Abstract: We conducted a genome-wide association study to identify novel associations between genetic variants and circulating plasminogen activator inhibitor-1 (PAI-1) concentration, and examined functional implications of variants and genes that were discovered. A discovery meta-analysis was performed in 19 599 subjects, followed by replication analysis of genome-wide significant (P 〈 5 × 10−8) single nucleotide polymorphisms (SNPs) in 10 796 independent samples. We further examined associations with type 2 diabetes and coronary artery disease, assessed the functional significance of the SNPs for gene expression in human tissues, and conducted RNA-silencing experiments for one novel association. We confirmed the association of the 4G/5G proxy SNP rs2227631 in the promoter region of SERPINE1 (7q22.1) and discovered genome-wide significant associations at 3 additional loci: chromosome 7q22.1 close to SERPINE1 (rs6976053, discovery P = 3.4 × 10−10); chromosome 11p15.2 within ARNTL (rs6486122, discovery P = 3.0 × 10−8); and chromosome 3p25.2 within PPARG (rs11128603, discovery P = 2.9 × 10−8). Replication was achieved for the 7q22.1 and 11p15.2 loci. There was nominal association with type 2 diabetes and coronary artery disease at ARNTL (P 〈 .05). Functional studies identified MUC3 as a candidate gene for the second association signal on 7q22.1. In summary, SNPs in SERPINE1 and ARNTL and an SNP associated with the expression of MUC3 were robustly associated with circulating levels of PAI-1.

Abstract: Abstract 82 Introduction: Plerixafor is thought to enhance chemotherapy mediated myeloid leukemia cell death by promoting mobilization from the protective bone marrow niche and disrupting CXCL12-mediated survival signals. We conducted a Phase 1, open-label, multi-center, dose escalation study in patients (pts) with newly diagnosed AML to determine the maximum tolerated dose (MTD) and safety of plerixafor when combined with cytarabine and daunorubicin. Methods: Adult pts ≤70 years old with newly diagnosed AML were eligible to participate in this ongoing study. Pts with M3 AML and those 〈 50 years old with core binding factor leukemias were excluded. Pts received IV cytarabine 100 mg/m2/day by continuous infusion on days 1–7 and IV daunorubicin 90 mg/m2/day on days 1–3 (7+3 regimen). Plerixafor was given as a 30-min IV infusion, 4–5 hours before daunorubicin beginning on day 2, and repeated at the same time on days 3–7, starting at 0.24 mg/kg, proceeding to dose levels of 0.32, 0.40, and 0.48 mg/kg. Three to 12 evaluable pts were enrolled in each cohort in a modified 3+3 design. Pts were observed for dose-limiting toxicities (DLT) from the first plerixafor dose through day 49. Responses were assessed using IWG response criteria. Serial peripheral blood (PB) samples were analyzed before and after the first and last plerixafor doses for circulating CD34+/CD117+ leukemia cells. Results: Twenty-three pts (median age 57 years) have been enrolled in 4 cohorts. Baseline characteristics are included in Table 1. Plerixafor infusion on day 2 caused a rise in PB AML blasts (mean 3.01-fold increase) peaking at 2–4 hours after administration. On day 7, there was a mean 1.51-fold increase in PB AML blasts but far fewer total cells were detected. Eighteen (86%) pts experienced adverse events (AEs) that were reported as at least possibly related to plerixafor. The majority were Grade 1/2 in severity and mainly included gastrointestinal disorders. Four (19%) pts experienced Grade 3 plerixafor-related AEs including febrile neutropenia (n=3), neutropenia (n=1), nausea (n=1), infections (n=2) and decreased appetite (n=1) commonly observed with 7+3 regimen. One (5%) pt (0.48 mg/kg cohort) experienced Grade 4 related AEs of thrombocytopenia and asymptomatic pulmonary embolism (while receiving medroxyprogesterone); the latter was the only possibly-related SAE reported. The median time to neutrophil (≥ 0.5 × 109/L) and platelet (≥100 × 109/L) recovery for responders was 19.5 (range 13–35) and 21 (range 17–37) days, respectively. There were 4 (17%) plerixafor unrelated deaths (0.24mg/kg): 1 within 30 days post induction due to an AE of acute respiratory distress syndrome and 3 due to disease progression 〉 3 months post induction. No DLTs have been reported. Of 21 pts with available data, 14 (67%) had complete response (CR), 2 had CR with incomplete count recovery (CRi), 2 had residual leukemia (RL), 2 had treatment failure (TF) due to resistant disease and 1 was not evaluable (NE) due to early death. Conclusions: The toxicity or hematopoietic recovery expected with the 7+3 regimen was not significantly altered by the addition of plerixafor. Transient mobilization of AML blasts into PB was observed immediately following plerixafor treatment. Sixteen of 21 patients, majority of who had intermediate or poor risk cytogenetics, achieved a CR or CRi, with responses observed across all plerixafor doses. Twice daily plerixafor dosing and addition of G-CSF to augment mobilization are being currently explored. Disclosures: Uy: Sanofi Oncology: Consultancy, Speakers Bureau. Off Label Use: Plerixafor (Mozobil®), a hematopoietic stem cell mobilizer, is approved by the US FDA in combination with granulocyte-colony stimulating factor (G-CSF) to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation in patients with non-Hodgkin's lymphoma and multiple myeloma. Avigan:Sanofi Oncology: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cortes:Ariad: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chemgenex: Membership on an entity's Board of Directors or advisory committees, Research Funding. Becker:Millenium: Research Funding; Glycomimetics: Research Funding; Sanofi Oncology: Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Consultancy. Hewes:Sanofi Oncology: Employment. Johns:Sanofi Oncology: Employment. Erba:Ambit: Research Funding; Ascenta: Research Funding; Celgene: Speakers Bureau; Chroma: Research Funding; Eli Lilly: Research Funding.

