study could compromise antibody-based strategies designed at augmenting NK cell tumor killing via checkpoint inhibition. Clin Cancer Res; 22(21); 5211–22. ©2016 AACR. See related commentary by Felices and Miller, p. 5161

Abstract: Human plasma routinely contains measureable quantities of cell free DNA (cf-DNA). Some is generated directly within the vascular space, and some is presumably transported into the circulation from cells dying at extravascular sites. Recent studies using highly sensitive and specific detection methods demonstrate that tumor-derived circulating cf-DNA can be a powerful predictor of total body tumor burden in patients with colon and breast carcinoma. While cf-DNA monitoring is clearly a promising new approach, its general applicability in tracking other malignancies, which may vary widely in their patterns of distribution, vascularity, and cell turnover, remains to be determined. We conducted a pilot study to address the utility of cell free tumor DNA in monitoring disease burden in myeloma patients receiving combination chemotherapy. Methods All patients were enrolled in NIH protocols for treatment using carfilzomib, lenalidomide, and dexamethasone (CRd) combination chemotherapy. Molecular studies were performed using DNA from bone marrow (BM) aspirates obtained before and after CRd treatment, and cf-DNA extracted from 0.5-1 ml samples of plasma and/or serum obtained before each CRd cycle. The frequency and immunophenotype of myeloma cells in BM and blood was assessed using an 8-color flow cytometric panel to analyze 〉 3 x106 events (sensitivity of 1 x 10-5). Clonal VDJ products were identified in pretreatment BM DNA using Biomed 2 primer “cocktails” targeting framework 1, 2, and 3 of the IgH chain and the variable region of the IgK light chain. Monoclonal VDJ or VJ products identified by capillary electrophoresis were cloned into pCR2.1 plasmid and sequenced. Quantitative rt-PCR assays employing patient-specific primer/Taqman probe combinations and linearized VDJ-plasmid DNA as a standard were used to measure VDJ in BM and cf-DNA. VDJ levels in BM DNA were normalized based on total actin copy number and expressed as % VDJ DNA. Cell free VDJ levels (cf-VDJ) were expressed in copies/ml of plasma or serum. Results To date, 6 patients with newly diagnosed multiple myeloma (NDMM) and 3 with smoldering myeloma (SMM) have been studied. BM infiltration with CD138+ plasma cells varied from 15% to 60% and VDJ DNA levels in BM varied from 13% to 61% in this group. Circulating cf-VDJ levels before therapy were 〉 50 copies/ml in 3 patients (444, 200, and 70 copies), detectable but 〈 50 copies/ml in 4 patients, and undetectable in 2 patients. Cf-VDJ levels, where measurable, decreased rapidly in parallel with the decline in monoclonal M-protein concentration after CRd therapy. Unlike M-protein concentrations, which were often more persistent, cf-VDJ levels became undetectable in all cases within 1-2 cycles. By comparison, VDJ levels in BM DNA often remained detectable at low levels even in patients with complete remission by conventional clinical and laboratory criteria. Of interest, there was no correlation between the pretreatment level of cf-VDJ and disease burden estimated based on the % CD138+ plasma cells in BM, the proportion of VDJ DNA in BM, or the M-protein concentration in blood/urine. There was however, a statistically significant relationship between the level of cf-VDJ and the number of circulating myeloma cells in peripheral blood. Conclusions In this pilot study, cf-VDJ is detected in the blood of many patients with untreated myeloma and levels fall precipitously in patients responding to highly effective CRd therapy. In some untreated patients, cf-VDJ copy numbers in peripheral blood are low, limiting assay sensitivity. Our observation of a statistical association between the level of cf-VDJ and the number of circulating myeloma cells in peripheral blood (defined by flow cytometry) suggests that circulating myeloma cell lysis potentially accounts for, at least a portion of, the observed levels of cf-VDJ. Future studies are needed to assess the potential of cf-VDJ DNA in peripheral blood and VDJ DNA in BM for tracking disease before and after anti-myeloma therapy. Disclosures: Off Label Use: The abstract discussess off-label use of carfilzomib and lenalidomide.

