ALBERT

Description: The optimal source of donor hematopoietic stem cells (HSC) is controversial. Granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood (G-PB) has replaced bone marrow (BM) as the most common allograft source in adults but is associated with donor morbidity and higher rates of chronic graft versus host disease (GVHD) compared to BM. The CXCR4 antagonist plerixafor (Px) mobilizes HSC into the PB (Px-PB) faster than G-CSF and preliminary data suggest both quantitative and qualitative differences in allograft content that may impact clinical outcomes. We sought to assess the efficacy and safety of transplanted allografts collected following mobilization with Px alone in HLA-identical sibling transplantation. This was a Phase II, two-strata, multi-center prospective trial (NCT01696461) to evaluate Px-PB allografts prior to reduced intensity conditioning (RIC) and myeloablative conditioning (MAC) based hematopoietic cell transplantation (HCT). Patients aged 18-65 years with an HLA-ID sibling donor and a hematological malignancy suitable for HCT were eligible. The primary objective was to determine the proportion of donors whose cells could be successfully mobilized and collected with a sufficient CD34+ cell dose using Px as the sole mobilizing agent. Px mobilization was considered successful if ≥ 2.0x10^6 CD34+ cells/kg recipient weight were collected in no more than two leukapheresis (LP) collections. All donors receiving Px were included in the analysis of the primary objective based on the intention-to-treat principle. Secondary objectives included the incidence of acute and chronic adverse events in donors, rates of hematopoietic engraftment, donor chimerism, rates of acute and chronic GVHD, non-relapse mortality (NRM), progression free survival (PFS) and overall survival (OS) for the recipients. From July 2013 to December 2014, 64 donor/recipient pairs were enrolled at 12 centers. Donors received Px at 240μg/kg subcutaneously 4 hours prior to LP. LP was performed processing at least 4X blood volume for up to two consecutive days (a third day was allowed for low CD34+ cell yields after 2 LP procedures) to achieve a target CD34+ cell dose of ≥ 4.0 x 10^6/kg recipient weight with a minimum goal of ≥ 2.0 x 10^6/kg. All allografts were cryopreserved. GVHD prophylaxis included cyclosporine or tacrolimus in combination with methotrexate, mycophenolate mofetil, or sirolimus. G-CSF was given routinely post HCT only to MAC recipients. Patient demographics are provided in Table 1. The median donor age was 56 years (18-65). 64% of the donors were male. Donors underwent one (23%), two (72%), or three (5%) LP procedures. 63 of 64 (98%) donors achieved the primary objective. The median total CD34+ cell dose/kg recipient weight collected within 2 days was 4.6 (0.9-9.6). Maximal donor toxicity following Px injection and LP was grades 0 (30%), 1 (52%), 2 (17%), and 3 (2%). Bloating, flatulence, abdominal pain, headache, paresthesisas, injection site reaction, and dizziness were the most commonly observed toxicities. Bone pain was not observed. The one grade 3 toxicity was a vasovagal episode felt related to LP and unlikely to Px. Toxicities typically resolved within a week of LP. The median follow up is 6.3 months. Median days to ANC (〉0.5 x10^9/L) and Platelet count (〉20 x 10^9/L) recovery were 13.5 (10-148) and 19 (1-76) after MAC and 14.5 (0-25) and 18 (0-141) after RIC, respectively. The cumulative incidence of acute GVHD grades 2-4 and 3-4 at day 100 were 47% (95% CI: 30-64) and 9% (95% CI: 2-22) after MAC and 19% (95% CI: 6-38) and 5% (95% CI: 0-18) after RIC. Probability of NRM at day 100 was 4% (95% CI: 0-13) and 0% after MAC and RIC, respectively. The probability of OS at day 100 was 97% (95% CI: 88-100) and 90% (95% CI: 78-98) after MAC and RIC, respectively. In conclusion, this is the first multi-center trial to demonstrate that as an alternative to G-CSF, Plerixafor rapidly, safely, and effectively mobilizes sufficient numbers of CD34+ cells from HLA-ID sibling donors for HCT following both RIC and MAC regimens. Engraftment was generally prompt and early results of secondary endpoints in recipients are encouraging. Longer follow-up and more extensive analysis of donor allografts and recipient outcomes will be presented at the time of the meeting. Research support was provided in part by Genzyme, a Sanofi Company. Table 1. Characteristics of recipients Table 1. Characteristics of recipients Disclosures Chen: Bayer: Consultancy, Research Funding. Devine:Genzyme: Research Funding.