Abstract: Myelodysplastic syndromes (MDS) are clonal disorders of aberrant hematopoietic stem cells. The role of the hematopoietic microenvironment (HMEV) in the initiation and maintenance of MDS remains unclear, though model systems have provided strong evidence for the potential of specific mesenchymal subsets, mostly osteoprogenitor cells, within the HMEV as a driver of the disease. Distinct behavior of various models emphasizes the importance of analyzing specific and highly defined populations rather than bulk mesenchymal cells when investigating patient-derived bone marrow (BM) samples. We have developed a platform that allows us to prospectively isolate specific human BM mesenchymal subsets with a comparable transcriptional profile to mouse osteoprogenitor cells that have been shown to drive hematological malignancies when perturbed (Raaijmakers et al., 2010; Dong et al, 2016). Using a combination of three cell surface markers (CD271, CD146, CD106) that were previously shown to prospectively enrich for mesenchymal cells in human BM, we isolated and performed transcriptional and functional analysis of four distinct subsets within the non-hematopoietic (CD45, CD235), non-endothelial (CD31) compartment of the human BM. The four populations are labeled 1A (CD271+CD106-), 1B (CD271+CD106+), 2A (CD271+CD146+CD106-), and 2B (CD271+CD146+CD106+) (Figure 1A). Comparative analysis of differentially expressed gene sets based on RNA-seq within the human and mouse mesenchymal populations identified population 2B to be the most similar to osterix (OSX)-labeled mouse osteoprogenitor cells, whereas population 1A was more similar to osteocalcin (OCN)-labeled mouse mature osteoblasts. In animal models, genetic perturbations specifically in OSX but not OCN labeled cells resulted in hematological malignancy. Based on that observation, we hypothesized that population 2B is the cell subset most likely to harbor molecular perturbations in the BM of MDS patients. We analyzed populations 2A, 2B, 1(1A+1B) and another population 3 which is devoid of colony forming cells and encompasses all CD271-CD146- non hematopoietic stromal cells in 16 human MDS patients. BM from 11 patients undergoing hip replacement surgeries was used as a source of age- and gender-matched normal controls. Despite the heterogeneity of the disease, principal component analysis revealed that the transcriptional profile of normal samples clearly separated from MDS patients mostly in populations 2B and 1 (Figure 1B). Functional annotation of the differentially expressed genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) demonstrated minimal overlap of enriched terms between the four populations. Thus our data demonstrate transcriptionally distinct subsets of BM mesenchymal cells and identify signatures of transcriptional deregulation which differentiate these cell subsets in MDS patients from normal individuals. Osteopontin, an extracellular matrix protein expressed by osteoblasts among other cells, was the most significantly differentially expressed gene in population 2B of MDS patients.To study the effect of the gene expression perturbations on MDS evolution in vivo, we created a chimeric model where BM cells expressing the Nup98/HoxD13 (NHD13) translocation were transplanted in competition with wild type cells in wild type (WT) or osteopontin knock out (Opn-KO) recipients. Animals were conditioned using immunotoxin to avoid the damaging effect of irradiation on the microenvironment. Following chimerism of donor cells in peripheral blood over 24 weeks demonstrated a competitive advantage of NHD13 cells in the Opn-KO recipients leading to a significant difference in survival when compared to NHD13 cells transplanted in wild type mice (Figure 1C, D). Altogether, our data provide evidence that specific mesenchymal progenitor subsets in the BM of MDS patients harbor molecular perturbations that contribute to the competitive advantage of MDS over normal hematopoietic cells. Disclosures Sykes: Clear Creek Bio: Equity Ownership, Other: Co-founder.