Abstract: Background: State of the art treatment for patients with NDMM involves induction with triplet regimens utilizing newer therapies including combinations of immunomodulatory (IMiD) drugs and proteasome inhibitors (PI) which improve progression-free survival (PFS) over doublet regimens. CFZ is a selective PI that inhibits the chymotrypsin-like activity of the 20S proteasome and has been approved in combination with LEN and DEX for the treatment of patients with relapsed or refractory MM who have received 1-3 lines of therapy. Furthermore, it has been associated with decreased neuropathy and increased clinical benefit over bortezomib-based combinations. This phase 2 study of 45 patients demonstrated that deep and durable responses with CRd-R can be achieved in the NDMM setting (Korde et al. JAMA Onc 2015). Here, we expand on our initial results in assessing minimal residual disease negativity (MRDneg) at complete response (CR) and after 1 and 2 years of LEN maintenance. We also characterize depth of response by age and cytogenetic risk profile. Methods: Treatment-na•ve patients with MM were treated for 8 cycles (28-day cycles) with CFZ 20/36 mg/m2 IV days 1, 2, 8, 9, 15, 16; LEN 25 mg PO days 1-21, and DEX 20/10 mg IV/PO days 1, 2, 8, 9, 15, 16, 22, 23. Transplant eligible patients underwent stem cell collection after ≥4 cycles of CRd and then continued CRd treatment (i.e. by-default-delayed high-dose melphalan with autologous stem cell transplant; HDM-ASCT). After 8 cycles of combination therapy, patients with SD or better received 2 years of LEN 10 mg PO maintenance. The primary objective of the study was to estimate the rate of ³ Grade 3 peripheral neuropathy with secondary objectives of response (ORR), MRDneg, PFS, and response duration (DoR) assessed after every cycle during induction and subsequently after every 90 days of maintenance therapy. Assessment of MRDneg by multi-color flow cytometry (bone marrow aspirate; 10-5 sensitivity) was performed after 8 cycles of induction, and 1 and 2 years of LEN maintenance. Results: Forty-five patients meeting eligibility criteria were enrolled (60% male; median age 60, range 40-89; race: 82% White, 13% Black, 4% Asian; isotypes: 51% IgG kappa, 16% IgG lambda, 13% IgA kappa, 9% IgA lambda, 9% free kappa, and 4% free lambda). The proportion of patients who obtained a complete response (CR) with MRDneg after 8 cycles of induction, 1 year of maintenance and 2 years of maintenance was 44%, 54% and 46%, respectively (Table 1). These deep responses of MRDneg CR were observed regardless of age group or cytogenetic-based risk profile (Table 2). ORR was 98% with a DoR at 48 months of 81%. PFS at 48 months was 82% and overall survival at 58 months was 86% with a median duration of follow-up of 31 months. Toxicities, in general, were manageable, with Grade 3-4 events occurring in 〉 1 patient including lymphopenia (69%), neutropenia (27%), thrombocytopenia (20%) anemia (18%), hypophosphatemia (18%), leukopenia (16%), maculo-papular rash (13%), alanine aminotransferase elevation (11%), fatigue (9%), dyspnea (9%), hyponatremia 7%, thromboembolism (7%), lung infection (4%), hypoalbuminemia (4%). Serious adverse events included thromboembolism (13%), dyspnea (9%), anemia (7%), lung infection (4%), thrombocytopenia (4%), hyponatremia (4%), and pleural effusion (4%). Conclusions: Upfront treatment of NDMM with modern, highly efficacious CRd-R therapy with by-default-delayed HDM-ASCT led to high rates of sustained MRDneg CR (as defined by the updated 2016 IMWG response criteria) which is correlated with longer PFS and OS. Clinically important, these deep responses were observed regardless of the age of patients or cytogenetic risk. Our findings stress the importance of utilizing highly efficacious triplet-based regimens for the treatment of patients with NDMM regardless of age or cytogenetic risk. Updated results will be presented at the Annual Meeting. Disclosures Korde: Medscape: Honoraria. Bhutani:Prothena: Research Funding; Takeda Oncology: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Onyx, an Amgen subsidiary: Speakers Bureau. Landgren:Takeda: Honoraria; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Medscape Myeloma Program: Honoraria; Merck: Honoraria; BMS: Honoraria.