Description: Introduction: MRD positivity after induction/consolidation therapy in pts with de novo ALL has been shown to carry a very negative impact on outcome. However, the significance of MRD status in the salvage setting has not been extensively studied. Methods: We evaluated 130 pts with R/R B-cell ALL pts who received first (n=68), or second (n=62), salvage therapy between 2010 and 2015. Salvage therapies included single agent inotuzumab ozogamicin (INO; n=75), blinatumomab (n=20), or INO in combination with mini-hyper-CVD (n=35) [Jabbour E et al; EHA 2015]. Of the 130 pts treated, 78 (60%) responded and were assessed for MRD by six-color flow cytometry on marrow samples with a sensitivity of 0.01%. Morphologic responses were defined as follows, complete response (CR); disappearance of all disease with neutrophils ≥ 1.0 X 109/L, platelet 〉 100 X 109/L and blasts ≤ 5%, CRp; CR without platelet recovery, CRi; CR without platelet and/or neutrophil recovery. Results: The clinical characteristics of the 78 responding pts with R/R ALL are summarized in Table 1. Overall, MRD negativity was achieved at response in 41 pts (53%). Among the 41 pts who responded to single agent INO (12 CR, 26 CRp, 3CRi), 17 (41%) achieved a negative MRD status. Among the 11 pts who responded to blinatumomab (9 CR, 2Cri), 8 (73%) achieved a negative MRD status. Among the 26 pts who responded to INO in combination with mini-HCVD (21 CR, 4 CRp, 1CRi), 16 (62%) achieved a negative MRD status. Forty-four pts received allogeneic stem cell transplantation (ASCT): of those, 21 pts were MRD negative and 23 pts were MRD positive at the time of response. Median follow-up was 19 months (2-55). Overall, there was a trend for more durable morphologic responses in MRD negative pts compared with MRD positive pts: the median complete remission durations (CRD) were 17 months and 8 months, with a 2-year CRD rate of 47% and 28% respectively (Table 2). The median event-free survival (EFS) was 12 and 6 months, respectively; the 2-year EFS rates were 32% and 8%, respectively. Similarly, there was a trend for better overall survival (OS) with a median of 17 months and 9 months for pts with negative and positive MRD, respectively; the 2-year OS rates were 36% and 27%, respectively. No difference in outcome was reported whether pts were censored or not at the time of ASCT. Conclusion: In pts with R/R ALL, the achievement of negative MRD in addition to the morphologic response confers an improvement, although not statistically significant, in response duration and survival. Larger number of pts with longer follow-up is needed to validate these findings.Table 1.Clinical Characteristics of Pts (n=78)N (%)/Median [range]Age (years)38 [18-87]Sex (Male)50 (64)Performance Status1 [1-3]WBC (x 109/L)3 [0.3-38]% PB Blasts0 [0-83]% BM Blasts60 [8-97]CytogeneticsDiploid20 (26)t(9;22)4 (5)t(4;11)7 (9)Miscellaneous39 (50)Not done/ Insufficient metaphases8 (10)SalvageInotuzumab Ozogamicin41 (53)Blinatumomab11 (14)Inotuzumab Ozogamicin + mini-HCVD26 (33)Number of prior therapies1 prior therapy46 (59)2 prior therapies32 (41) Table 2. Response Rates and Survival by MRD Status MRD Negative (n=41) MRD Positive (n=37) p CR 24 18 n/a CRp 16 14 n/a CRi 1 5 n/a CRD, median (m) 17 8 0.63 2-year CRD rate (%) 47 28 EFS, median (m) 12 6 0.06 2-year EFS rate (%) 32 8 OS, median (m) 17 9 0.18 2-year OS rate (%) 36 27 Disclosures Cortes: Teva: Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BerGenBio AS: Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. DiNardo:Novartis: Research Funding.

Description: Introduction: Red blood cell (RBC) transfusion is the cornerstone of management in many patients with sickle cell disease (SCD). However, RBC transfusion can be complicated by alloimmunization and hemolytic transfusion reactions in this population despite providing extended phenotype-matched RBC transfusions. This is due to heterogeneity of RBC antigens, unique variant mutations in this population, and genetic mismatch between the blood donor pool and SCD patients in North American settings. In this study, we evaluated the level of discrepancy between RBC antigen genotyping and traditional phenotyping methods and the association of these discrepancies with the presence of RBC alloantibodies in SCD patients at our centre in Canada. Methods: Commencing in January 2015, RBC antigen genotyping has been included in the care for patients with SCD treated at our Hemoglobinopathy Clinic in an academic medical centre. Patient blood samples are sent to a reference laboratory to perform genotyping of RhCE, Kell, Kidd, Duffy, and S antigens. RBC antigen phenotyping was performed locally using both tube and automated solid phase assays. Additional clinical data, demographic and transfusion-related data were obtained from a local transfusion registry databse and thorough clinical chart reviews. Approval from our centre's research ethics board was obtained prior to commencement of data collection. Results: To date, RBC antigen genotyping has been performed on 45/88 SCD patients treated at our centre. The mean age of these patients was 25, and 58% were female. The majority of patients had HbSS SCD genotype (64.4%), or HbSC (26.7%). Overall, 32/45 (71%) of patients had variant mutations detected by genotyping, including 9 (20%) patients with more than one variant mutation. The most common mutation detected was the GATA mutation (n= 23; 51%) resulting in loss of Fyb antigen expression on RBCs, but associated with expression of Fyb on non-erythroid tissues. The RhCE system showed variant mutations resulting in partial expression of antigens in 9 (20%) patients. Alloantibodies were found in 9/36 (25%) patients with either a GATA mutation or no variant mutations. Alloantibodies were found in 2/9 (22.2%) patients with mutations resulting in partial antigen expression. The proportion of patients with any discrepancy between genotyping and phenotyping was 34/45 (75.6%). The largest rates of discordance were seen in the RhCE system, with the c antigen having a kappa of 0.68 and e antigen having a kappa of 0.32 (Table 1). Conclusion: Our results showed a high prevalence of variant mutations and significant discrepancies between genotyping and phenotyping methods, most notably in the RhCE antigen system. Mutations resulting in partial antigen expression were associated with development of alloantibodies in 22.2% of patients in our study, which may have been prevented with a genotype-based antigen-matching strategy. Additionally, knowledge of presence of GATA mutation will enhance feasibility of antigen matching for affected patients, who may have otherwise required RBC units negative for Fyb based on local policies. To our knowledge these results represent the first published data from a Canadian centre, showing similar rates of discrepancy between traditional phenotyping methods and RBC antigen genotyping as reported in other regions. Although phenotype-based matching strategies are used in many centres, these strategies can place patients with partial RBC antigen variant mutations at a direct increased risk of alloimmunization. Thus genotype-based antigen-matching strategies should be considered for transfusion of matched RBCs in patients with SCD. Table 1. Blood Group Antigen Frequency In SCD Patients By Phenotyping/Genotyping with Level of Agreement Between Both Methods Antigen Phenotype Genotype Kappa Positive Negative Positive Negative Partial C 37.78 62.22 28.89 62.22 8.89 0.82 c 88.89 11.11 80.00 11.11 8.89 0.68 E 13.33 86.67 13.33 86.67 1.00 e 97.78 2.22 88.89 2.22 8.89 0.32 Fya 13.33 86.67 13.33 86.67 1.00 Fyb 22.22 68.89 26.67 73.33 0.94 Jka 80.00 20.00 82.22 17.78 0.93 Jkb 51.11 48.89 55.56 44.44 0.91 S 28.89 42.22 46.67 53.33 1.00 s 51.11 6.67 86.67 13.33 1.00 Disclosures No relevant conflicts of interest to declare.

Description: Key Points Reticulocyte maturation involves the release of intact, inside-out autophagic vesicles with PS exposed on their surface. Elevated levels of autophagic vesicles on circulating reticulocytes cause PS exposure in patients with SCD.

Description: Key Points Decitabine priming increases antileukemic effects of selinexor in AML in vitro and in vivo. Decitabine priming allows for decreasing the dose of selinexor in patients, thus increasing tolerability without affecting antileukemic activity.

Description: Background: Midostaurin (M) is a multi-targeted small molecule FLT3 inhibitor which has single agent activity in both internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutant FLT3 AML. The objective of this global rand phase III trial was to determine if the addition of M to ind and consol therapy followed by one year of maint would improve overall survival (OS) compared to standard chemotherapy in younger adults with activating FLT3 muts. Methods: Between May 2008 and October 2011, 3279 previously untreated AML pts age 18-60 (exclusive of acute promyelocytic leukemia) in 225 sites/17 countries were screened for FLT3 muts at one of 7 academic labs (subject to extensive assay cross-validation). Hydroxyurea was allowed for up to 5 d prior to beginning ind therapy while awaiting results of mut testing. Pts were rand for the duration of therapy to M or P stratified by FLT3 mut subtype (TKD v ITD high allelic mut fraction (〉0.7) vs low mut fraction (0.05-0.7). Ind therapy consisted of D 60 mg/m2 IV d1-3 and C 200 mg/m2 d1-7 CIV plus M or P (50 mg po bid, d 8-22). Re-treatment with a second blinded course was allowed if residual AML was noted on a d 21 marrow exam. Pts achieving complete remission (CR) received 4 cycles of C 3g/m2 over 3h q 12h on days 1, 3, and 5 plus M or P (50 mg po bid, d 8-22) followed by a year of maint therapy with M or P (50 mg po bid). Transplantation (SCT) was allowed. With a sample size of 717 pts, the trial was powered to detect an improvement from 16.3 (P) to 20.9 (M) months in median OS (HR = 0.78) using a one-sided alpha of 0.025 and power of 84%. The final analysis was to occur after 509 deaths, but given the slow rate of events (359 deaths by April 2015), the trial was amended to change the timing of the OS analysis, and promote event free survival (EFS, defined as the earliest of death, relapse, or no CR within 61 d of the start of ind) as a key secondary endpoint. The critical value for this primary analysis is set at 0.02286 (1-sided) accounting for the alpha spent at the interim analysis (0.5%). Support: U10CA180821, U10CA180882, CA31946, Novartis Results: 717 pts (341 FLT3 ITD-Low, 214 FLT3 ITD-High; 162 FLT3 TKD) were rand to either M (n=360) or P (n=357). There were no significant differences between the arms in age (median, 48y), race, FLT3 subtype, or baseline CBC except for gender (M, 48.2% male; P, 40.6% male; p=.04). All pts are off active treatment, with a median follow-up of 57 months for surviving pts. No statistically significant differences were observed in the overall rate of grade 3 or higher hematologic or non-hematologic adverse events (AEs) between M and P (regardless of attribution). A total of 37 grade 5 AEs were reported (M, 5.3%; P, 5.0%; p=1.0). No differences in treatment-related grade 5 AEs were observed (M, 3.1%; P, 2.5%; p=0.82). CR rate is 59% (M) and 54% (P) (p=0.18). The HRs comparing M to P for OS is 0.77 (one-sided p = 0.007; Figure 1), and for EFS is 0.80 (one-sided p = 0.004; Figure 2). 402/717 (57%) pts received an allogeneic SCT (M, 58%; P, 54%) at any time; 177/717 (25%) in CR1 (M, 27%; P, 22%). Median time to allogeneic SCT was similar on each arm (M, 5.0 months; P, 4.6; p=0.23). Secondary analyses for OS and EFS censoring at the time of SCT provided similar results (Table). The benefit of M was consistent across all FLT3 subgroups for both EFS and OS (Figure 3). Conclusions: The C10603 trial demonstrated that a prospective trial in a pre-therapy genetically defined subgroup of AML pts was feasible and that the addition of the multi-kinase inhibitor M to standard chemotherapy and for one year of maint therapy significantly improved EFS and OS (in both uncensored and censored for transplant analyses) in pts whose blasts had a TKD or ITD (low or high FLT3 mut burden). These findings may lead to improved outcomes through the use of M as a component of therapy in younger adults with mutant FLT3 AML. Table.ArmMedian, mos (95% CI)p-value 15-year Event rate% (95% CI)HR2(95% CI)OSM74.7 (31.5, * )0.00750.8 (45.4-55.9)0.77 (0.63, 0.95)P26.0 (18.5, 46.5)43.1 (37.6-48.4)OS, SCT censoredM* (*,*)0.04762.6 (54.6-69.7)0.77 (0.56,1.05)P* (36.9, *)54.9 (46.2-62.8)EFSM8.0 (5.3, 10.6)0.004426.7 (22.2-31.5)0.80 (0.67, 0.95)P3.0 (1.9, 5.8)19.1 (15.1-23.6)EFS, SCT censoredM8.2 (5.5, 10.7)0.02524.2 (18.9-29.8)0.84 (0.70, 1.0020)P3.0 (1.9, 5.8)21.8 (16.8-27.3)1Stratified on FLT3 subtype; one-sided, log-rank p-value.2Cox model stratified on FLT3 subtype.*= not attained Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Stone: Celgene: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Novartis: Research Funding; Amgen: Consultancy; Agios: Consultancy; Roche/Genetech: Consultancy; Merck: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Celator: Consultancy; Juno: Consultancy; Abbvie: Consultancy; Karyopharm: Consultancy. Off Label Use: midostaurin- FLT 3 inhibitor. Thiede:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AgenDix GmBH: Equity Ownership. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Medeiros:Celgene: Honoraria, Research Funding; Agios Pharmaceuticals: Honoraria. Schlenk:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Research Funding; Arog: Honoraria, Research Funding; Teva: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding. Larson:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy, Research Funding; Pfizer: Consultancy.

Description: Background: Monoclonal antibodies (mAbs) are an emerging therapeutic class for MM patients (pts). Elotuzumab, a mAb in late-phase clinical development, targets the SLAMF7 receptor expressed highly on MM cells. While its primary mechanism of action is through CD16-mediated ADCC, elotuzumab can also directly activate SLAMF7-expressing NK cells. Gaining a greater understanding of phenotypic and functional changes in NK cells over the course of the disease, and how these changes impact capacity for ADCC, may help identify profiles that can better select pts likely to benefit from elotuzumab or other mAb therapies. Methods: We prospectively performed a comprehensive flow cytometry-based analysis of lymphocyte subsets, focusing on expression of NK cell activating and inhibitory receptors, activation and maturation markers, and degranulation in 30 MM pts (12 newly-diagnosed (ND), 18 relapsed/refractory (RR)) and 19 aged-matched healthy donors (HD). Over 140 immune parameters were analyzed, with differences in expression between HD and pt subsets compared by Wilcoxon rank-sum test. We analyzed correlations between expression of certain markers with each other, and with elotuzumab-induced NK cell degranulation against MM cell targets (MM1R) in a 2-hour co-culture assay. We also compared NK cell parameters in blood and bone marrow (BM) from pts with matched samples available. Results: Within the blood, there was no difference in relative NK cell frequency between the groups, and little difference phenotypically between HD and ND pt NK cells, except for decreased DNAM1 expression in ND. In contrast, in comparison to HD, CD56dim NK cells in RR pts were less mature with a higher CD56bright to CD56dim NK cell ratio and reduced expression of the terminal differentiation/maturation markers, CD57 and KLRG1. RR pts also showed increased expression of the activation marker CD69 on all NK cells, and their CD56dim NK cells had increased levels of the natural cytotoxicity receptors, NKp30 and NKp46 and decreased expression of activating receptors DNAM1 and NKG2D. SLAMF7 expression was also increased in RR pts, but only on the CD56bright subset. Consistent changes in NK cell expression of checkpoint/co-stimulatory molecules (eg. PD-1, Tim3, LAG3, CD137) were not seen. Despite these phenotypic changes, no significant differences between groups were noted for elotuzumab-induced ADCC against MM1R targets, as measured by CD107a degranulation by CD56dim NK cells, with significant variability noted within groups. Interestingly, the expression levels of SLAMF7 on CD56dim NK cells directly correlated with CD16 levels, particularly within RR pts (Fig.), suggesting cooperative interactions between these receptors that may be beneficial in MM patients treated with elotuzumab. In addition, degranulation toward elotuzumab-treated MM1R targets was significantly associated with surface expression levels of both SLAMF7 and CD16 on the CD56dim NK cells. The status of NK cells was also compared between matching blood and BM samples from ND (n=7) and RR (n=8) pts. NK cell phenotype and degranulation in blood and BM were similar in ND pts, but in RR pts, expression of CD69 and SLAMF7 were higher on BM-derived NK cells, and CD56dim NK cells from BM demonstrated greater degranulation toward elotuzumab-treated MM1R targets. DNAM1 expression was reduced, but NKG2D, NKp30, and NKp46 were upregulated on various NK cell populations in BM from RR pts compared to peripheral blood. Conclusions: Taken together, our data indicate that NK cells in RR MM pts had increased activation, reduced maturation status, and distinct changes in activating receptor expression levels that are often further enhanced in the BM microenvironment. Furthermore, CD56dim NK cells in many RR pts had parallel increased expression levels of CD16 and SLAMF7, which correlated with enhanced degranulation toward elotuzumab-treated MM target cells. The fact that these changes are seen primarily in RR pts rather than untreated ND pts implies a significant impact of disease evolution and prior therapy on the NK cell compartment, and supports further exploration of these parameters as potential biomarkers of activity of elotuzumab and other therapeutic mAbs in myeloma. Figure 1. Figure 1. Disclosures Campbell: Bristol-Meyers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cohen:Bristol-Meyers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.

Description: Acute myeloid leukemia (AML) is a lethal malignancy because patients who initially respond to chemotherapy eventually relapse with treatment-resistant disease. Leukemia stem cells (LSCs) reestablish the disease by self-renewal: the ability of a stem cell to reproduce itself and give rise to progeny. LSC self-renewal is therefore critical to relapse. Most anticancer therapies are designed to inhibit proliferation. Yet, the mechanisms that direct hematopoietic stem cell (HSC) proliferation are distinct from the mechanisms that allow HSCs to self-renew (Li et al. Nature 2013). Consequently, targeting proliferation may explain the failure of traditional chemotherapy to eradicate this disease. To study leukemia self-renewal, we use a manipulatable, transgenic mouse model of AML with an Mll-AF9 fusion and a tetracycline repressible, activated NRAS (NRASG12V, Kim et al. Blood 2009). Doxycycline abolishes NRASG12V expression leading to leukemia remission. We demonstrated that expression of NRASG12V is required for self-renewal in this AML model and that NRASG12V -mediated signaling is distinct among leukemic subsets (Sachs et al. Blood 2014). We hypothesize that NRAS-activated pathways required for LSC self-renewal are limited to a subpopulation of cells with the LSC immunophenotype. Defining the mechanisms of self-renewal has been a challenge because cancer cells are highly heterogeneous and because disengaging proliferation from self-renewal can be difficult experimentally. To overcome these obstacles, we use single-cell technologies (single-cell, whole transcriptome, RNA sequencing and mass cytometry, CyTOF) to define the signaling and transcriptional profiles of individual cells. We performed single-cell RNA sequencing on unsorted leukemia cells and on a sorted, LSC-enriched population. The single-cell transcriptional profile of LSCs was distinct from the bulk population (Fig. 1A). The 100 most differentially expressed genes between these groups are involved in hematopoietic cell fate and differentiation, confirming the biological validity of this technique. Next, we sought to identify an NRASG12V -mediated self-renewing subpopulation among the LSCs. Unsupervised, two-dimensional, hierarchical clustering of LSC single-cell data identified three discrete subpopulations among the LSCs, each expressing a unique gene expression profile (Fig. 1B). Comparing the single-cell transcriptional profiles of NRASG12V -expressing LSCs to those of LSCs treated with doxycycline to extinguish NRASG12V ("RAS-On" and "RAS-Off" LSCs) revealed that two of the three LSC-expression profiles seen in RAS-on cells (Groups 1 and 3, Fig. 1B) are lost when NRASG12V is withdrawn (Fig. 1C). These data suggest that these two profiles (Groups 1 and 3) are NRASG12V -dependent, consistent with an earlier report that activated NRAS exerts bimodal effects on HSCs (Li et al., Nature 2013). Gene set enrichment analysis of these profiles, modified for single-cell data, revealed that Group 1 preferentially expresses genes associated with leukemia self-renewal. On the basis of this gene expression data, we identfied cell surface markers (CD36 and CD69) that delineate the two NRASG12V -responsive LSC-subpopulations (Groups 1 and 3). We sorted LSCs based on CD36 and CD69 status and found that CD36- CD69+ LSCs (consistent with Group 1 gene expression) harbor nearly all of the colony-forming capacity of the LSCs, forming an average of 13 colonies versus 0.33 colonies for CD36+CD69- LSCs (Group 3) and versus 0.11 colonies for non-LSCs (per 10,000 cells plated, p 〈 0.00001 for each comparison). We have previously shown that colony-forming capacity is an accurate surrogate for in vivo leukemia reconstituting ability and self-renewal in our model (Sachs, et al. Blood 2014). These experiments characterize the NRASG12V -mediated self-renewal transcriptional signature and suggest that single-cell RNA sequencing data may be an effective tool for delineating the self-renewing subpopluation among immunophenotypically-defined LSCs. Using mass cytometry to query the activation status of signaling pathways simulteneously with multiple immunophenotypic markers, we show that Ki67Low LSCs (the putative self-renewing LSCs) preferentially express increased levels of b-catenin and Myc. These data implicating AML self-renewal pathways can provide precise molecular targets for treating this deadly disease. Disclosures Largaespada: NeoClone Biotechnology, Inc.: Consultancy, Other: stockholder; Genentech Inc: Honoraria, Research Funding; Discovery Genomics Inc.: Consultancy, Other: stockholder; B-MoGen Biotechnologies Inc.: Consultancy, Other: stockholder; Orbimed Inc: Consultancy.

Description: Background: Telemedicine (TM) is the exchange of medical information from one site to another via electronic communications to improve patients' health status and access to care. We used telemedicine to provide comprehensive hematology services to patients with blood disorders at a variety of sites that included medical homes, hospital specialty clinics, HTC, patients home and teleconsultation with out of state HTC, all located at a distance from the Michigan State University Center for Bleeding and Clotting Disorders (MSU CBCD). The goals were to increase access to family centered and culturally competent specialty care and increase the numbers of patients with blood/bleeding/clotting disorder that were timely and accurately diagnosed, managed and referred for specialty care. Objectives: 1) To understand the feasibility of telemedicine between specialists and a variety of remote sites for children with bleeding/blood disorders 2) To assess the acceptability by patients, families, primary care physicians, remote site staff, specialist physicians, and specialist staff and 3) To assess the cost of telemedicine visits versus traditional visits from the societal perspective 4) To provide education and resources to distant providers. Methods: Telemedicine sites were 1) two medical homes-one at a pediatrician's office, Upper Great Lakes Family Health Center (UGLFHC)/Portage Health in Houghton MI and the other at a family medicine office, Iron Mountain/Iron River Upper Peninsula, MI 2) specialty clinics at Marquette General Hospital, Marquette MI 3) Hemophilia Treatment Centers (HTC) at Traverse City MI and Rush University IL 4) and a patient's home in MI. PolycomTM and/or VidyoTM systems were used to deliver HIPAA (Health Insurance Portability and Accountability Act) regulated bidirectional videoconferencing technology. Records of patient visits seen by local providers and via TM by MSU CBCD pediatric hematologists, nurse and social worker were obtained. Data included patient diagnosis, distance traveled, estimated time and travel costs saved, and education of distance provider. Measurable objectives included feasibility, acceptability. Technology issues were addressed. Site visits with Portage and Iron River Health Department were conducted to discuss local barriers and needs. Results: Beginning 1998, a total of 68 patients, ages 2 weeks to 17 years and their families were seen by TM at various sites. There were 97 TM visits and frequency of clinics varied monthly to as needed. Average distance travelled by patients to local clinics ranged from 12 - 70 miles (range 2-170 miles). In addition to coagulation disorders (hemophilia, von Willebrands disease, factor deficiencies, patients with bleeding symptoms such as epistaxis, menorrhagia etc.), other diagnosis such as cytopenias, spherocytosis, hemoglobinopathies, lymphadenopathy, Hereditary Hemorrhagic Telangiectasia, Ehlers Danlos Syndrome, and pediatric oncology patients in follow-up phase were also seen and in some cases family members tested. Physician and staff were educated regarding disease management; guidelines and educational materials shared. Estimated costs per patients to fly or drive to MSUCBCD were $999 ($579 -$1275) and $1653 ($1405-1887) respectively compared to obtaining care locally $87.6 ($73-117) via TM. This resulted in cost savings per patient of $911.4 and $1565.7 for driving and flying respectively. The MSU CBCD team also incurred significant cost savings by avoiding travel/lodging costs. Interviews with staff and physicians resulted in improvement in TM acceptance and delivery. A majority of laboratory testing were send-outs and local testing for platelet function defects were not available. Technology issues addressed included adjustments in bandwidth, firewall and network settings. It was feasible to do consultations with out-of-state HTCs as well as follow patients at home. Conclusion: Besides improving access to specialty care, TM with remote areas is feasible and acceptable in a variety of settings with considerable time and cost savings for both patients as well as the specialty care treatment team. Our study is in accordance with the American Academy of Pediatrics recent policy statement (Pediatrics 2015;136:202-209) recommending that TM services be coordinated through the medical homes thereby involving local primary care providers. Disclosures Kulkarni: Biogen: Research Funding, Speakers Bureau; Baxter: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Kedrion: Membership on an entity's Board of Directors or advisory committees; BPL: Membership on an entity's Board of Directors or advisory committees. Witkop:Pfizer: Other: Advisory Board, Research Funding; Baxter Bioscience: Other: Advisory Board; Novo Nordisk: Other: Advisory Board, Speakers Bureau.

Description: Multiple myeloma bone disease (MMBD) is a paradigm for uncoupled bone remodeling and is characterized by non-healing lytic bone lesions. Osteoblast (OB) function is highly suppressed or absent, persists in the absence of MM cells, and remains a significant cause of skeletal-related events. The persistence of OB suppression in MMBD suggests that MM cells induce repressive epigenetic changes at the Runx2 gene, the key transcription factor required for OB differentiation of bone marrow stromal cells (BMSC), which are preOB. We reported that TNFα is a major suppressor of OB in MMBD that reduces Runx2 levels by inducing Gfi1, a transcriptional repressor of Runx2. Using ChIP analysis of MM-exposed BMSC, we showed that Gfi1 directly binds the Runx2 promoter and recruits the chromatin corepressor HDAC1 to the Runx2 promoter in MM-exposed BMSC, reducing transcriptionally permissive euchromatin marks such as H3K9ac. Recently, we reported that p62 (sequestosome-1) in BMSC is critical for the formation of MM cell-induced signaling complexes that mediate OB suppression and IL-6 production. We found that XRK3F2, a novel inhibitor of the p62 ZZ domain (p62-ZZ), blunted MM cell-induced repression of Runx2 and induction of Gfi1 in BMSC, and induced new bone formation and remodeling in mice with established MMBD, but did not alter normal bone. In the current study, we further evaluated the specificity of XRK3F2's inhibition of p62-ZZ interactions and investigated the mechanism by which blocking p62-ZZ prevents MM-induced suppression of key osteogenic transcription factors in BMSC. We previously showed that TNFα and MM cell-induced IL-6 production by BMSC are both p62 dependent and independent. XRK3F2 blocked TNFα-induced NFκB signaling in BMSC and MM cells, and partially inhibited TNFα-induced IL-6 production by BMSC. We now report that XRK3F2 blunted the TNFα upregulation observed in BMSC after MM cell coculture but did not alter TNFα production in p62 knockout (p62KO) BMSC following coculture with MM cells. TNFα treatment of p62KO BMSCs resulted in minimal induction of IL-6, which was not altered by XRK3F2. Transfection of p62KO BMSC with full-length p62 constructs restored TNFα-induced IL-6 production and XRK3F2's capacity to reduce TNFα-induced IL-6. In contrast, XRK3F2 had no effect on TNFα-induced IL-6 production by p62KO BMSC transfected with p62 constructs lacking p62-ZZ. To further investigate XRK3F2's mechanism of action, we tested if XRK3F2 prevents Gfi1-induced epigenetic suppression of Runx2 by preventing Gfi1's upregulation and binding to Runx2. ChIP analysis of MM exposed BMSC treated with XRK3F2 demonstrated that XRK3F2 reduced MM-induced Gfi1 occupancy of the Runx2 promoter and prevented MM-induced reduction of H3K9ac. Since the region proximal to the Runx2 promoter contains putative C/EBPβ binding sites, we tested if MM-induced p62-ZZ signaling activates C/EBPβ, a transcription factor that regulates OB lineage maturation and IL-6 production in BMSC and plays a role in the upregulation of Gfi1 expression. MM-exposed BMSC had increased enrichment of C/EBPβ at both the Gfi1 gene and the C/EBPβ-regulated Il6 gene, and XRK3F2 reduced upregulation of MM-induced C/EBPβ binding at both the Gfi1 and Il6 genes. We conclude that XRK3F2's reduction of the MM-induced C/EBPβ binding in BMSC results in decreased Gfi1 mRNA expression, and thereby reduces MM-induced Gfi1 occupancy and HDAC1 recruitment at the Runx2 promoter. These results suggest that targeting p62-ZZ in MMBD may reverse C/EBPβ mediated Gfi1 activation, resulting in rescue of the MM cell-induced epigenetic suppression of Runx2 in BMSC, and that next-generation derivatives of XRK3F2 should impact BMSC-supported MM cell survival. Disclosures Silbermann: Celgene: Research Funding; Amgen: Consultancy. Xie:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Roodman:Eli Lilly: Research Funding; Amgen: Consultancy.