Abstract: Recent genome wide association studies (GWAS) of Hodgkin lymphoma (HL) have identified several associations at both HLA and non-HLA loci. However, much of HL heritability remains unexplained. Methods To identify novel risk loci, we performed a meta-analysis of 3 HL GWAS including a total of 1,810 cases and 7,879 controls. Results were replicated in an independent set of 1,163 cases and 2,580 controls, for a total of 3,097 and 11,097 cases and controls combined, respectively. participants in discovery and replication stages were of European descent. quality control and imputation we conducted a meta-analysis addressing 1,004,829 variants (λ= 1.10, λ1000= 1.03). Associations between SNP genotypes and HL risk were evaluated under a log-additive model of inheritance adjusting for sex, study center and significant principal components to control for population stratification. We performed an analysis with all HL cases and then conducted stratified analyses by histological subtype (classical, nodular sclerosis and mixed cellularity), age at diagnosis (nodular sclerosis among those diagnosed at 15- 35 years in all studies, and those diagnosed at 35 and older years in the European Study only) and EBV tumor status (negative and positive). We then used a bioinformatic approach (FunciSNP) to identify potential functional variants associated with HD risk correlated with risk loci of interest. We extracted publically available ENCODE data on biofeatures to identify potential functional motifs associated with the index SNP or correlated SNPs. Finally, we measured expression levels of the two alternative mRNA transcripts in lymphoblastoid cell lines (LCLs) from 49 post-therapy HL patients and 25 unaffected controls. RT-PCR was carried out in triplicate. Relative expression levels were calculated relative to TBP as housekeeping gene. Linear models were used to assess correlation between genotype and TCF3expression levels. Results The meta-analysis identified a novel susceptibility variant at chromosome 19p13.3 (rs1860661) associated with HL risk (Odds Ratio [OR]= 0.78, P=2.0*10-8, I2=0%). variant is located in intron 2 of TCF3 (also known as E2A), a regulator of B- and T-cell lineage commitment. was also significantly associated with HL (OR= 0.85, P=0.002) in the replication series of 1,281 cases and 3,218 controls. the combined analysis consisting of the discovery and replication sets, rs1860661was strongly associated with HL (OR=0.81,=3.5*10-10), with no evidence of heterogeneity between contributing studies (Phom=0.41, I2=0%). The number of G alleles defined by rs1860661 was significantly associated with a reduced risk of each HL subtype except EBV positive HL. rs1860661 and two correlated SNPs, rs10413888 (r2=0.90) and rs8103453 (r2=0.89) identified by FunciSNP analysis map in or near marks of open chromatin and in DNAse hypersensitivity sites in TCF3 in CD20+ B cell lines., the protective minor alleles of these SNPs as defined by the G-G-G haplotype map to the binding sites for ZBTB7a (rs10413888 and rs1860661) and (rs8103453) transcription factors, likely improving the binding efficiency to the sites which may result in increased transcription rates of TCF3. TCF3 is encoded by two alternative transcripts (E12 and E47). Higher expression levels of TCF3-E47, whose transcription start site is located close to rs1860661, was associated with the rs1860661-G allele in controls (P=0.02), but not in HL patients (P=0.22). Conclusion/Discussion TCF3 is essential for the commitment of lymphoid progenitors to both B-cell and T-cell lineage development. A molecular and phenotypic hallmark of classical HL is the loss of the B-cell phenotype in HRS cells, including lack of demonstrable B-cell receptor and most B-cell specific markers such as CD19 or CD20. HRS cells have a low level of TCF3, particularly homodimers of the isoform E47, due to expression of the ABF-1 and ID2 inhibitors that bind to TCF3. Thus, higher TCF3 levels in HRS precursor cells may lead to enhanced retention of the B cell phenotype, thereby conferring a protective effect. These data suggest a link between the 19p13.3 locus including TCF3 and HL risk, indicating that TCF3 could be relevant to HL etiology and pathogenesis. Disclosures: Link: Genentech: Consultancy; Millenium: Consultancy; Pharmacyclics: Consultancy; Spectrum: Consultancy.

Abstract: Background: This phase II trial evaluated the response rate of sequential bortezomib (VELCADE®), cyclophosphamide (Cytoxan®), and dexamethasone ([VCD]; 3 cycles) followed by bortezomib, thalidomide (Thalomid®), and dexamethasone ([VTD] ; 3 cycles) as first-line therapy for patients with multiple myeloma. The primary endpoints were overall response, achievement of „d very good partial response (VGPR), as well as assessment of safety and tolerability. Methods: Patients with newly diagnosed, untreated symptomatic myeloma were eligible. Treatment consisted of three 21-day cycles of bortezomib 1.3mg/m2 days 1, 4, 8, and 11, cyclophosphamide 300mg/m2 IV days 1 and 8 and dexamethasone 40mg p.o. or IV days 1, 2, 4, 5, 8, 9, 11, 12; followed by three 21-day cycles of bortezomib 1.0mg/m2 days 1, 4, 8, and 11; thalidomide 100 mg p.o. daily and dexamethasone same as cycles 1–3. Patients received thrombosis prophylaxis with ASA 325mg daily during cycles 4–6. Upon completion of the 6 cycles, patients proceeded to autologous stem cell harvest, transplant and/or maintenance therapy. Responses were defined as a decrease in serum and/or urine monoclonal (M) protein by 50% or greater. VGPR and other responses were as per the International Response Criteria. Results: This report provides results for all 44 eligible patients: median age 59 years; 68% male; 61% Stage III (D/S); documented ISS Stage II/III 59%; IgG 66%, IgA 17%; 17% light chain only. To date the first 30 patients are fully evaluable for response with 28-day post therapy follow up. The overall response rate (ORR; ≥PR) is 90% with 18/30 (60%) achieving CR + VGPR (CR 33%); 9/30 (30%) PR and 3/30 (10%) stable disease or not evaluable. Overall, both components of the sequential regimen were very well tolerated. One patient had a ruptured colonic diverticulum possibly related to dexamethasone, but recovered well and achieved VGPR on trial. There have been 49 therapy attributed toxicity events which have required drug/schedule adjustments. Of these, 9 events were Gd 3/4: 3 attributed to cyclophosphamide, 3 to dexamethasone, 2 attributed to bortezomib and 1 to thalidomide. DVT occurred in one patient who was at high risk because of prior bilateral hip surgery. Fourteen patients have proceeded to successful stem cell harvest and autologous transplant. Post transplant follow up as well as response information for all 44 patients will be presented. Conclusion: This bortezomib/cyclophosphamide/dexamethasone (VCD) based combination induction therapy followed by VTD is a promising addition to the treatment armamentarium for previously untreated patients. The response rates: ORR 90%; □ VGPR (60%) and CR (33%) are extremely encouraging and improve over our bortezomib/dexamethasone 2-drug experience which produced 90% □ PR and 38% □ VGPR. This very well tolerated new regimen is potentially an important step forward in induction therapy with presentation of more mature data. Supported by the Aptium Oncology Research Network and a research grant from Millennium Pharmaceuticals, Inc.