Abstract: Introduction Defining high risk (HR) smoldering multiple myeloma (SMM) is becoming increasingly important as multiple clinical trials are actively investigating the role of early treatment. On average, patients with SMM progress to multiple myeloma (MM) at a rate of 10% per year for the first 5 years (Kyle 2007). Several classification systems have been developed to identify patients with a higher rate of progression, including two commonly used models: the 2008 Mayo Clinic model and the PETHEMA (Programa de Estudio y Tratamiento de las Hemopatias Malignas) model. The 2008 Mayo Clinic model incorporates M-protein ( 〉 3 g/dL), bone marrow plasma cell percentage (BMPC%) 〉 10%, and a ratio of involved to uninvolved serum free light chains (sFLCr) 〉 8. Patients with all three characteristics had a 76% risk of progression to MM in 5 years (Dispenzieri 2008). The PETHEMA model uses the proportion of BMPCs with aberrant plasma cell phenotype on flow cytometry ( 〉 95%) and reduction in uninvolved immunoglobulins (immunoparesis) to identify HR patients. Patients with both risk factors had a 5-year rate of progression to MM of 72% (Perez-Persona 2007). The 2008 Mayo Clinic model was validated prior to the International Myeloma Working Group reclassification of MM in 2014. Therefore, in 2018, Mayo Clinic proposed a new model to define HR SMM referred to as "2/20/20": M-protein 〉 2 g/dL, BMPC% 〉 20%, and sFLCr 〉 20 (Lakshman 2018). The median time to progression for the HR group (2-3 risk factors) was 29 months, compared to 110 months in the low risk (LR) group (0 risk factors). Previously, a high discordance rate among the 2008 Mayo model and the PETHEMA model was reported (Cherry 2013). In this study, we aim to define the concordance among patients defined as HR SMM by the aforementioned models in an independent sequential patient cohort. Methods The medical records of patients sequentially assigned a diagnosis of SMM by the myeloma program at the NIH Clinical Center between April 2010 to July 2019 were reviewed. Patients with myeloma defining events were excluded (i.e. MM). Each patient was assigned a risk score based on the 2008 Mayo Clinic model, the 2018 Mayo Clinic model, and the PETHEMA model. The distribution of patients in the LR, intermediate (IR), and HR groups were compared between the models. Concordance ratios were calculated between the three models. Results A total of 236 patient records were reviewed and per the 2014 IMWG criteria, 138 patients were identified as having SMM. Two patients did not have bone marrow flow cytometry samples and thus could not be classified by the PETHEMA model. Therefore, 136 patients were stratified by risk based on all three models (Table 1,2). The rate of concordance between the 2008 Mayo Model and the PETHEMA model was 31.6% (95% CI: 24.4-39.8%), similar to previously published results. The concordance between the 2018 Mayo Model and the PETHEMA model was slightly higher at 44.8% (95% CI: 36.7-53.2%; P=0.0337). There was significant discordance between the models in classifying patients as HR versus non-HR (Table 3). However, the 2018 Mayo Clinic model had a higher concordance with the PETHEMA model (27.2%; 95% CI: 20.4-35.3%) than the 2008 Mayo Clinic model (4.4%; 95% CI:1.8-9.5%). Conclusions The accurate identification of SMM patients at highest risk of developing MM remains elusive and no one model has been found to be superior than the other. As this study indicates, a significant number of patients may be classified as "HR" according to the PETHEMA model while simultaneously be defined as "LR" based on the Mayo Clinic models. While the 2018 Mayo Clinic model has a higher concordance rate to the PETHEMA model, it remains significantly discordant. These results indicate that the current clinical variables used to determine risk are not reliable. This is likely due to the fact that they are markers of disease burden rather than biology and risk is subject to increase over time (Landgren 2019). It is time for genomic signatures which signify varying biology to be incorporated into risk models. The treatment of HR SMM is currently being investigated in multiple clinical trials. As the results from these trials are published, the data will need to be scrutinized as to how patients were defined as "HR" in order to compare results. At this time, it remains unclear which patients warrant early intervention and it is imperative that patients with SMM be exclusively treated on clinical trials. Disclosures Mailankody: Juno: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Takeda Oncology: Research Funding; CME activity by Physician Education Resource: Honoraria. Landgren:Merck: Other: IDMC; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Theradex: Other: IDMC; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract: Multiple myeloma (MM) therapies are becoming increasingly effective with deeper responses. Indeed, recent prospective clinical trials based on carfilzomib, lenalidomide and dexamethasone (CRd) show complete response (CR)/near (n)-CR rates of over 75%. Consequently, newer techniques are needed to detect minimal residual disease (MRD). We are conducting a prospective Phase II clinical trial studying response to CRd in newly diagnosed MM patients. We assessed treatment responses obtained by traditional IMWG response criteria, MRD measurement by 8-color multiparameter flow cytometry (MFC) based on a minimum of 3 million events (maximum detection rate: 0.0005%), and MRD measurement by high throughput sequencing in 14 newly diagnosed MM patients who achieved very good partial response (VGPR)/CR/nCR. Methods Bone marrow (BM) and/or plasma samples were obtained from 14 newly diagnosed MM patients receiving CRd therapy. Samples were subjected to deep sequencing using the LymphoSIGHT™ platform, which has a sensitivity to detect one cancer cell per million leukocytes in peripheral blood (Faham et al, Blood 2012). Briefly, using universal primer sets, we amplified immunoglobulin heavy and kappa chain (IGH and IGK) variable, diversity, and joining gene segments from genomic DNA obtained from CD138+ BM cell lysate at baseline, as well as post-treatment plasma samples. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency within the B-cell repertoire in the CD138+ cell lysate BM sample. The presence of the myeloma-specific clonotype was then quantified in plasma samples obtained post-treatment. A quantitative and standardized measure of clone level among all leukocytes in the sample was determined using internal reference DNA. For patients who achieved VGPR/CR/nCR, we compared the results from MFC of the BM with those obtained by sequencing the cell free DNA in plasma samples. Results We detected a high frequency myeloma-specific gene rearrangement in 13 of 14 (93%) CD138+ BM cell lysate samples obtained at diagnosis. We assessed for MRD by flow cytometry and deep sequencing for the seven patients who were in VGPR/CR/nCR based on traditional protein response criteria. One patient was MRD positive by flow cytometry of the marrow and negative by deep sequencing in blood. Another patient was MRD positive by deep sequencing but negative by flow cytometry. Additionally, in the diagnostic BM sample of one patient, we observed two distinct high-frequency IGH clones that were related through a process of somatic hypermutation. The specific mutation pattern observed is consistent with a branched model of evolution where the two observed clones did not evolve from each other but rather from a common ancestor. We are currently analyzing samples collected at baseline and post-treatment in an additional 23 MM patients enrolled on the CRd trial, and results will be presented. Conclusions The development of sensitive, non-invasive MRD assays is becoming increasingly important to assess the impact of modern anti-myeloma therapies. One novel approach for MRD detection, termed the LymphoSIGHT™ platform, relies on high-throughput sequencing of VDJ rearrangements at the immunoglobulin locus. Based on our CRd clinical trial for newly diagnosed MM patients, we found sequencing of cell free tumor DNA in bone marrow aspirates and peripheral blood (plasma) to be technically feasible. Among 6 patients who obtained VGPR/CR/nCR and were found to be MRD negative by MFC of the BM, we found one patient to be MRD positive by sequencing in the peripheral blood (plasma compartment). One patient was MRD positive by flow cytometry but negative by deep sequencing. These preliminary results suggest that a sequencing-based MRD blood test may be more sensitive than standard protein response criteria and complementary to MFC-based BM tests. Analysis of additional samples will be presented. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Weng:Sequenta, Inc.: Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Research Funding.