Abstract: Background: Although the median survival for patients with multiple myeloma has improved dramatically, almost all patients will eventually relapse and become resistant to standard therapies. Chimeric antigen receptor T cells (CAR T cells) targeting B cell maturation antigen (BCMA) have shown early promise in MM, with high initial response rates. Responses are often incomplete and durability has been a key concern, with most patients relapsing within 1 year (Raje N et al NEJM 2019). We have previously demonstrated that gamma secretase inhibitors (GSI) increase BCMA surface density, decrease soluble BCMA levels and augment anti-tumor efficacy of BCMA CAR T cells in preclinical models. In a phase I first-in-human trial (NCT03502577), we combined CAR T cells expressing a fully human BCMA scFv with an orally administered gamma secretase inhibitor (JSMD194). Methods: Eligible patients had relapsed/refractory MM, with ≥ 10% plasma cells in the bone marrow by CD138 IHC, and measurable disease by IMWG criteria. BCMA was measured on CD138+ plasma cells by flow cytometry. CD8+ and CD4+ T cells were isolated via positive selection. The T cells were stimulated in separate cultures and transduced with lentiviral vector encoding a fully human BCMA scFv in conjunction with 41BB and CD3 zeta signaling domains. Following expansion, the cell product was formulated in a 1:1 ratio of CD4+:CD8+ BCMA CAR T cells. To assess the discreet impact of the GSI on plasma cell BCMA expression, patients received a GSI (JSMD194) monotherapy "run-in" involving three oral doses (25 mg) administered 48 hours apart over 5 days. A bone marrow aspirate was obtained on day 5 and BCMA expression on tumor cells was compared to baseline. Then, after lymphodepleting chemotherapy, BCMA CAR T cells were infused at a total starting dose of 5 x 10^7 EGFRt+ cells, in combination with JSMD194 dosed at 25 mg thrice weekly for three weeks, starting on the day of CAR infusion. Results: Eight patients, with a median age of 64.5 (range, 50-70 years) and a median of 10 prior regimens (range, 4-23), were screened, and seven patients have been treated. One patient had not responded to prior treatment with BCMA CAR T cells using a different construct, and another had progressed on a clinical trial employing a BCMA bispecific antibody. Median bone marrow plasma cell involvement by IHC was 32.5% (range, 10-80%) at enrollment. High-risk features were present in 75% of patients. Median involved serum free light chain at screening was 68.7 mg/dL (range18.45 - 365.61 mg/dL) and median monoclonal protein was 2.55 g/dL (range 0.1 - 5.1 g/dL). Following 3 oral doses (run in) of JSMD194, the percent of plasma cells expressing BCMA increased from 75% to 99% (7.6 to 98% pre, 75 to 100% post), soluble BCMA decreased by 2.0 fold (range 1.6 to 2.6 fold) after 3 oral doses, and BCMA antigen binding capacity increased from a median of 718 receptors to 13355 receptors per cell, or a median of 20-fold (range, 7.55-fold to 156.68-fold). Among 6 assessable patients, the best overall response rate was 100% (5 VGPR, 1 PR), with 5/6 patients MRD negative by flow. At data cutoff of July 15, 2019, no patient has relapsed, with a median follow-up of 5 months (range 1-11 months). One patient died at day 33 post-CAR T cell in the setting of cytokine release syndrome and concurrent fungal infection. The most common non-hematologic ≥ Grade 3 AE was neutropenic fever in 70%. CRS occurred in 100% of patients, primarily grades 1-2 (Lee Criteria), and neurotoxicity in 70%. Conclusions: Although BCMA CAR T cell therapy has demonstrated potent anti-tumor efficacy in multiple myeloma, a significant proportion of patients relapse. The mechanism of myeloma recrudescence requires further study, however BCMA antigen loss has been observed after CAR T cell therapy and is a putative pathway for tumor escape. In this study we demonstrate that gamma secretase inhibition with JSMD194 routinely increases BCMA surface density on myeloma cells in treated patients and reduces soluble BCMA. The combination of a gamma secretase inhibitor with BCMA CAR T cells leads to rapid responses including in patients that have failed prior BCMA targeted therapy. These responses are achieved with low CAR T cell doses. Longer follow up is required to determine if the durability of response is improved. Disclosures Cowan: Cellectar: Consultancy; Juno: Research Funding; Sanofi: Consultancy; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Abbvie: Research Funding. Pont:Fred Hutchinson Cancer Research Center: Other: Inventor on a patent. Sather:Lyell Immunopharma: Employment. Turtle:T-CURX: Membership on an entity's Board of Directors or advisory committees; Allogene: Other: Ad hoc advisory board member; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Humanigen: Other: Ad hoc advisory board member; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, Research Funding; Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Other: Ad hoc advisory board member; Novartis: Other: Ad hoc advisory board member. Till:Mustang Bio: Patents & Royalties, Research Funding. Libby:Alnylam: Consultancy; Abbvie: Consultancy; Pharmacyclics and Janssen: Consultancy; Akcea: Consultancy. Becker:AbbVie, Amgen, Bristol-Myers Squibb, Glycomimetics, Invivoscribe, JW Pharmaceuticals, Novartis, Trovagene: Research Funding; Accordant Health Services/Caremark: Consultancy; The France Foundation: Honoraria. Blake:Celgene: Employment, Equity Ownership. Works:Celgene: Employment, Equity Ownership. Maloney:Juno Therapeutics: Honoraria, Patents & Royalties: patients pending , Research Funding; Celgene,Kite Pharma: Honoraria, Research Funding; BioLine RX, Gilead,Genentech,Novartis: Honoraria; A2 Biotherapeutics: Honoraria, Other: Stock options . Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; Lyell Immunopharma: Equity Ownership, Patents & Royalties, Research Funding. Green:Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding; GSK: Consultancy; Celgene: Consultancy; Seattle Genetics: Research Funding; Cellectar: Research Funding. OffLabel Disclosure: Gamma secretase inhibitor to increase BCMA expression in multiple myeloma

Abstract: Background: Despite advances in the treatment of multiple myeloma (MM) almost all patients relapse and high risk features continue to portend a short median survival. The adoptive transfer of B-Cell Maturation Antigen (BCMA) chimeric antigen receptor (CAR) T cells is demonstrating early promise in MM, but the durability of response has not been established. The infusion of genetically modified CD8+ and CD4+ T cells of a defined composition facilitates the evaluation of each subset's function and has contributed to reproducible efficacy and safety in clinical trials with CD19-specific CAR T cells. In this phase I first-in-human study employing a human scFv containing BCMA CAR T cell construct, we report rapid and deep objective responses at a low CAR T cell dose level (5 x 107) suggesting that construct specific features and differences in product formulation may substantially impact efficacy. Methods: Eligible patients had relapsed or treatment refractory MM, ≥10% CD138+ bone marrow (BM) plasma cells (PC), and ≥5% BCMA expression by flow cytometry (FC). Patients were stratified by tumor burden (CD138+ IHC) into two cohorts; 10-30% MM cells [cohort A] or 〉 30% BM involvement [cohort B] to facilitate assessment of impact of disease burden on outcome. Eligible patient's CD8+ and CD4+ T cells were isolated via positive selection, enriched separately by immunomagnetic selection and cryopreserved. The CD8+ and CD4+ T cells were stimulated in independent cultures with anti-CD3/anti-CD28 paramagnetic beads and transduced with a 3rd generation lentiviral vector encoding a fully human BCMA scFv and 4-1BB and CD3 zeta signaling domains. After in vitro expansion, the cell product for infusion was formulated in a 1:1 ratio of CD4+:CD8+ BCMA CAR T cells. A truncated non-functional human epidermal growth factor receptor (EGFRt) was encoded in the transgene cassette to identify transduced T cells. Lymphodepleting chemotherapy preceded infusion of EGFRt+ CAR T cells at a starting dose of 5 x 107 EGFRt+ cells (n=5) for each cohort. Results: Seven patients (median age of 63 years; range 49 to 76) with a median of 8 prior regimens (range 6 to 11) have received treatment. The median %PC in BM (IHC) at enrollment was 58% (range 20% to 〉 80%). In cohort B the dose has been escalated to 15 x107 EGFRt+ cells (n=2). All patients (7/7) had at least one high risk cytogenetic feature (17p- [n=4], t(4;14) [n=2] , t(14;16) [n=1]), 71% had ≥ 2 high risk cytogenetic features, 71% had prior autologous stem cell transplant, 43% had prior allogeneic transplant, and one patient (14%) had PCL. The median involved free light chain (FLC) at enrollment was 180 mg/dL (range 40.37 to 502.96 mg/dL; n=5) and the median monoclonal protein was 3.8 g/dL (range 1.6 to 6.5 g/dL; n=5). The overall response rate at 28 days was 100%; at this time all evaluable patients (n=6) had no detectable abnormal BM PC clone by both IHC and high sensitivity FC. Within 28 days of treatment the involved FLC was normal or sub-normal in all patients and the M-protein had decreased by a median of 73% (range 56.25 to 83% reduction). BCMA CAR T cells remained detectable 90 days after infusion, representing up to 41.5 percent of CD3+ lymphocytes. All patients were surviving at a median of 16 wks (range 2 to 26 wks). One patient relapsed (day +60) and a tumor biopsy demonstrated the presence of a BCMAneg PC population, a 70% reduction in the fraction of MM cells expressing BCMA by FC and a fivefold reduction in BCMA antigen binding capacity on MM cells retaining target expression. A cytotoxic T lymphocyte response to the trans-gene product was not identified in this patient. No dose limiting toxicity has been observed during the 28 day monitoring window and treatment has been well tolerated with no cytokine release syndrome (CRS) observed in one patient and grade 2 or lower CRS (Lee Criteria) for all other patients. No neurological toxicity has been observed. Conclusion: BCMA CAR T cells harboring a fully human scFv with a defined composition of CD4+:CD8+ T cells were well tolerated and potent, demonstrating complete objective responses in heavily pretreated high-risk MM at total cell doses as low as 5 x 107. Next generation sequencing and multiparameter high sensitivity flow cytometry studies to evaluate for minimal residual disease are ongoing. Peak expansion levels and persistence of the CAR T cells are being monitored with early findings suggesting an absence of transgene product immunogenicity. Disclosures Green: Juno Therapeutics: Patents & Royalties, Research Funding. Sather:Juno Therapeutics: Employment. Cowan:Janssen: Research Funding; Abbvie: Research Funding; Juno Therapeutics: Research Funding; Sanofi: Research Funding. Turtle:Caribou Biosciences: Consultancy; Gilead: Consultancy; Bluebird Bio: Consultancy; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptevo: Consultancy; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Nektar Therapeutics: Consultancy, Research Funding; Juno Therapeutics / Celgene: Consultancy, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy. Till:Mustang Bio: Patents & Royalties, Research Funding. Becker:GlycoMimetics: Research Funding. Blake:Celgene: Employment, Equity Ownership. Works:Juno Therapeutics: Employment. Maloney:GlaxoSmithKline: Research Funding; Juno Therapeutics: Research Funding; Seattle Genetics: Honoraria; Roche/Genentech: Honoraria; Janssen Scientific Affairs: Honoraria. Riddell:Cell Medica: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; NOHLA: Consultancy.