Abstract: Background: State of the art treatment for patients with NDMM involves induction with triplet-based regimens utilizing combinations of immunomodulatory drugs and proteasome inhibitors (PI) which improve time to progression (TTP), progression-free survival, and overall survival (OS) over doublet regimens. Carfilzomib is a selective PI with FDA approval in the KRd combination regimen for the treatment of patients with relapsed or refractory MM. Carfilzomib-based combinations are associated with increased clinical benefit over bortezomib-based combinations and carfilzomib does not cause neuropathy. This phase 2 study of 45 patients demonstrated that deep responses with KRd-r is achieved in the NDMM setting (Korde et al. JAMA Onc 2015). Here, we expand on our initial results in assessing response to present the long-term durability of minimal residual disease negativity (MRDneg) complete response (CR) and time to progression. We also characterize TTP by depth of response, age, and cytogenetic risk profile. Methods:Treatment-naïve patients with MM were treated for 8 cycles (28-day cycles) with carfilzomib 20/36 mg/m2 IV days 1, 2, 8, 9, 15, 16; lenalidomide 25 mg PO days 1-21, and dexamethasone 20/10 mg IV/PO days 1, 2, 8, 9, 15, 16, 22, 23. Transplant eligible patients underwent stem cell collection after ≥4 cycles and then continued KRd treatment (i.e. without default autologous stem cell transplant (ASCT)). After 8 cycles of KRd, patients received 2 years of lenalidomide 10 mg PO maintenance on days 1-21. The primary objective of the study was to estimate the rate of ≥ Grade 3 peripheral neuropathy with secondary objectives of International Myeloma Working Group criteria for overall response rate (ORR), MRDneg CR, TTP, and response duration (DoR) assessed after every cycle during induction and subsequently after every 90 days of maintenance therapy. Assessment of MRDneg CR by multi-color flow cytometry (bone marrow aspirate; 10-5 sensitivity) was performed after 8 cycles of induction, 1 and 2 years of lenalidomide maintenance, and then annually. Results: Forty-five patients meeting eligibility criteria were enrolled (60% male; 42% ≥ age 65, range 40-89; race: 82% White, 13% Black, 4% Asian; isotypes: 51% IgG kappa, 16% IgG lambda, 13% IgA kappa, 9% IgA lambda, 9% free kappa, and 4% free lambda; 33% high risk cytogenetics, del(17p), t(4;14), t(14;16)or t(14;20)). The median potential follow up was 5.7 years (68.3 months). The ORR was 97.8% (95% Confidence Interval (CI): 88.2-99.9%) with a median DoR of 65.7 months (95% CI: 55.6-not reached (NR) months). Strikingly, 28 of the 45 patients, 62.2%, (95% CI: 46.5-76.2%) attained deep responses of MRDneg CR; durability of MRDneg CR was observed up to at least 70 months with a median duration of over 4 years (52.4 months; 95% CI: 35.3-61.6 months). Moreover, the median TTP was over five and a half years (67.3 months; 95% CI: 51.0-NR months) and the median OS was NR, however, at 80 months, 84.3% of patients were still alive. As expected, patients who attained MRDneg CR, by cycle 8, had a 78% reduction in the risk of progression (Hazard Ratio (HR): 0.22 (95% CI: 0.07-0.69); p=0.005) (Figure 1). Importantly, these deep responses of MRDneg CR and long progression free durations were observed regardless of age group or cytogenetic-based risk profile (Table 1). Toxicities have been previously reported and were generally manageable with no Grade ≥ 3 neuropathy or death due to toxicity. Conclusions: Upfront treatment of NDMM with the modern and highly efficacious KRd-r regimen incorporating a "by-default-delayed" ASCT strategy led to high rates of MRDneg CR (10-5 sensitivity) which even more importantly were sustained with a median duration of over 4 years. Moreover, attaining MRDneg CR, was strongly associated with a delay in progression. Clinically important, we observed that these deep responses and long progression-free durations are observed regardless of age or cytogenetic risk and stress the importance of utilizing highly efficacious triplet-based regimens for these sub-categories of NDMM. Lastly, our results with KRd-r in NDMM compare favorably to ASCT-based regimens and question the use of upfront ASCT for all patients. Our observed median TTP of 67 months is approximately 17 months longer than published data using the regimen of bortezomib, lenalidomide, and dexamethasone with ASCT (Attal et al. NEJM 2017). Updated results will be presented at the Annual Meeting. Disclosures Korde: Amgen: Research Funding. Mailankody:Janssen: Research Funding; Juno: Research Funding; Takeda: Research Funding; Physician Education Resource: Honoraria. Landgren:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Pfizer: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract: BACKGROUND: Early treatment with lenalidomide and dexamethasone delays progression and increases overall survival in patients with high-risk smoldering multiple myeloma. The addition of the selective proteasome inhibitor carfilzomib to a lenalidomide and dexamethasone backbone has proven effective in patients with newly-diagnosed multiple myeloma; this combination may allow patients with high-risk smoldering multiple myeloma to obtain deep and durable responses. METHODS: In this phase 2 pilot study, patients with high-risk smoldering multiple myeloma received eight 28-day cycles of induction therapy with carfilzomib (at a dose of 20/36 mg per square meter on days 1, 2, 8, 9, 15, and 16), lenalidomide (at a dose of 25 mg on days 1–21), and dexamethasone (at a dose of 10 or 20 mg on days 1, 2, 8, 9, 15, 16, 22, and 23). Patients achieving stable disease or better after combination therapy received 2 years of maintenance therapy with lenalidomide. Minimal residual disease was assessed with multi-color flow cytometry, next-generation sequencing by the LymphoSIGHT method, and fluorodeoxyglucose-positron emission tomography-computed tomography (FDG-PET/CT). Myeloma clonotypes were identified in genomic DNA obtained from CD138+ bone marrow cell lysate or cell-free bone marrow aspirate at baseline for each patient based on their high frequency within the B-cell repertoire. Per study protocol, minimal residual disease assessment by next-generation sequencing, multi-color flow cytometry and FDG-PET/CT was repeated when patients achieved a complete response or completed 8 cycles of induction treatment. A sample size of 12 evaluable patients was calculated as being minimally necessary based on the following probability calculations: If the true probability of a very good partial response was 20% or 50%, we calculated that there would be a 7.3% or 80.6% probability, respectively, if 5 or more patients exhibiting a very good partial response (VGPR). Thus, if 5 or more patients out of 12 achieved a very good partial response, there would be strong evidence that the true probability of a VGPR was 50% or more. RESULTS: Twelve patients were enrolled. All 11 patients (100%) who completed 8 cycles of combination therapy obtained VGPR or better (primary end point). Minimal residual disease assessment by next-generation sequencing was performed on bone marrow supernatant to detect cell-free myeloma clonotypes, while flow cytometry analysis utilized bone marrow cells. Overall (N=12), 100% of patients achieved a complete response or better over the study period, including 11 patients (92%) negative for minimal residual disease based on multi-color flow cytometry. Based on next-generation sequencing, two of the 12 patients were positive for minimal residual disease in the bone marrow supernatant; one of these two patients was also positive for minimal residual disease based on multi-color flow cytometry in the bone marrow cells. Information regarding longitudinal minimal residual disease status will be available and presented at the meeting. Adverse events were manageable. CONCLUSIONS: Early treatment with carfilzomib, lenalidomide, and dexamethasone was associated with high rates of complete response and minimal residual disease negativity by multi-color flow cytometry, next-generation sequencing, and FDG-PET/CT in patients with high-risk smoldering multiple myeloma. Disclosures Landgren: Onyx Pharmaceuticals: Consultancy; Medscape: Consultancy; Millennium Pharmaceuticals: Independent Data Monitoring Committee (IDMC), Independent Data Monitoring Committee (IDMC) Other. Off Label Use: Carfilzomib and lenalidomide for high-risk smoldering multiple myeloma.

Abstract: Introduction: Treatment combinations involving CD38 targeted monoclonal antibodies have significantly prolonged the median duration and depth of response in myeloma (MM), reflected in minimal residual disease-negativity (MRD-) rates of over 70% in newly diagnosed patients (Landgren et al. JAMA Onc 2021). Key to improving our understanding of treatment failures is the combined use of single cell analysis of the microenvironment with genome wide assessment of tumor genetics to decipher the mechanisms of disease resistance. Methods: We isolated malignant plasma cells from bone marrow (BM) samples using CD138+magnetic or flow (CD38, CD138, and CD45) sorting from 60 newly diagnosed MM patients treated with KRd with daratumumab (D-KRd n=46; NCT03290950) and without daratumumab combination therapy (KRd, n=14; NCT01402284). Fifty-five baseline samples underwent whole genome sequencing (WGS), median coverage of 80x using somatic DNA as a normal comparator. The BM cellular content of 22 patients (44 samples-5 failed) treated with D-KRd (10 MRD+ and 12 MRD-) underwent 5'single cell RNA-sequencing with an additional capture of the TCR and surface protein markers (CITEseq) to interrogate the single cell composition of the immune microenvironment at baseline (T1, n=20) and at the end of induction (T2, n=19). Paired (T1/T2) single-cell data were obtained in 17 patients and paired WGS and single cell data (T1) were available in 15 patients. MRD-, sustained MRD- (defined as two MRD- results, the first at the end of the induction (T2) and the subsequent at the first year of follow-up (T3)) and progression/loss