Abstract: Introduction: Primary mediastinal B cell lymphoma (PMBL) is clinically and biologically distinct from the other subtypes of diffuse large B cell lymphoma (DLBCL), typically affecting young female patients (pts) with a bulky mediastinal mass. Standard treatment (TRT) is a combination of anti-CD20 antibody (Ab) and anthracycline-based chemotherapy. We aimed to compare patients' outcomes after CHOP delivered every 21 days (CHOP21) or 14 days (CHOP14) or ACVBP combined with anti-CD20 Ab in real life. Methods: All Pts treated in LYSA centers were eligible in this retrospective analysis. Inclusion criteria were as follow: age ≥18 years (yrs), newly diagnosed PMBL, TRT with CHOP or ACVBP plus anti-CD20 Ab between 2006 and 2017, and patient non-opposition statement. Primary endpoint was progression-free survival (PFS), secondary endpoints were: overall survival (OS), response rate (Lugano 2014) and total metabolic tumor volume (TMTV). Results: 313 pts were enrolled from 25 LYSA centers in France and Belgium. In all, median age at diagnosis was 32 (18-88) yrs. Majority of pts were female pts (60.7%) and presented at diagnosis with a good performance status (0-1: 81.8%), stage I-II (57.5%), elevated LDH (85%), Bulk & gt;10cm (58.5%) and low/low-intermediate aaIPI 0-1 (56.7%). Pts in the CHOP21 (n=57) group were older (median 40 yrs, vs 33 vs 29.5, p & lt;0.001), had more frequent low/low-intermediate risk aaIPI (71.4%, vs 55.6% vs 52.5%, p=0.044) and displayed a lower proportion of elevated LDH (66.7%, vs 88.2% vs 83.9%, p=0.014) as compared to CHOP14 (n=76) and ACVBP (n=180), plus anti CD20 Ab (Rituximab: n=296, obinutuzumab: n=17). 229 pts (73.2%) received intrathecal prophylaxis. Median number of chemotherapy cycles for CHOP21, CHOP14 and ACVBP groups were: 6 (1-8), 7.5 (1-8) and 4 (1-4), respectively. Consolidation ASCT was performed for 1 (1.8%), 24 (31.6%) and 46 (25.6%) pts, (p & lt;0.001) and mediastinal RT was delivered to 2 (3.5%), 11 (14.5%) and 4 (2.2%) pts, respectively (p & lt;0.001). ASCT + RT was done for 0, 5 (6.6%) and 3 (1.7%) pts, respectively (p=0.043). Baseline TMTV assessment was available for 233 pts (74.4%). Complete metabolic response rates at end of TRT were comparable between CHOP21, CHOP14 and ACVBP groups: 81.1%, 90.9%, and 85.5%, respectively (p=0.46). 37 (11.8%) pts progressed including 32 (10.2%) who displayed primary refractory disease and 6 (16.2%) pts who relapsed after consolidation ASCT. CNS relapse occurred in 9 (2.9%) pts. Median time between ASCT and relapse was 3 (2-58) months. Patients received the following salvage TRTs: high dose chemotherapy (HDC: R-ICE/R-DHAOX-like) (n=30) followed by second-line consolidation ASCT (n=11/30) and post-ASCT mediastinal RT (n=5/11); salvage RT without chemotherapy (n=1); other regimens (R-CHOP, R-GEMOX) (n=3); none (n=3). 2-yrs second PFS (PFS2) rates in pts who had previously received CHOP21, CHOP14 and ACVBP were: 20.5% vs 62.5% vs 18.8% (p=0.43). Only HDC + ASCT strategy granted disease control (2-yrs PFS2: 32.3%). Median follow up was 44 (1-153) months and the CHOP21, CHOP14, ACVBP 5-yrs PFS and 5-yrs OS were: 74.7% (95%CI: 64-97.1%), 89.4% (82.7-96.6%), 89.4% (84.8-94.2%) (p=0.018, Figure A); and 81% (70-94.4%), 100% (100-100%), 92.4% (88.4-96.7%) (p=0.0036, Figure B), respectively. In a multivariate model including TRT group, consolidation ASCT and/or RT, aaIPI, Bulk & gt;10cm and TMTV≥360cm3, CHOP14 was not associated with better PFS as compared to ACVBP and CHOP21 (p=0.1548). Baseline higher TMTV (≥ 360 cm3) was associated with lower PFS in multivariate analysis, independently of TRT (HR=0.41 [0.2-0.85], p=0.02). All grades TRT-related adverse events were similar between the groups, except for an excess of febrile neutropenia (5.3% vs 5.3% vs 24.4%, p & lt;0.001) and mucositis (1.8% vs 3.9% vs 22.8%, p & lt;0.001) in the ACVBP group. Twenty-two pts died (CHOP21: n=8, ACVBP: n=14) mainly due to lymphoma progression (n=15; 68.2%). 2 toxic deaths were observed (CHOP21: n=1, ACVBP: n=1). Secondary malignancies appeared in 7 pts (CHOP21: n=2, ACVBP: n=5), including 3 cases of acute myeloid leukemia. Conclusion These results confirm the favorable outcome of PMBL pts treated with CHOP14 and ACVBP plus anti CD20 Ab. The toxicity of ACVBP was more pronounced and CHOP14 was associated with a better OS. Baseline TMTV≥360cm3 is a highly predictive factor of unfavorable outcome in PMBL pts, independently of TRT. We recommend R-CHOP14 as standard of care in PMBL. Figure Disclosures Camus: ROCHE: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); PFIZER: Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); AMGEN: Honoraria; JANSSEN: Honoraria. Decazes:Bayer: Other: travel, accomodations, expenses. Bernard:Janssen: Other: Travel and accommodation . Gandhi Laurent:Abbevie, Pfizer, Takeda, Roche: Other: Travel; Roche, Takeda, Iqone, Accord: Consultancy; Roche, Takeda, Accord: Honoraria. Laribi:amgen: Research Funding; novartis: Honoraria, Research Funding; takeda: Research Funding; abbvie: Honoraria, Research Funding. Houot:Gilead: Honoraria; Kite: Honoraria; Roche: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; MSD: Honoraria. Tilly:BMS: Honoraria.

Abstract: Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses and exhibit innate memory following stimulation with IL-12, IL-15, and IL-18, thereby differentiating into cytokine-induced memory-like (ML) NK cells. ML NK cells have well-described enhanced anti-tumor properties; however, the molecular mechanisms underlying their enhanced functionality are not well-understood. Initial reports of allogeneic donor ML NK cellular therapy for relapsed/refractory (rel/ref) acute myeloid leukemia (AML) demonstrated safety and a 47% CR/CRi rate (PMID32826231). In this setting, allogeneic ML NK cells are rejected after 3 weeks by recipient T cells, which precludes long-term evaluation of their biology. To address this limitation, we conducted a clinical trial for rel/ref AML patients that added adoptive transfer of same-donor ML NK cells on day +7 of a reduced-intensity conditioning (RIC) MHC-haploidentical HCT, followed by 4 doses of IL-15 (N-803) over 2 weeks (NCT02782546). Since the ML NK cells are from the HCT donor, they are not rejected, but remain MHC-haploidentical to the patient leukemia. Using samples from these patients, we profiled the single cell transcriptomes of NK cells using multidimensional CITE-seq, combining scRNAseq with a custom NK panel of antibodies. To identify donor ML NK cells in an unbiased fashion, we developed a CITE-seq ML NK classifier from in vitro differentiated paired conventional NK (cNK) and ML NK cells. This classifier was applied via transfer learning to CITE-seq analyzed samples from the donor (cNK cells) and patients at days +28 and +60. This approach identified 28-40% of NK cells as ML at Day +28 post-HCT. Only 1-6% of donor peripheral blood NK cells and 4-7% of NK cells in comparator leukemia patients at day +28 after conventional haplo-HCT alone were identified as ML NK cells (Fig 1A). These ML NK cells had a cell surface receptor profile analogous to a previously reported mass cytometry phenotype. Within the CITE-seq data, ML NK cells expressed a transcriptional profile consistent with enhanced functionality (GZMK, GZMA, GNLY), secreted proteins (LTB, CKLF), a distinct adhesome, and evidence of prior activation (MHC Class II and interferon-inducible genes). ML NK cells had a unique NK receptor repertoire including increased KIR2DL4, KLRC1(NKG2A), CD300A, NCAM1(CD56) , and CD2 with decreased expression of the inhibitory receptor KLRB1(CD161). Furthermore, ML NK cells upregulated HOPX, a transcription factor implicated in memory T cells and murine CMV adaptive NK cells. Additionally, ML NK cells downregulated transcription factors related to terminal maturation (ZEB2) and exhaustion (NR4A2). We next sought to identify changes during ML differentiation in patients post-HCT from day +28 to +60 post-HCT. Trajectory analysis identified a ML NK cell state distinct from cNK cells that was present at least 60 days post-HCT (Fig 1B). The ML transcriptional phenotype continued to modulate during late differentiation, including downregulation of GZMK and NCAM1, and upregulation of maturation related transcription factors, while maintaining high expression of HOPX. ML NK cells retained their enhanced functionality during in vivo differentiation, as patient ML NK cells had significantly increased IFNγ production compared to cNK cells after restimulation with leukemia targets or cytokines using mass cytometry (Fig. 2). Subsequently, we confirmed the ML CITE-seq profile in an independent clinical trial treating pediatric AML relapsed after allogenic HCT with same-donor ML NK cells (NCT03068819). In this setting, ML NK cells expressed a similar transcriptional signature and persisted for at least 2 months in the absence of exogenous cytokine support. Thus, ML NK cells possess a distinct transcriptional and surface proteomic profile and undergo in vivo differentiation while persisting within patients for at least 2 months. These findings reveal novel and unique aspects of the ML NK cell molecular program, as well as their prolonged functional persistence in vivo in patients, assisting in future clinical trial design. Figure 1 Figure 1. Disclosures Foltz: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Bednarski: Horizon Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Fehniger: Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; ImmunityBio: Research Funding; Kiadis: Other; Affimed: Research Funding; Compass Therapeutics: Research Funding; HCW Biologics: Research Funding; OrcaBio: Other; Indapta: Other.

Abstract: Background: Autologous stem cell transplantation (ASCT) is the standard of care for eligible patients with newly diagnosed multiple myeloma (MM). Typically, patients receive several cycles of induction therapy to achieve appropriate cytoreduction prior to ASCT. Since the introduction of lenalidomide into induction therapy, there have been conflicting reports about its impact on subsequent autologous peripheral blood stem cell (PBSC) mobilization. Early reports showed a trend of patients failing PBSC mobilization and collection after lenalidomide when granulocyte colony stimulating factor (GCSF) was employed as a single agent. More recent reports suggested that using chemotherapy in combination with growth factors including plerixafor can overcome failures of mobilization. To address these conflicting reports, we evaluated the impact of prior lenalidomide in a large cohort of MM patients undergoing mobilization and collection at a tertiary stem cell transplant center. Methods: The primary aim of this study was to identify the impact of prolonged lenalidomide therapy on mobilization of autologous PBSCs. We examined all patients 18 years of age and older, with a confirmed diagnosis of MM who attempted stem cell mobilization and collection between January 2012 and July 2015 at the Fred Hutchinson Cancer Research Center/Seattle Cancer Care Alliance. Collected information included: demographics, staging according to the International Staging System, results of cytogenetics by conventional karyotype or FISH, prior treatments, number of cycles of lenalidomide received, mobilization method, receipt of plerixafor, number of stem cells collected, days of collection, and receipt of prior radiation therapy. Patients were also scored as successful or failed mobilization (defined as collecting 〈 2.5 x 106 CD34+ cells/kg). The patients were categorized into 3 groups (based on our recommendations for induction duration ≤ 6 cycles) for analysis: 1) Those patients with prior receipt of 〉 6 cycles lenalidomide, 2) Those patients with prior receipt of ≤ 6 cycles of lenalidomide, and 3) Patients without prior lenalidomide exposure. Results: We identified 297 patients with MM who underwent mobilization of PBSCs. Of these, 35 received 〉 6 cycles of lenalidomide (median of 8 cycles, range of 7 - 25), 156 received ≤ 6 cycles of lenalidomide (median of 4 cycles, range 1 - 6), and 106 received no lenalidomide (Table 1). Similar proportions of patients in each group of lenalidomide exposure received chemotherapy mobilization, chemotherapy with plerixafor, GCSF alone, and GCSF with plerixafor. There was no significant difference in receipt of plerixafor between these groups (p=0.41). A second mobilization attempt was made in 1 patient (2.9%) who received 〉 6 cycles of lenalidomide, 1.9% of patients who received ≤ 6 cycles of lenalidomide, and 0% of patients with no prior lenalidomide exposure. Only one patient, who had not received lenalidomide previously, failed to collect. In a multivariate linear regression analysis for association between receipt of 〉 6 cycles of lenalidomide with total number of PBSCs collected, with other covariates including prior radiation therapy, age, and number of prior regimens received, we did not observe a significant effect (regression coefficient of -3.37, p=0.166). We then examined whether receiving 〉 6 cycles of lenalidomide was associated with days of stem cell collection in a multivariate regression; other covariates included prior radiation therapy, age, mobilization regimen, and number of prior regimens, and did not see any association (regression coefficient -0.053, p=0.697). Discussion: In this retrospective analysis of MM patients undergoing autologous PBSC mobilization at a single center, we show that prior lenalidomide exposure did not meaningfully impact the total number of PBSC collected or the number of days of apheresis after controlling for clinically relevant features in a multivariate model. These data suggest that longer courses of induction therapy with lenalidomide-containing regimens to achieve a maximum response before transplant should not adversely impact the ability to collect PBSC, and that arbitrarily limiting lenalidomide use pre-mobilization does not appear warranted. The extensive use of chemotherapy with G-CSF and plerixafor for mobilization at our center may account in part for these findings. Disclosures Cowan: Abbvie: Research Funding; Janssen: Research Funding; Juno Therapeutics: Research Funding; Sanofi: Research Funding. Becker:GlycoMimetics: Research Funding. Gopal:Incyte: Consultancy; Aptevo: Consultancy; Janssen: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; BMS: Research Funding; Teva: Research Funding; Brim: Consultancy; Asana: Consultancy; Merck: Research Funding; Pfizer: Research Funding; Spectrum: Research Funding; Takeda: Research Funding. Holmberg:Millenium-Takeda: Research Funding; Sanofi-Genzyme: Research Funding; Seattle Genetics: Research Funding; Merck Sharpe: Research Funding; Jazz Pharmaceuticals: Consultancy; NCCN: Consultancy.