of MRD- were used as clinical endpoints for this study. Results: After a median follow up of 29 months, 36 (54%) patients achieved MRD-; 34 (51%) had sustained MRD- & gt;1 year after completion of combination therapy. Overall, 10 (15%) patients had clinical progression and two conversions from MRD- to MRD+. A comprehensive catalogue of MM-genomic events associated with these three clinical endpoints was defined. Deletion (del) 13, biallelic loss CYLD, del XBP1, del 20q13.12 (CD40), and 8q gains were associated with MRD+ and failure to achieve sustained MRD-. Presence of del RPL5 and multiple chromothripsis events significantly correlated with early progression and loss of MRD-. Interestingly, structural variants (SV) involving IKFZ3 were seen in all three negative clinical endpoints (p & lt;0.05) and also associated with early progression in the CoMMpass data set (p=0.01, n=12). Trisomy 21 emerged as a favorable subset (11/13 sustained MRD- cases p=0.02) and was also seen in CoMMpass (p=0.003). Overall, these data highlight potential novel genomic drivers associated with resistance to D-KRd. We interrogated the BM microenvironment at baseline and correlated its composition with the tumor genomic architecture. Across 15 evaluable patients, del XBP1 were associated with fewer memory B-cells (p=0.03), naïve B-cell (p=0.01) and dendritic cells (p=0.03) compared to the wild type. Also, low dendritic cell at baseline cases were observed in patients with del 20q13.12 (CD40) (p=0.03). Interestingly, low level of plasmacytoid dendritic cells at baseline was associated with failure to achieve MRD- and sustained MRD-. Patients with 6p24 amplification showed a reduced number of CD8 effectors 1 and 2 (p & lt;0.05). Overall, these data suggest that distinct genomic lesions are associated with distinct immune-microenvironment composition. When comparing baseline (T1) and end of induction (T2), significant differences were seen between sustained MRD+ and MRD-. We identified significantly depleted NK, and naïve and memory B-cell after D-KRd MRD- patients had significantly more CD14+ monocytes both at T1 and T2 than their MRD+ counterparts (Fig. 1). Differential expression suggests that inflammatory response genes including IL1B are upregulated in the absence of sustained MRD- whereas genes implicated in IL2, IL6, and IFNα response as well as adipocyte differentiation are associated with sustained MRD response. Conclusion: We show, for the first time, evidence of complex interplay between MM tumor genetics and the microenvironment in the context of D-KRd treated patients. Our results highlight the importance of genomic-based mechanisms in the persistence of disease (IKZF3, XBP1) as well as heterogeneity in the composition of the BM microenvironment, with the monocytes pointing towards the importance of inflammation. Figure 1 Figure 1. Disclosures Maura: OncLive: Honoraria; Medscape: Consultancy, Honoraria. Hassoun: Celgene, Takeda, Janssen: Research Funding. Mailankody: Jansen Oncology: Research Funding; Allogene Therapeutics: Research Funding; Bristol Myers Squibb/Juno: Research Funding; Legend Biotech: Consultancy; Takeda Oncology: Research Funding; Fate Therapeutics: Research Funding; Physician Education Resource: Honoraria; Plexus Communications: Honoraria; Evicore: Consultancy. Hultcrantz: Intellisphere LLC: Consultancy; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Curio Science LLC: Consultancy; Amgen: Research Funding; Daiichi Sankyo: Research Funding. Scordo: Omeros Corporation: Consultancy; i3 Health: Other: Speaker; Kite - A Gilead Company: Membership on an entity's Board of Directors or advisory committees; Angiocrine Bioscience: Consultancy, Research Funding; McKinsey & Company: Consultancy. Kazandjian: Arcellx: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Shah: Janssen Pharmaceutica: Research Funding; Amgen: Research Funding. Landau: Takeda, Janssen, Caelum Biosciences, Celgene, Pfizer, Genzyme: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Genzyme: Honoraria. Giralt: SANOFI: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; JANSENN: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Dogan: Physicians' Education Resource: Honoraria; Seattle Genetics: Consultancy; EUSA Pharma: Consultancy; Roche: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Peer View: Honoraria. Lesokhin: Behringer Ingelheim: Honoraria; pfizer: Consultancy, Research Funding; Janssen: Honoraria, Research Funding; Iteos: Consultancy; Genetech: Research Funding; bristol myers squibb: Research Funding; Trillium Therapeutics: Consultancy; Serametrix, Inc: Patents & Royalties. Davies: Janssen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Korde: Amgen: Research Funding; Medimmune: Membership on an entity's Board of Directors or advisory committees. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Landgren: Janssen: Other: IDMC; Celgene: Research Funding; Amgen: Honoraria; Janssen: Research Funding; Janssen: Honoraria; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria.