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Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for both malignant and nonmalignant hematologic disorders. However, graft-versus-host disease (GVHD) and malignant relapse limit its therapeutic success. We previously demonstrated that the blockade of interferon-gamma receptor (IFNGR) signaling in donor T cells resulted in a reduction in GVHD while preserving graft-versus-leukemia (GVL) effects. However, the underlying molecular mechanisms remain inconclusive. In this study, we found that S100A9 is a novel GVHD suppressor upregulated when IFNGR is blocked in T cells. Both Ifngr1−/− and S100a9-overexpressing T cells significantly reduced GVHD without compromising GVL, altering donor T-cell trafficking to GVHD target organs in our mouse model of allo-HSCT. In addition, in vivo administration of recombinant murine S100A9 proteins prolongs the overall survival of recipient mice. Furthermore, in vivo administration of anti-human IFNGRα neutralizing antibody (αhGR-Nab) significantly upregulates the expression of S100A9 in human T cells and improved GVHD in our mouse model of xenogeneic human peripheral blood mononuclear cell transplantation. Consistent with S100a9-overexpressing T cells in our allo-HSCT model, αhGR-Nab reduced human T-cell trafficking to the GVHD target organs. Taken together, S100A9, a downstream molecule suppressed by IFNGR signaling, functions as a novel GVHD suppressor without compromising GVL.

Historically, Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) has been associated with poor outcomes, and allogeneic hematopoietic cell transplantation (allo-HCT) is recommended in first complete remission (CR1). However, in the tyrosine kinase inhibitor (TKI) era, rapid attainment of a complete molecular remission (CMR) is associated with excellent outcomes without allo-HCT, suggesting transplant may not be required for these patients. To test this hypothesis, we retrospectively identified adult patients with Ph+ ALL treated with induction therapy, including TKIs, and attained CMR within 90 days of diagnosis at 5 transplant centers in the United States. We compared outcomes of those who did and did not receive allo-HCT in first remission. We identified 230 patients (allo-HCT: 98; non-HCT: 132). The allo-HCT cohort was younger with better performance status. On multivariable analysis (MVA), allo-HCT was not associated with improved overall survival (adjusted hazard ratio [aHR]: 1.05; 95% CI, 0.63-1.73) or relapse-free survival (aHR: 0.86; 95% CI, 0.54-1.37) compared with non-HCT treatment. Allo-HCT was associated with a lower cumulative incidence of relapse (aHR: 0.32; 95% CI, 0.17-0.62) but higher non-relapse mortality (aHR: 2.59; 95% CI, 1.37-4.89). Propensity score matching analysis confirmed results of MVA. Comparison of reduced-intensity HCT to non-HCT showed no statistically significant difference in any of the above endpoints. In conclusion, adult patients with Ph+ ALL who achieved CMR within 90 days of starting treatment did not derive a survival benefit from allo-HCT in CR1 in this retrospective study.

Natural killer (NK) cells are a promising alternative to T cells for cancer immunotherapy. Adoptive therapies with allogeneic, cytokine-activated NK cells are being investigated in clinical trials. However, the optimal cytokine support after adoptive transfer to promote NK cell expansion, and persistence remains unclear. Correlative studies from 2 independent clinical trial cohorts treated with major histocompatibility complex-haploidentical NK cell therapy for relapsed/refractory acute myeloid leukemia revealed that cytokine support by systemic interleukin-15 (IL-15; N-803) resulted in reduced clinical activity, compared with IL-2. We hypothesized that the mechanism responsible was IL-15/N-803 promoting recipient CD8 T-cell activation that in turn accelerated donor NK cell rejection. This idea was supported by increased proliferating CD8+ T-cell numbers in patients treated with IL-15/N-803, compared with IL-2. Moreover, mixed lymphocyte reactions showed that IL-15/N-803 enhanced responder CD8 T-cell activation and proliferation, compared with IL-2 alone. Additionally, IL-15/N-803 accelerated the ability of responding T cells to kill stimulator-derived memory-like NK cells, demonstrating that additional IL-15 can hasten donor NK cell elimination. Thus, systemic IL-15 used to support allogeneic cell therapy may paradoxically limit their therapeutic window of opportunity and clinical activity. This study indicates that stimulating patient CD8 T-cell allo-rejection responses may critically limit allogeneic cellular therapy supported with IL-15. This trial was registered at www.clinicaltrials.gov as #NCT03050216 and #NCT01898793.

Natural killer (NK) cells are a promising cellular immunotherapy for cancer. Cytokine-induced memory-like (ML) NK cells differentiate after activation with interleukin-12 (IL-12), IL-15, and IL-18, exhibit potent antitumor responses, and safely induce complete remissions in patients with leukemia. However, many cancers are not fully recognized via NK cell receptors. Chimeric antigen receptors (CARs) have been used to enhance tumor-specific recognition by effector lymphocytes. We hypothesized that ML differentiation and CAR engineering would result in complementary improvements in NK cell responses against NK-resistant cancers. To test this idea, peripheral blood ML NK cells were modified to express an anti-CD19 CAR (19-CAR-ML), which displayed significantly increased interferon γ production, degranulation, and specific killing against NK-resistant lymphoma lines and primary targets compared with nonspecific control CAR-ML NK cells or conventional CAR NK cells. The 19-CAR and ML responses were synergistic and CAR specific and required immunoreceptor tyrosine-based activation motif signaling. Furthermore, 19-CAR-ML NK cells generated from lymphoma patients exhibited improved responses against their autologous lymphomas. 19-CAR-ML NK cells controlled lymphoma burden in vivo and improved survival in human xenograft models. Thus, CAR engineering of ML NK cells enhanced responses against resistant cancers and warrants further investigation, with the potential to broaden ML NK cell recognition against a variety of NK cell–resistant tumors.

Introduction: Allogeneic hematopoietic cell transplantation (HCT) in first complete remission (CR1) is the standard of care for adult patients with Philadelphia Chromosome Positive (Ph+) Acute Lymphoblastic Leukemia (ALL). The addition of tyrosine kinase inhibitors (TKI) to therapy have resulted in significantly higher rates of complete molecular remissions (CMR) and better overall outcomes. Given the increased toxicity associated with HCT, we aimed to study the benefit of HCT in adult patients with Ph+ ALL in CR1 in CMR. Methods: We performed a multi-institutional, retrospective analysis of 186 patients ≥18 years of age who received induction therapy including TKI for Ph+ ALL from January 2001 through December 2018 and achieved a CMR CR1. Patients achieving CMR within 3 months of diagnosis were included. Sixty-six patients underwent HCT consolidation (HCT group) and 120 patients did not receive HCT in CR1 (no HCT group). Primary outcomes of interest were overall survival (OS), relapse free survival (RFS), cumulative incidence of relapse (CIR), non-relapse mortality (NRM), and GVHD free relapse free survival (GRFS). GRFS was defined as being alive without grade III-IV acute GVHD, extensive or systemic chronic GVHD requiring therapy, or relapse. Although GRFS in no HCT group should be exactly as RFS, GRFS comparison was done to compare the composite outcome of quality of life in addition to survival in two groups. Landmark analysis was performed at 3 months to ensure CMR in all subjects at time 0. Survival end points were estimated using Kaplan-Meier method and analyzed with the log-rank test and Cox proportional hazard regression models. Gray's test was used for the comparison of cumulative incidence between cohorts. Results: Patient characteristics at diagnosis are summarized in Table 1. Compared to the non-transplanted patients, HCT patients were younger (median age 45 years vs. 56 years, p 90% in 90% of patients vs. 60%, p In both univariate and multivariate analysis, there was no statistically significant difference in OS or RFS between the two treatment groups (Figure 1A and 1B). Compared to the non-transplanted patients, HCT patients had higher rates of NRM (HR: 3.57; 95% CI: 1.62-7.85), lower rates of CIR (Figure 1C), and a trend toward lower GRFS (Figure 1D). Five-year estimates of the probabilities of OS, RFS, CIR, and GRFS were 65%, 59%, 21%, and 38% for allo-HCT group and 58%, 54%, 28%, and 54% for no allo-HCT group, respectively. Conclusions: Comparing transplant versus chemotherapy only consolidation in CR1, this multicenter retrospective study shows that adult patients with Ph+ ALL in CMR have similar estimates of OS and RFS. Lower CIR in the HCT group was offset by higher NRM, resulting in similar RFS. Results of this study need to be confirmed in a prospective randomized trial. Disclosures Ghobadi: Kite: Consultancy, Research Funding; BMS: Consultancy; Amgen: Consultancy, Research Funding; EUSA: Consultancy; WUGEN: Consultancy. Kantarjian:Janssen: Honoraria; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz: Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Honoraria, Research Funding; Sanofi: Research Funding; Oxford Biomedical: Honoraria; BMS: Research Funding; Amgen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Ascentage: Research Funding; BioAscend: Honoraria; Aptitute Health: Honoraria; Adaptive biotechnologies: Honoraria; Delta Fly: Honoraria. Jabbour:AbbVie: Other: Advisory role, Research Funding; Adaptive Biotechnologies: Other: Advisory role, Research Funding; Genentech: Other: Advisory role, Research Funding; BMS: Other: Advisory role, Research Funding; Amgen: Other: Advisory role, Research Funding; Takeda: Other: Advisory role, Research Funding; Pfizer: Other: Advisory role, Research Funding. Short:Astellas: Research Funding; Amgen: Honoraria; AstraZeneca: Consultancy; Takeda Oncology: Consultancy, Honoraria, Research Funding. Uy:Daiichi Sankyo: Consultancy; Astellas Pharma: Honoraria; Agios: Consultancy; Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Genentech: Consultancy. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Champlin:Actinium: Consultancy; Johnson and Johnson: Consultancy; DKMS America: Membership on an entity's Board of Directors or advisory committees; Genzyme: Speakers Bureau; Takeda: Patents & Royalties; Cytonus: Consultancy; Omeros: Consultancy. Ravandi:Macrogenics: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Orsenix: Consultancy, Honoraria, Research Funding; Xencor: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding. Kebriaei:Amgen: Other: Research Support; Jazz: Consultancy; Pfizer: Other: Served on advisory board; Kite: Other: Served on advisory board; Novartis: Other: Served on advisory board; Ziopharm: Other: Research Support.

Aging is associated with an expansion of phenotypic hematopoietic stem cells (HSCs) with reduced self-renewal capacity and myeloid-skewed lineage differentiation. Signals from commensal flora support basal myelopoiesis in young mice; however, their contribution to hematopoietic aging is largely unknown. Here, we characterize hematopoiesis in young and middle-aged mice housed under specific pathogen free (SPF) and germ-free (GF) conditions. We did not analyze older mice due to the difficulty in maintaining mice in a gnotobiotic facility for more than one year. Consistent with prior studies, there is a shift in hematopoiesis in aged SPF mice towards granulopoiesis, with a significant increase in the percentage of granulocytic cells and a decrease in B lineage cells in the bone marrow. The marked shift from lymphopoiesis to myelopoiesis that develops during aging of SPF mice is mostly abrogated in GF mice. Compared with aged SFP mice, there is a marked expansion of B lymphopoiesis in aged GF mice, which is evident at the earliest stages of B cell development. To investigate the impact of microbiota signals on multipotent HSPCs, we first quantified HSPCs by flow cytometry (Figure 1A-B). In aged SPF mice, the number of lineage - Sca1 + cKit + CD150 + CD48 - (LSK-SLAM) cells and CD34 - LSK-SLAM cells is increased 6.4 ± 1.7-fold and 3.4 ± 1.2-fold, respectively. Similar increases were observed in aged GF mice, with LSK-SLAM increasing 5.3 ± 1.6-fold (p=NS compared to SPF mice) and CD34 - LSK-SLAM cells increasing 2.8 ± 0.31-fold (p=NS). To quantify functional HSCs, limiting dilution transplantation experiments using unsorted bone marrow cells was performed. Although on a per cell basis the repopulating activity of aged HSCs is reduced, due to the large increase in phenotypic HSCs, the number of functional HSCs actually increases with aging, with similar increases in functional HSCs in aged SPF and GF mice (Figure 1C). Finally, to assess lineage-bias, we transplanted a limiting number of sorted HSCs and assessed lineage output. As expected, in young SPF mice, the majority of HSCs displayed a balanced myeloid/lymphoid lineage output, with a significant increase in myeloid-biased HSCs observed with aging (Figure 1D). In young GF mice, the majority of HSCs are lymphoid-biased. Moreover, although the myeloid output increased modestly with aging, the majority of HSCs in aged GF remained lymphoid-biased or balanced. Consistent with these data, RNA expression profiling of phenotypic HSCs from aged GF mice show enrichment for non-myeloid biased HSCs. Surprisingly, the RNA expression profiling data also suggest that inflammatory signaling is increased in aged GF HSCs compared with aged SPF HSCs. Collectively, these data suggest that microbiota-related signals suppress the lymphoid potential of HSCs, contributing to the expansion of myeloid-biased HSCs that occurs with aging. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Haploidentical hematopoietic cell transplantation (haplo-HCT) is an increasingly utilized therapy for a variety of hematologic malignancies. Determining which donor characteristics affect transplant outcomes is of particular interest in haplo-HCT, as there are often multiple donors available for a given patient. A survival benefit with younger donors has been reported in some recent observational studies (DeZern et. al., Blood Advances, March 2021); (Canaani et. al., AJH, Sep. 2017). A decrease in non-relapse mortality (NRM) and increase in relapse with no overall survival difference associated with younger donors has also been observed (Mariotti et. al., Blood Advances, June 2020). These previous studies have utilized populations with bone marrow as the predominant stem cell source. Solomon et al. (BBMT Sep. 2018) observed poorer survival, increased relapse, and worse NRM with parent donors relative to children in a largely peripheral blood population. HLA DR and DP mismatch were noted to be associated with improved survival. Here we describe outcomes in peripheral blood haplo-HCT and their association with potentially selectable donor characteristics including age and relationship to the patient. Patients and Methods: We performed a retrospective review of patients who underwent peripheral blood haplo-HCT with PtCy from July 2009 through May 2021. A total of 323 patients were identified with AML (205), ALL (43), MDS (26), and other (49). Univariate and multivariate analyses (MVA) were conducted examining the effect of donor characteristics on overall survival (OS), NRM, relapse, acute and chronic GVHD. Donor characteristics included age, relationship, ABO status, CMV status, and HLA match grade. We controlled for patient characteristics known to affect outcomes including disease type, DRI, HCT CI, KPS, active disease at transplant, myeloablative conditioning, and prior HCT. Results: Median donor age was 40 (range 15-71) with male predominance (64%). Most were ABO compatible (63%) - 12% had major ABO mismatch, 20% minor, and 4% bidirectional. Donor-recipient CMV status matched in 61% of pairs, 13% were donor positive-recipient negative, 26% donor negative-recipient positive. Most were 5/10 HLA matched (51%) with 20% 6/10 and 13% 7-9/10. Univariate analysis revealed that increasing donor age was associated with higher NRM (HR 2.29, p=0.005 for donors age 30-44; HR 2.06, p=0.012 age > 44) but lower relapse risk (HR 0.56, p=0.012 age 30-44; HR 0.69, p=0.10 age > 44). There were no differences in aGVHD or cGVHD based on donor characteristics in univariate analysis. In MVA, relapse risk was lower in patients with older donors , p=0.046). In contrast, NRM was higher in patients with older donors (HR 1.73 age 30-44, HR 1.69 age > 44, p=0.010). There was no difference in overall survival based on donor age (HR 1.23 age 30-44, HR 1.38 age > 44, p=0.11). We next examined the effect of donor relationship on outcomes while controlling for donor age, patient age, and patient disease risk factors. We found no difference in outcomes between parent, sibling, or child donors. Conclusions: Increasing donor age was associated with lower relapse risk but higher NRM. These competing effects resulted in no difference in OS based on donor age. Other donor factors including relationship (parent / sibling / child), CMV status, ABO mismatch, donor sex, and HLA match grade were not associated with outcomes. Solomon et al. reported better outcomes with child compared to parent donors, a finding not replicated here, however our analysis controlled for donor age which could have been a proxy for relationship in their study. These data suggest that in peripheral blood haplo-HCT, younger donors may be preferred in patients with high risk of transplant related complications. In contrast, older donors may be preferred in patients where relapse risk is high. Data on HLA-DR and DP match is being analyzed and will be presented at the ASH 2021 meeting. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: Up to 70% of allogeneic stem cell transplant (alloSCT) recipients who are cytomegalovirus (CMV) seropositive experience CMV reactivation and up to a third are complicated by end-organ disease. Letermovir, an antiviral agent targeting CMV, is FDA-approved for prophylaxis of CMV reactivation in CMV seropositive recipients of alloSCT. 1-4 In CMV seropositive recipients, letermovir use up to week 14 after alloSCT has demonstrated a significantly lower incidence of clinically significant CMV infection (csCMVi) and an overall mortality benefit as far as 24 weeks after transplant. 5-8 However, data on csCMVi beyond 24 weeks is lacking. Additionally, more information is needed on letermovir use in patients who had prior alloSCT, are CMV seronegative, have a detectable CMV viral load but without csCMVi, and/or are high risk*. Methods: This is a single-center, retrospective, comparative cohort analysis of 524 patients who received alloSCT at Barnes-Jewish Hospital from January 2016 to June 2019. Of those, 191 patients were excluded because both the recipient and donor were seronegative for CMV. Patient information was obtained from the electronic medical record systems after IRB approval of the protocol. Gray's sub-distribution methods (while account for death as competing risk) were used to calculate the incidence of csCMVi and to assess the effect of letermovir on csCMVi. Univariate and multivariate Cox proportional hazards models were fitted for the effect of letermovir on overall survival (OS), and time-dependent Cox model was used to determine the effect of csCMVi on OS. Results: Out of 333 patients, 149 received letermovir. The median follow-up period was 13.38 months (0.033 to 63.8 months). A univariate analysis demonstrated that csCMVi was associated with worse OS (HR 2.173, 95% CI 1.602-2.948). Among those who received letermovir, there were reductions in csCMVi for the overall cohort and for high-risk patients at 100 days, 180 days, and 365 days after alloSCT. In seropositive recipients receiving seropositive alloSCT (CMV +/+), there were reductions in csCMVi at days 100, 180, and 365 after transplant. In seropositive recipients receiving seronegative alloSCT (CMV +/-), there was a reduction in csCMVi at 100 days after transplant. There was a reduction in CMV-related mortality at day 180 post-transplant (p=0.03) but not at day 365 (p=0.46). Additionally, for the overall cohort, the letermovir group showed worse OS from days 180-365 (HR 1.938, 95% CI 1.143-3.285). In CMV seronegative patients receiving seropositive SCT (CMV -/+), no difference in csCMVi was detected at days 100, 180, and 365. A landmark analysis of Day 100 IgG level effect on csCMVi incidence demonstrated that lower IgG level ( Conclusion: Letermovir prophylaxis reduces csCMVi overall and in high risk and CMV seropositive patients. We observed a reduction in CMV-related mortality at 180 days after transplant with letermovir prophylaxis. A worsening of OS after day 180 and a trend towards worse non-relapse mortality was surprising and may be related to a higher degree of immunosuppression associated with post-transplant cyclophosphamide (PTCy) use, haploidentical SCT, thymoglobulin use, prior SCTs, and CMV high-risk* patients. Additionally, letermovir was stopped at day 100 at which point survival worsens and the incidence of csCMVi precipitously increases. Our results suggest that there may be additional benefit to extending letermovir therapy past day 100 in the high-risk groups, haploidentical SCT group, patients with low IgG levels at day 100, and subclinical CMV viremia. *High risk patients were defined as those receiving haploidentical, mismatched related, mismatched unrelated, umbilical cord blood, T-cell depleted graft stem cell transplants or immunosuppression with methylprednisolone > 1 mg/kg or 2 or more immunosuppressants. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

To the editor: Quantitative response evaluation in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDSs) relies on the morphologic quantification of bone marrow (BM) blasts. This process is subject to the operator-dependent quality of BM collection and the interobserver variability

T-cell acute lymphoblastic leukemia (T-ALL) is diagnosed in approximately 2,500 individuals in the United States each year. The five-year event-free survival rate for this aggressive hematologic malignancy declines with age, ranging from 70-75% for pediatric patients to 21.7% for patients aged 60-79. Thus, there is an unmet clinical need for better therapies for T-ALL. Mutations that result in Notch activation are present in the majority of T-ALL and are thought to induce transformation, in part, through activation of c-Myc. Increased expression of c-Myc induces apoptosis through a p19(ARF)-Mdm2-p53 tumor suppressor pathway. Thus, it is not surprising that over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). Of note, greater than 95% of primary T-ALL cases and 75% of relapsed cases maintain wild type p53. Based on these observations, we hypothesize that treatment with MDM2 inhibitors, by bypassing the loss of ARF, will increase p53 expression and lead to T-ALL cell apoptosis. We further hypothesize that inhibition of pro-survival BH3 domain proteins, in particular BCLXL, will synergize with MDM2 inhibition to kill T-ALL cells. Of note, a prior study showed that most T-ALL (~62%) respond to treatment with ABT-263 (navitoclax), an inhibitor of BCL2, BCLXL, and BCLW, although most responses were transient. To test this hypothesis, we assessed the response of four different T-ALL xenografts to treatment with idasanutlin (a second generation MDM2 inhibitor) alone, ABT-263 (navitoclax) alone, or their combination. In each case, human T-ALL cells were injected into immunodeficient NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl) mice. Leukemic burden was measured in the blood by flow cytometry for human CD45+ cells. Once engraftment of T-ALL was observed, mice were randomized to one of four treatments: 1) ABT-263 100 mg/kg by oral gavage daily for 14 days; 2) idasanutlin 40 mg/kg by oral gavage daily on a 5-days-on 2-days-off schedule for 14 days; 3) combined idasanutlin and ABT-263; and 4) vehicle alone. Two independent experiments were performed with cohort sizes ranging from 15 (3-4 per treatment) to 31 (7-8 per treatment). Treatment with ABT-263 alone induced a significant decrease in T-ALL burden in all 4 xenotransplants (Figure 1). Treatment with idasanutlin alone induced a significant decrease in T-ALL burden in 3 of 4 xenotransplants. The non-responding xenograft was an ETP T-ALL. Finally, a marked response to combination treatment with idasanutlin and ABT-263 was seen in all four T-ALL xenografts. Indeed, the combination was found to be synergistic in each case based on a modified Bliss Independence test, analyzing average daily change for synergy interaction. In addition, overall survival was significantly increased in the combination treatment group. These data show that the combination of idasanutlin and ABT-263 is highly active in T-ALL and merits consideration for clinical testing. Disclosures Johansson: Roche: Other: Roche provided Idasanutlin free of charge. No compensation was provided, and Roche was not involved in design, conduction, or analysis of experiments.. Gauthier:Roche: Other: Roche provided Idasanutlin free of charge. No compensation was provided, and Roche was not involved in design, conduction, or analysis of experiments.. Link:Roche: Other: Roche provided Idasanutlin free of charge. No compensation was provided, and Roche was not involved in design, conduction, or analysis of experiments..

Background: Maintenance therapy with lenalidomide post-autologous stem cell transplantation (ASCT) has shown to improve progression-free survival (PFS) in multiple myeloma (MM), and has largely become the standard of care. However, toxicity leads to early discontinuation in nearly one-third of patients and additional options are needed (McCarthy, et al, JCO, 2017). Ixazomib is another maintenance option that has been shown to improve PFS; however, studies comparing lenalidomide and ixazomib are lacking. In this randomized phase 2 study, we analyzed the safety and efficacy of using lenalidomide and ixazomib as part of consolidation and maintenance therapies after ASCT (NCT02253316). Methods: Eligible patients, age 18-70 with newly diagnosed MM undergoing ASCT during first-line treatment, were consented prior to ASCT. Approximately 4 months following ASCT, patients received 4 cycles of consolidation therapy with IRd [ixazomib 4 mg on days 1, 8 and 15 of a 28-day cycle, lenalidomide 15 mg on days 1 through 21, and dexamethasone 40 mg on days 1, 8 and 15]. Primary data on IRd consolidation were presented at ASH 2018 (Abstract 109920). One month after the last consolidation cycle, patients were randomized (1:1) to maintenance therapy with single-agent ixazomib (4 mg on days 1, 8 and 15 of a 28-day cycle) or lenalidomide (10 mg daily months 1-3 followed by 15 mg for months 4+). The arms were stratified based on MRD-status post-consolidation. In total, 237 patients were enrolled from 10 US centers. This abstract coincides with planned interim analysis 3 which is the first comparison of ixazomib and lenalidomide maintenance. While the study was not powered to compare PFS between the two arms, the sample will provide a reasonable power to estimate non-inferiority. There is a planned stopping rule for non-inferiority set at a hazard ratio of >1.3 in favor of lenalidomide. Secondary end-points include MRD-negativity following 12 cycles and toxicity. Results: At time of abstract submission, 215 patients had completed IRd consolidation and 191 had begun maintenance. 90 were randomized to ixazomib and 94 to lenalidomide. 7 patients were not randomized due to toxicity during consolidation; data from these patients are not included in the analyses. The characteristics of the two arms are summarized in Table 1. Hematologic toxicity has been infrequent with ixazomib with neutropenia and thrombocytopenia occurring in 11% and 23% of patients. In comparison, neutropenia and thrombocytopenia occurred in 45% and 35% of patients on lenalidomide. The most common non-hematologic toxicities in both arms have been GI-related and infections, both expected events. 16% of patients on ixazomib have experienced Grade 3-4 non-hematologic toxicity compared to 34% on lenalidomide. No grade 3 or higher peripheral neuropathy has been reported in either arm. 11% of patients on ixazomib have discontinued due to toxicity and another 9% have required a dose reduction to 3mg. Lenalidomide toxicity has led to discontinuation in 15% of patients and another 12% were dose reduced to 5mg. Only 45% of patients receiving 4+ cycles of lenalidomide were able to titrate to the 15mg dose. After a median follow-up of 11.2 months from randomization (19.7 months post-ASCT), 30% of patients on ixazomib have discontinued treatment due to disease progression. After a median follow-up of 12.3 months from randomization (20.2 months post-ASCT), 18% patients on lenalidomide have discontinued treatment due to disease progression. Conclusion: Ixazomib and lenalidomide maintenance have been well tolerated to date. A comparison of PFS is currently being conducted as part of interim analysis 3 and final results will be presented, representing the first report directly comparing lenalidomide and ixazomib maintenance. Table 1: Disclosures Vij: Genentech: Honoraria; Karyopharm: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Janssen: Honoraria; Sanofi: Honoraria. Martin:Amgen, Sanofi, Seattle Genetics: Research Funding; Roche and Juno: Consultancy. Fiala:Incyte: Research Funding. Deol:Novartis: Other: Advisory board; Kite: Other: Advisory board; Agios: Other: Advisory board. Kaufman:Celgene: Consultancy; Winship Cancer Institute of Emory University: Employment; Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy; Janssen: Honoraria; Incyte: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy; Takeda: Consultancy. Hofmeister:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees. Gregory:Poseida: Research Funding; Celgene: Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau. Berdeja:AbbVie Inc, Amgen Inc, Acetylon Pharmaceuticals Inc, Bluebird Bio, Bristol-Myers Squibb Company, Celgene Corporation, Constellation Pharma, Curis Inc, Genentech, Glenmark Pharmaceuticals, Janssen Biotech Inc, Kesios Therapeutics, Lilly, Novartis, Poseida: Research Funding; Poseida: Research Funding; Amgen Inc, BioClinica, Celgene Corporation, CRISPR Therapeutics, Bristol-Myers Squibb Company, Janssen Biotech Inc, Karyopharm Therapeutics, Kite Pharma Inc, Prothena, Servier, Takeda Oncology: Consultancy. Chari:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium/Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees; Array Biopharma: Research Funding; GlaxoSmithKline: Research Funding; Novartis Pharmaceuticals: Research Funding; Oncoceutics: Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Rosko:Vyxeos: Other: Travel support.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the major curative immunotherapy for hematologic malignancies. The therapeutic benefits of allo-HSCT are primarily derived from an anti-leukemia effect or graft-versus-leukemia (GvL) that is mediated by mature T cells in donor grafts. Unfortunately, these T cells could react against recipient tissues resulting in graft-versus-host disease (GvHD), a life-threatening complication post allo-HSCT. Due to this strong association between GvL and GvHD, developing a more effective therapeutic strategy to selectively suppress GvHD while preserving or enhancing the GvL effects remains a fundamental yet unmet medical need. We have previously published that co-blockade of interferon gamma receptor (IFNGR) and interleukin-6 receptor (IL6R) results in a complete prevention of GvHD (Choi et al., Leukemia, 2018). Moreover, in vivo administration of baricitinib, an inhibitor of Janus kinases 1 and 2 (JAK1/JAK2) which are downstream of IFNGR and IL6R, also prevents GvHD while preserving GvL in our preclinical murine model. The possible mechanistic pathways by which baricitinib mitigates GvHD include robust upregulation of regulatory T cells (Tregs), inhibition of GvHD-inducing Th1/Th2 cell differentiation, an altered T cell trafficking away from GvHD organs, and reduction of antigen presenting cells' allo-reactivity. Given the potent modulatory effect of baricitinib on GvHD and GvL, we next sought to determine the minimum duration of baricitinib treatment for the complete prevention of GvHD. Since we have found that baricitinib significantly increases Tregs during the first two weeks of the treatment, we hypothesized that two weeks of baricitinib administration is sufficient to completely prevent GvHD. We performed allo-HSCT in which B6 (H-2b) T cell-depleted bone marrow cells (5x106) along with pan T cells (5x105) were intravenously injected into lethally irradiated Balb/c mice (H-2d). Baricitinib was subcutaneously administered to the recipient mice once a day (5 days/week) for a period of one, two, three, or four weeks following allo-HSCT. Overall survival rates, clinical GvHD scores, complete blood counts, immunophenotyping of blood cells and histopathological scores were assessed to determine whether the GvHD was prevented. We have found that two to four-week treatment groups demonstrate significantly better overall survival rates, blood count recovery and histopathological scores than vehicle control or the one-week treatment groups, while no such significant differences were observed among the other groups (Fig. A). All of these data suggest that two weeks of baricitinib treatment is sufficient for complete prevention of GvHD. Lastly, we have previously reported that baricitinib reverses established GvHD (Choi et al., Leukemia, 2018) and we have recently found that both JAK1/JAK2 inhibitors, ruxolitinib and baricitinib, significantly enhance human intestinal organoid growth in vitro (Fig. B). Of note, the intestines are frequently diagnosed and severely damaged GvHD target organs in both human patients and our mouse models of GvHD. Thus, we hypothesized that baricitinib not only modulates immune cell functions as described above, but also promotes restoration of tissues damaged by GvHD. To test this hypothesis, we delayed baricitinib administration until mice developed clinically apparent GvHD and administered baricitinib starting on days 10, 17, or 31 after allo-HSCT for a period of two weeks. Despite this latency, the day 10 group showed 100% overall survival rate (5% in control, p In conclusion, our studies demonstrate that baricitinib-induced blockade of IFNGR and IL6R represents a promising therapeutic strategy for both prevention and treatment of GvHD while preserving GvL. Molecular mechanisms underlying baricitinib-mediated reversal of established GvHD are being currently investigated and will be presented. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Background: High dose post-transplant cyclophosphamide has improved and expanded the use of haploidentical hematopoietic cell transplantation (haplo-HCT). The impact of HLA disparity in this setting, however, is unclear and traditionally measured at the antigen or allele level. The HLA Matchmaker tool more precisely quantifies HLA disparity by calculating the number of mismatched three-dimensional amino acid patches, or epitopes, on the surface of two mismatched HLA antigens. Previous research has shown that class II epitope mismatch (EM), which includes the combination of DRB1 and DQB1 EM, is associated with reduced relapse and delayed engraftment in haplo-HCT (Rimando et al, ASBMT 2018 Abstract ID#53). The individual impact of DRB1 and DQB1 EM on clinical outcomes is currently unclear. Methods: 148 patients who received a peripheral blood T cell-replete haplo-HCT at a single center between July 2009 and November 2017 were retrospectively analyzed. All patients age ≥ 18 were included regardless of diagnosis. The HLA EM load for total class II disparity, DRB1, and DQB1 was quantified using the HLA Matchmaker software and a python script in a dose-dependent and vector-stratified fashion. The primary outcome was the incidence of relapse. The secondary outcomes included relapse-free survival (RFS), time to neutrophil engraftment, and time to platelet engraftment. The association between HLA EM and outcome was analyzed using the Cox proportional hazard model or Gray's sub-distribution method for competing risk as appropriate. The hazard ratios calculated report the hazard rate for a single unit increase in EM. This study was approved by the Institutional Review Board. Results: In this updated cohort, class II graft-versus-host (GvH) EM was again associated with reduced incidence of relapse (HR 0.966; 95% CI 0.938-0.995; p=0.023) and improved RFS (HR 0.978; 95% CI 0.957-0.999; p=0.037) (Table 1). Class II host-versus-graft (HvG) EM was again associated with delayed neutrophil (HR 0.976; 95% CI 0.955-0.997; p=0.027) and platelet (HR 0.971; 95% CI 0.950-0.993; p=0.010) engraftment. Neither DRB1 nor DQB1 GvH EM was independently associated with relapse or RFS. DRB1 HvG EM was associated with worse relapse-free survival (HR 1.038; 95% CI 1.002-1.076; p=0.040). DRB1 HvG EM was associated with delayed time to neutrophil engraftment (HR 0.965; 95% CI 0.934-0.997; p=0.033), while DQB1 HvG EM was not. DQB1 HvG EM was associated with delayed time to platelet engraftment (HR 0.967; 95% CI 0.938-0.997; p=0.032), while DRB1 HvG EM was not. Summary: HLA Matchmaker software enables the quantification of HLA EM in haplo-HCT patients. Neither DRB1 nor DQB1 GvH EM was independently associated with relapse or RFS. DRB1 HvG EM was associated with neutrophil but not platelet engraftment, while DQB1 HvG EM was associated with platelet but not neutrophil engraftment. HLA EM represents a novel strategy to predict clinical outcome in haplo-HCT. The mechanism for the divergent roles of DRB1 EM and DQB1 EM on neutrophil and platelet engraftment are currently unknown and warrant further investigation. Disclosures No relevant conflicts of interest to declare.

T cell malignancies represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults for which there are currently no effective or targeted therapies. Despite intensive multi-agent chemotherapy regimens, fewer than 50% of adults and 75% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage chemotherapy regimens induce remissions in 20-40% of cases. Allogeneic stem cell transplant, with its associated risks and toxicities, is the only curative therapy. T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. Such targeted therapies have shown great potential for inducing both remissions and even long-term relapse-free survival in patients with B cell leukemia and lymphoma7-9. Thus, a targeted therapy against T cell malignancies represents a significant unmet medical need. However, several challenges have limited the clinical development of CAR-T cells against T cell malignancies. First, the shared expression of target antigens between T effector cells and T cell malignancies results in fratricide, or self-killing, of CAR-T cells. Second, harvesting adequate numbers of autologous T cells, without contamination by malignant cells is, at best, technically challenging and prohibitively expensive. Third, the use of genetically modified CAR-T cells from allogeneic donors may result in life-threatening graft-vs.-host disease (GvHD) when infused into immune-compromised HLA-matched or mismatched recipients. We hypothesized that deletion of CD7 and the T cell receptor alpha chain (TRAC) using CRISPR/Cas9 in CAR-T targeting CD7 (UCART7) would result in the efficient targeting and killing of malignant T cells without significant effector T cell fratricide or induction of GvHD. To generate the CD7 CAR, the anti-CD7 single chain variable fragment (scFv) was created using commercial gene synthesis and cloned into the backbone of a 3rd generation CAR with CD28 and 4-1BB internal signaling domains. The construct was modified to express CD34 via a P2A peptide to enable detection of CAR following viral transduction. Human primary T cells were activated using anti-CD3/CD28 beads for 48 hours prior to bead removal and electroporation with CD7 gRNA, TRAC gRNA, and Cas9 mRNA. On day three, T cells were transduced with lentivirus particles encoding either CD7 CAR or CAR CD19 control and allowed to expand for a further 6 days. Transduction efficiency and ablation of CD7 and TRAC were confirmed by flow cytometry. Multiplex CRISPR/Cas9 gene-editing resulted in the simultaneous bi-allelic deletion of both CD7 and TRAC in 72.8%±1.92 of cells, as determined by both non-homologous end joining (NHEJ) and FACS analyses. To prevent alloreactivity, CD3+ CAR-T were removed from the product by magnetic depletion. Of particular importance is that by using two distinct methods for assessing "off-target" nuclease activity across the entire human T cell genome (Guide-seq and probe capture), we could only detect one gene, an intronic modification of RMB33, that was inappropriately targeted using this approach. No obvious genomic rearrangements were detected by these analyses. UCART7 effectively expanded and killed T-ALL cell lines (CCRF-CEM, MOLT3, and HSB2) and human primary T-ALL blasts in vitro. Next, we tested the capacity of UCART7 to kill primary T-ALL in vivo without xenogeneic GvHD. Considerable expansion of alloreactive T cells, severe GvHD (mean clinical GvHD score = 5.66), and a robust graft vs. leukemia effect were observed in recipients of WT T cells. In contrast, GvHD was completely absent, T cells were undetectable, and considerable tumor burden was observed in mice receiving TRACΔ T cells. Mice receiving UCART7, however, had no GvHD and, unlike UCART19 controls, effectively cleared primary human T-ALL in NSG mice. Fratricide-resistant and allo-tolerant 'off-the-shelf' UCART7 signifies a novel strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphomas without a requirement for autologous T cells and represents the first clinically feasible adoptive T cell therapy for T cell malignancies. Disclosures No relevant conflicts of interest to declare.

T-cell-directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123(+) AML.

Introduction: The introduction of reduced intensity conditioning (RIC) regimens, associated with less NRM, has significantly broadened transplant eligibility to older patients. However, graft-versus-host disease (GVHD) continues to be a major problem, even with RIC allo-HCTs. At our institution, we have been routinely adding mycophenolate mofetil (MMF) to the standard methotrexate (MTX) plus Tacrolimus combination in the majority of RIC allo-HCT recipients from unrelated donors. However, there is a paucity of data on whether this approach further decreases GVHD rates without increasing NRM and risk of relapse. To answer these questions, we retrospectively analyzed data from 177 patients who received MMF, MTX and Tacrolimus (MMF + Tac/MTX) as part of their GVHD prophylaxis and compared their outcomes to those of 117 patients who received MTX and Tacrolimus alone (Tac/MTX). Patients and methods: The study included a total of 294 consecutive patients who underwent RIC allo-HCT at Washington University Medical Center between January 2006 and December 2013. The data was analyzed for patients who received GVHD prophylaxis with either tacrolimus/methotrexate (Tac/MTX) or tacrolimus/mycophenolate mofetil/methotrexate (MMF+Tac/MTX). Cumulative incidence of relapse was estimated treating death in remission as competing risk event. Cumulative incidence of NRM was calculated considering death in relapse as competing risk event. Univariate analysis was performed to evaluate variables, and multivariate Cox models with a backward selection procedure were done to assess whether GVHD prophylaxis was an independent predictor after adjusting other factors significant in the univariate analysis. All analyses were two-sided, and significance was set at a p-value of 0.05. Statistical analyses were performed using library cmprsk in statistical package R for competing risk analysis and SAS 9.4 (SAS Institutes, Cary, NC) for all other analyses. Results: 177 patients (60.2%) received MMF+Tac/MTX whereas 117 patients (39.8%) received Tac/MTX. Median age was 61 years in the MMF+Tac/MTX group and 58 years in the Tac/MTX group, and the most common diagnosis was AML(31%) followed by NHL (26%) and MDS (19%) in the MMF+Tac/MTX group vs. AML (45%) followed by MDS (14%) and NHL (13%) in the Tac/MTX group. There were more transplants from unrelated and mismatched donors, higher intermediate ASMBT RFI risk group patients and more frequent use of conditioning with Flu/Bu/Thymo regimen in the MMF+Tac/MTX cohort. In particular 160 (90.4%) patients in MMF+Tac/MTX group received thymoglobulin as part of their conditioning, compared to 64 (54.7%) patients in the Tac/MTX group (p < 0.001). The cumulative incidence of grade 2-4 aGVHD at day 100 was 31.0% in the MMF+Tac/MTX group vs. 36.2% in the Tac/MTX group (p=0.191) while as grade 3-4 aGVHD at day 100 and 1 year were 16.8% and 25.9% in the Tac/MTX group compared to 8.0% and 12.4% in the MMF+Tac/MTX group, respectively (p=0.010). However, after adjusting for the thymoglobin use there was no significant difference in the incidence of grade 3-4 aGVHD in these two groups (p=0.129). To eliminate the confounding effect of thymoglobulin, we performed a subgroup analysis of patients without thymoglobulin. Given the small number of samples (n=17 in MMF+Tac/MTX and n=53 in Tac/MTX), propensity score matching was used and the results again showed no significant difference in severe aGVHD between two groups (p=0.219). The incidence of cGVHD at 1 year was 15.3% and 21.6% in MMF+Tac/MTX and Tac/MTX, respectively (p=0.645). Although patients in the MMF+Tac/MTX group had more unfavorable disease risk, the cumulative incidence of relapse at 3 years was not significantly different: 40.8% in the MMF+Tac/MTX group and 39.8% in the Tac/MTX group (p=0.941). 5-year OS was 33.7% in the MMF+Tac/MTX group, compared to 35.1% in the Tac/MTX group (p=0.769). Conclusion: Here we report transplant outcomes of RIC allo-HCT patients who received MMF in addition to Tac/MTX for GVHD prophylaxis. Interestingly we did not see any increase in NRM or relapse rates with additional immune-suppression from adding MMF in these patients. However, addition of MMF to the Tac/MTX regimen did not result in any significant improvement in acute or chronic GVHD outcomes in our patients. Based on these results we do not recommend adding MMF to Tac/MTX regimen for GVHD prophylaxis in RIC allo-HCT patients. Disclosures Uy: Novartis: Research Funding. Schroeder:Incyte: Consultancy; Celgene: Other: Azacitidine provided for this trial by Celgene. Abboud:Pfizer: Research Funding; Merck: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Teva Pharmaceuticals: Research Funding; Gerson Lehman Group: Consultancy. Cashen:Celgene: Speakers Bureau.

Introduction: CD34+-selected peripheral blood stem cell infusions without conditioning have recently been utilized for poor graft function (PGF) with promising results. Unfortunately, many patients have been unable to receive the boost infusion as their donors were unwilling or unable to undergo an additional stem cell collection. Therefore, we conducted this pilot trial utilizing either fresh or cryopreserved peripheral blood stem cell products to create CD34+-selected peripheral blood stem cell infusions for the treatment of PGF. Additionally, to explore relationship of CD34+ dose and response we included a cohort of donors mobilized with plerixafor in addition to the standard G-CSF. Methods: Poor graft function (PGF) was defined as ANC < 0.5k/μL, platelets < 30k/μL, or platelet or RBC transfusion dependence despite full donor chimerism and in the absence of GVHD more than 60 days following allogeneic stem cell transplant (allo-SCT). Three different donor products were used for CD34+ selection: (1) fresh mobilized product using G-CSF only (SC at a dose of 10 μg/kg daily for 5 days), (2) fresh mobilized products using G-CSF (SC at a dose of 10 μg/kg daily for 5 days, days-4 through day 0) and plerixafor (IV at a dose of 320 μg/kg on day 0), and (3) cryopreserved cells mobilized with G-CSF (SC at a dose of 10 μg/kg daily for 5 days). Seventeen donor-recipient pairs were enrolled onto this prospective study. The original allo-SCT donor either underwent an additional peripheral blood stem cell (PBSC) mobilization and collection with G-CSF only (n=5) or G-CSF plus plerixafor (n=9) or a cryopreserved product (n=3) from a previous collection (using G-CSF only) was used to create the CD34+ cells for infusion. CD34+ cell selection was performed using a CliniMACS. The infusion was not preceded by administration of any chemotherapy or conditioning regimen. Neutrophil improvement was defined as neutrophil count > 500/μl without growth factor support for >7 days; platelet improvement was defined as platelet count ≥ 50,000/µl without platelet transfusion support for > 7 days; and RBC improvement was defined as hemoglobin > 9 g/dL and transfusion independence. Complete response was defined as improvement of all involved cells lines; partial response was defined as improvement of platelets and/or neutrophils with continuing RBC transfusion dependence. Results: The mean post-selection CD34+ count per kg of recipient weight was 3.7x106, 12x106, and 1.7x106 for G-CSF only, G-CSF plus plerixafor, and cryopreserved products, respectively. Mean CD34+ yields (defined as the number of CD34+ cells after selection/CD34+ cells prior to CD34 selection) was 58%, 67%, and 31% for G-CSF only, G-CSF plus plerixafor, and cryopreserved products, respectively (Table 1 and Figure 1). The mean number of CD3+ T cells/kg infused in each product was 0.004 x 106, 0.032 x 106, and 0.015 x 106 for G-CSF only, G-CSF plus plerixafore and cryopreserved groups respectively (Table 1). Following the CD34+-selected stem cell infusion, 12 of the 17 recipients (71%) had a complete response including all 3 who received cryopreserved products; 3 had a partial response (17%) and 2 patients (12%) did not respond. One year relapse-free and overall survival was 71% and 65%. There was no treatment related toxicity reported other than graft-versus-host-disease (GVHD); three (18%) developed acute GVHD (1 grade I localized to the skin, 2 grade II with gut involvement), 6 chronic GVHD (2 limited and 4 extensive) (Table 1). Conclusion: Cryopreserved products seem to be a viable alternative to create CD34+ -selected peripheral blood stem cell infusions for the treatment of PGF. When collecting fresh products is an option, the addition of plerixafor increases CD34+ yield over G-CSF alone. Disclosures Abboud: Teva Phamaceutical: Research Funding. Uy:Novartis: Research Funding.

Introduction: Pre-transplant remission status in patients with acute myeloid leukemia (AML) is one of the most important factors determining their outcomes after allogeneic hematopoietic cell transplantation (allo-HCT). Patients not in remission prior to undergoing allo-HCT continue to have a very poor prognosis. Complete remission with incomplete cell count recovery (CRi) following chemotherapy for acute myeloid leukemia (AML) has also been associated with reduced overall survival and increased risk of relapse. However, there is currently a paucity of data on transplant outcomes in AML patients with ablated marrows after prior chemotherapy. In these patients, waiting for count recovery for an extended time period leads to increased infectious complications, often making them ineligible for allo-HCT. We hypothesized that outcomes of AML patients with pre-transplant ablated marrows who undergo allo-HCT would be similar to those of patients who are in complete remission (CR) prior to allo-HCT. To answer this question, we retrospectively analyzed the outcomes of AML patients from a single institution who underwent allo-HCT in CR, CRi, or with an ablated marrow. Patient and methods: For this retrospective study, we analyzed the outcomes of 270 AML patients (median age 54 years, range 17 to 74 years, 146 males and 124 females) who underwent 6/6 HLA-matched sibling or 10/10 HLA-matched unrelated donor transplantation at Washington University School of Medicine in St. Louis between 2006 and 2013. Patients with persistent disease based on bone marrow biopsy immediately prior to transplant were excluded. Data was collected on baseline characteristics including patient and donor sex, AML type, induction therapy, conditioning regimen, stem cell source, and graft-versus-host disease (GVHD) prophylaxis. Based on their cell count and marrow recovery prior to allo-HCT, patients were divided into 3 categories: CR, CRi, and ablated marrow, using response criteria from the International Working Group. The primary outcomes were overall survival (OS) and disease-free survival (DFS), and the secondary outcomes were cumulative incidence of non-relapse mortality (NRM), cumulative incidence of relapse, acute graft vs. host disease (aGVHD), and chronic graft vs. host disease (cGVHD). Propensity-score matching was used to adjust for potential confounding effects of patient characteristics, and the between-group differences in OS or DFS were compared by Cox proportional hazards model. Results: Of the 270 patients, 206 were in CR, 45 in CRi, and 19 had ablated marrow prior to their allo-HCT. Median follow-up for the CR, CRi, and ablated groups were 524, 410, and 290 days, respectively. The patients with ablated marrows had a 3-year OS of 47% and DFS of 40%, as compared to patients in CR whose 3-year OS and DFS were 49% and 45%, respectively (Fig. 1). Using a 1:3 propensity-score matching (3 CR per 1 ablated), no significant differences were found in OS (HR=1.66, 95% CI 0.88-3.12; P = 0.12) or DFS (HR=1.39, 95% CI 0.75 - 2.58; P = 0.30). The cumulative incidence of NRM in our ablated marrow cohort was 58%, significantly higher than those in CR and CRi who had cumulative incidences of NRM of 22% (P = 0.0004) and 16% (P = 0.002), respectively (Fig. 2). The cumulative incidence of grade 2-4 aGVHD in patients with CR, CRi, and ablated marrow, were 38%, 38%, and 42%, respectively (P = 0.93). Likewise, the cumulative incidence of cGVHD in patients with CR, CRi, and ablated marrow, were 34%, 38%, and 16%, respectively (P = 0.22). Conclusions: Allo-HCT continues to be the curative therapy for patients with AML. Based on our results, overall survival and disease-free survival following transplant for patients with ablated marrows do not differ significantly from those in CR or CRi. Interestingly, there was a higher incidence of non-relapse mortality among patient with ablated marrows compared to those in Cr or CRi, which is most likely related to the use of myeloablative conditioning regimen in our ablated patients. These results could have an important bearing on patients with ablated marrows whom treating physicians might post-pone allo-HCT to wait for marrow recovery, thereby potentially putting them at increased risk for infectious and bleeding complications. Smaller cohort size is the major limitation of our study, and our results suggest investigating this important question in a large registry. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Background Autologous stem cell transplantation (ASCT) after high-dose chemotherapy improves remission rates and prolongs survival in a number of hematologic malignances including multiple myeloma (MM), non-Hodgkin’s lymphoma (NHL), and Hodgkin’s lymphoma (HL).The prerequisite to ASCT is the collection of an adequate number of CD34+ cells; the minimum dose needed to ensure hematopoietic recovery is 2.0x106cells/kg, but recent studies have suggested that 5.0x106cells/kg is the optimal dose as it results in more rapid and sustained engraftment. An estimated 15-30% of patients fail to mobilize the required number of cells for ASCT, although the number of mobilization failures is declining due to advances in mobilization regimens. Previously identified variables for predicting mobilization failure include: advanced age, the type of chemotherapy and number of cycles administered, and prior radiotherapy. Patients/Methods We performed retrospective chart review of 803 consecutive patients who underwent apheresis for autologous stem cell collection at the Siteman Cancer Center from January 2008 through December 2011. Patients were considered eligible for analysis if they had a diagnosis of MM, NHL or HL and had not undergone previous stem cell collection. Univariate analysis was performed to identify predictors associated with successful collection of > 2.0x106 cells/kg and > 5.0x106 cells/kg. Variables analyzed were: mobilization regimen; age; gender; BMI; weight/ideal body weight; diagnosis; prior radiation; number of chemo cycles administered; alkylating agent exposure; lenalidomide exposure; blood glucose level; blood triglycerides level; glucocorticoid use at apheresis; statin use at apheresis; and history of diabetes mellitus, coronary artery disease, hypertension, or hyperlipidemia. Results 706 patients met the eligibility criteria for analysis. The median age was 57.9 years (range 18.5-74.6) and 58% (411) were male. 53% (373) had MM, 38% (266) had NHL, and 9% (67) had HL. 63% (444) of patients were mobilized with plerixafor and G-CSF, 17% (117) with chemotherapy and G-CSF, and 21% (145) with G-CSF alone. After one apheresis procedure, 75% (531) of patients collected > 2.0x106 cells/kg and 50% (356) collected > 5.0x106 cells/kg. 42% (295) of patients underwent more than one apheresis procedure. During their mobilization, 94% (664) of patients were able to collect > 2.0x106 cells/kg and 68% (480) collected > 5.0x106 cells/kg. Data was available on >99% of the population for all variables with the exceptions of number of chemo cycles administered (n= 667), blood triglyceride level (n= 358), and glucocorticoid use at apheresis (n= 660). Significant results from univariate analysis are summarized in table 1. Conclusion The most significant predictors of mobilization failure identified were mobilization regimen, diagnosis, alkylating agent exposure, and number of chemo cycles. Lenalidomide exposure was associated with a reduced risk of mobilization failure, but this is likely attributed to the reduced risk of mobilization failure for MM, as 99% (255) of patients with lenalidomide exposure had MM. Interestingly, diagnosis of hypertension was associated with a reduced risk of mobilization failure. The mobilization failure rate in the study population is 6%, which is below the expected rate found in historical studies. This is likely a result of advances in mobilization regimens as only 21% of the study population was mobilized with G-CSF alone. Disclosures: Abboud: Novartis: Membership on an entity’s Board of Directors or advisory committees; Ariad: Membership on an entity’s Board of Directors or advisory committees; Alexion: Membership on an entity’s Board of Directors or advisory committees; Teva: Speakers Bureau. Schroeder:Celgene: Research Funding; Sanofi: Research Funding. Uy:Celgene: Research Funding; Sanofi: Research Funding. Vij:Celgene: Research Funding, Speakers Bureau; Onyx: Research Funding, Speakers Bureau; Millennium: Speakers Bureau; BMS: Honoraria; Lilly: Honoraria. Westervelt:Celgene: Honoraria, Research Funding. Stockerl-Goldstein:Celgene: Speakers Bureau; Millennium: Speakers Bureau.

Abstract 4945 Owing to serious side effects and multidrug resistance (MDR), systematic chemotherapy is limited in cancer treatment. On the one hand, most of side effects are result from non-selective for target cells of antitumor drugs. On the other hand, the main mechanism of MDR is the overexpression of P-glycoprotein (P-gp). Design and exploitation of novel clinical chemotherapy formulation with dual functional properties of targeted therapy and reversal of drug resistance is a promising approach in tumor therapy. Nanoparticles drug delivery system has been shown to improve cancer therapy by increased drug delivery to target sites because of the enhanced permeability and retention effect (EPR). In the present studies, we developed a new formulation, which magnetic nanoparticles (MNPs) coloaded with daunorubicin (DNR) and 5-bromotetrandrin (Br Tet), an anticancer drug and a MDR reversal agent, and studied the in vitro antitumor efficacy. The drug encapsulation efficiency of DNR and Br Tet (alone or in combination) in MNPs was 62–83%. The release of DNR and Br Tet from nanoparticles was sustained. The DNR and Br Tet loaded MNPs (DNR-Br Tet-MNPs) are provided with good magnetic responsibility. The drugs in combination incorporated in MNPs displayed well synergistic cytotoxicity in the MDR leukemia K562/A02 cells. Fluorescence intensity of intracellular DNR detection demonstrated that DNR-Br Tet-MNPs could be endocytosed by K562/A02 cells and sustained releasing DNR intracellular. The results of RT-PCR and western blot demonstrated that the expressions of MDR1 mRNA and G-pg of K562/A02 cells are decreased by treated with DNR-Br Tet-MNPs after 48 h. It was concluded that the antitumor efficacy of DNR-Br Tet-MNPs was due to the inhibition of Br Tet to P-gp and reducing the efflux of DNR. Our DNR-Br Tet-MNPs could be of high clinical value in the target treatment of many resistant tumors, such as haematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract 2148 Introduction: Myelodysplastic syndrome (MDS) and Acute Myeloid Leukemia (AML) are hematological disorders that exist on a spectrum of ineffective and malignant hematopoiesis. Hypomethylating agents have been recently shown to result in clinical response and improved survival in patients with either disease, although only in a minority of patients. Arsenic trioxide too has shown limited single agent activity in patients with MDS and AML, and ascorbic acid improves response to arsenic trioxide in patients with multiple myeloma. We therefore sought to assess the tolerability of combination decitabine, arsenic and ascorbic acid in patients with MDS and AML. Study: The primary object of this single institution, dose-escalation study was to establish the maximum tolerated dose and dose-limiting toxicities during four cycles of therapy. Arsenic trioxide was administered in three dose cohorts of 3–6 patients each: 0.1 mg/kg, 0.2 mg/kg, and 0.3 mg/kg IV on days 1–5 followed by weekly administration for 15 weeks. All patients received decitabine 20 mg/m2 IV on days 1–5 every 28 days and ascorbic acid 1000 mg IV following every administration of arsenic trioxide. Secondary objectives were to establish overall response rates and effect of therapy on bone marrow angiogenesis. Results: Thirteen patients were enrolled in three dose cohorts [9 men, 4 women; median age: 67 (range 24 – 77); 5 MDS, 7 AML; ECOG 0 (46%), 1 (38%), 2 (15%)]. Most patients were transfusion dependent [RBC dependent: MDS 4/5, AML 4/7; platelet dependent: MDS 2/5, AML 4/7]. Most patients had received prior therapy [MDS 1/5; AML 7/7]. Ten patients received at least 2 cycles with four patients completing four cycles. Dose limiting toxicities were pneumonia/infection, which occurred in the third dosing cohort. Other grade 3–4 toxicities occurring during 4 cycles of treatment were: infection (46%), hypotension (15%), hypoxia/pneumonia (20%), anemia (53%), neutropenia (38%), QTc prolongation (15% - all asymptomatic), pericardial effusion (8%), pleural effusion (8%), hyperglycemia (20%), hypokalemia (8%). According to IWG response criteria, 2/5 MDS patients achieved stable disease, 2/5 developed progressive disease, and 1/5 withdrew prior to reevaluation. Similarly, 3/7 AML patients achieved stable disease, 2/7 progressive disease, and 2/7 died without repeat bone marrow evaluation. No transfusion dependent patients achieved transfusion independence. The median overall survival of these cohorts was 207 days and four patients remain alive with a median follow-up of 490 days. We did not observe a correlation between dosing cohort and response or survival. Because arsenic trioxide has been proposed to inhibit angiogenesis, we assessed bone marrow microvessel density (MVD) using CD34 immunohistochemistry by two binded, independent reviewers. Paired bone marrow samples from eight patients surprisingly revealed an increase in the number of vessels per high powered field during therapy [pre-treatment: (ave 10.3, stdev 5.2); after cycle 2: (ave 18.0, stdev 7.0, p = 0.06); after cycle 4: (ave 17.1, stdev 3.2, p = 0.05)]. Increased MVD occurred independent of response, disease or bone marrow cellularity. Conclusions: This study demonstrates that decitabine can be safely combined with arsenic trioxide and ascorbic acid in a heavily pre-treated population of MDS and AML patients. Our maximum tolerated dose of arsenic was observed in cohort 2: 0.2 mg/kg. Combination therapy resulted in increased bone marrow microvessel density, independent of response and bone marrow cellularity. Phase 2 studies will be required to assess the efficacy of combination therapy. Disclosures: Off Label Use: Decitabine, Arsenic and Ascorbic acid for the treatment of AML. Vij: Eisai: Speakers Bureau; Cephalon: Speakers Bureau.

Abstract 3970 The objective of this paper is to study the biocompatibility and security of self-prepared magnetic nanoparticles Fe3O4 (Fe3O4-MNPs) loaded with daunorubicin (DNR), which has the potential application in hematologic malignancies therapy. Hemolysis test was carried out to estimate it s blood toxicity; Fe3O4-MNPs loaded with DNR were intra peritoneally injected into mouse to calculate the LD50; micronucleus (MN) assay was reckoned to identify its genotoxicity; acute toxicity testing was evaluated its influence to mouse hepatic and renal functions. The result of hemolysis rate (HR) of Fe3O4-MNPs loaded with DNR was 2.908%, far less than 5%. Therefore, we concluded that self-prepated Fe3O4-MNPs loaded with DNR nanoparticles had no hemolytic reaction, and they consistent with the requirement of hemolytic test of biomaterials. The LD50 of Fe3O4-MNPs loaded with DNR nanoparticles to the mice was 1009.71mg/kg (relative content of DNR was 10mg/kg) and the 95% confidence interval was 769.11∼1262.40mg/kg, it had no significant difference compared with LD50 of using DNR only, which data was 8.51mg/kg and the 95% confidence interval was 6.48∼10.37mg/kg, and it had wide safety value circumscription. In micronucleus assay, compared Fe3O4-MNPs loaded with DNR nanoparticles experimental groups with negative control group, we found that the result had no significant difference (P > 0. 05) in micronucleus formation rate, while compared experimental groups with positive control group, the result had significant difference (P < 0. 05). The result indicated that Fe3O4 magnetic nanoparticles had no cacogenesis and mutagenesis. Acute toxicity testing showed that mice body weigh of control group, Fe3O4-MNPs loaded with DNR nanoparticles experimental group and isodose DNR group had no significant difference in 24h, 48h, and 72h after intra peritoneally injection; they had normal activity, eating and evacuation; toxic reactions such as instability of gait, convulsion, paralysis and respiratory depression were not been found; the alanine transarninase (ALT), blood urea nitrogen (BUN), and creatinine clearance rate (CCr) of Fe3O4-MNPs loaded with DNR nanoparticles experimental group was 66.0±28.55u/L, 9.06±1.05mmol/L, and 18.03±1.84umol/L, respectively, which had no significant difference compared with control group and isodose DNR group. From the results of our experiment, we could consider that self-prepared Fe3O4-MNPs loaded with DNR nanoparticles is a kind of high biocompatibility and security materials and perhaps is suitable for further application in hematologic malignancies therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 4859 Objectives: Signal transducer and activator of transcription 5 (STAT5) protein is one of the concernful part of STATs families. The constitutive STATs activation has been especially showed in transformation by fusion genes in leukemias. Methods: In this study, we aimed to investigate whether the genetic polymorphisms in the STAT5 gene are associated with the treatment outcomes of Ara-C-based chemotherapy regimens in Acute Myeloid Leukemia (AML) patients. 152 AML patients in a Chinese sample were enrolled in our study. Peripheral blood samples were analyzed by matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS). Results: The results showed that the frequencies of the T/T genotype in rs2293157 and rs1135669 were higher in unfavorable and intermediate group respectively (P=0.023 and P=0.033). The frequency of patients with T/T genotype of rs1135669 was significantly higher in complete remission (CR) group. Conclusions: We concluded that the T/T genotype of rs1135669 might be an important marker for therapeutic efficiency of Ara-C-based chemotherapy in AML patients, especially in Chinese population. Disclosures: No relevant conflicts of interest to declare.

Abstract 4951 Objective To investigate the relevance of Inducing apoptosis and reversal effect of nilotinib in combination with Tet on multidrug reststahoe of K562/A02 cell line and its mechanism. Methods Methyl-thiazol tetrazolium(MTT) assay is employed to examine the pharmacological effect of Nilotinib or Tet alone on K562/A02 cell line, to calculate the inhibitor concerntration 50(IC50) of daunorubicin(DNR) in K562/A02 cell line and the resistance multiple of DNR to K562/A02 cell line. Flow cytometry(FCM) was employed to comple the effect on the inducing apoptosis of K562/A02. The expression of bax/survivin mRNA was determined by RT-PCR, and the expression of bax/survivin protein was assessed by Western blot. Results After 48 h 5 nmol/L nilotinib or 1.0μmoL/L Tet treatment, IC50 of DNR to K562/A02 was 5.71mg/L or 6.52 mg/L respectively;While on combinative treatment, its IC50 decreased to 3.12 mg/L. Nilotinib or Tet alone was able to increase the DNR induced apoptosis rate of K562/A02 cell (P Conclusion Nilotinib or Tet alone can pattially reverse drug resistance of K562/A02 cells. There is resistance phenomena of DNR to K562/A02 cells, and Nilotinib or Tet alone alone is able to reserve the resistance of DNR and enhance the inducing apoptasis effect of DNR. The mechanism may be associated with the increase of bax mRNA and protein expression and decrease of survivin mRNA and protein expression and increase of the apoptosis rate. And there is a synergistic action with these two agants in combination. Disclosures: No relevant conflicts of interest to declare.

Abstract 823 Background: Plerixafor, a reversible inhibitor of the interaction between stromal cell-derived factor 1 (SDF-1) and CXCR4, improves autologous stem cell collection in patients with non-Hodgkin lymphoma (NHL), multiple myeloma, and Hodgkin lymphoma (HL). In Phase II and III trials, plerixafor has been combined with the standard G-CSF regimen and administered as a subcutaneous (sc) injection of 0.24 mg/kg, given 9–11 hours before pheresis. Intravenous (IV) administration of plerixafor may result in a faster rise and higher peak in the peripheral CD34+ cell count, allowing administration of plerixafor the same day as pheresis and improving stem cell collection. Methods: The primary objectives of this Phase I/II study were to determine the maximum tolerated dose (MTD) of IV plerixafor, up to 0.40 mg/kg combined with G-CSF, and to determine the efficacy of IV plerixafor + G-CSF to mobilize ≥ 2 × 106 CD34+ cells/kg from patients with lymphoma. Patients were given an initial dose of plerixafor 0.24 mg/kg sc on day -5, to assess the mobilization of CD34+ cell subsets, and then started mobilization with G-CSF (10 ug/kg SC daily) on days -4 thru -1 and on each day of pheresis. IV plerixafor was given over 30 min. 4 hrs. before each pheresis, beginning day 0. Samples for CD34+ cell subsets were collected after treatment with plerixafor only on day -5, after 5 days of G-CSF on day 0 (G-CSF only), and 4 hrs after plerixafor administration on day 0 (G-CSF + plerixafor). Human CD34+ cells were purified by positive selection with a Magnetic Affinity Cell Selection CD34 isolation kit. Results: In the Phase I component of the study, 25 adult pts. (median age 49) with NHL (n=15) or HL (n=10) were treated with IV plerixafor at escalating doses (10 pts. at 0.16 mg/kg, 3 at 0.24 mg/kg, 6 at 0.32 mg/kg, and 6 at 0.40 mg/kg). One dose-limiting toxicity (grade 2 chest pain) was observed at 0.32 mg/kg, and no grade 3/4 toxicities occurred at 0.40 mg/kg. 24 of 25 pts. (96%) met the goal collection of ≥ 2.0 × 106 CD34+ cells/kg. 21 of 25 pts. (84%) collected ≥ 5.0 × 106 CD34+ cells/kg in a median 1 day of pheresis, including 6 of 6 pts. in the 400 ug/kg cohort. Peripheral blood CD34+ cell count increased median 27-fold (range, 4–174) after G-CSF + plerixafor IV and 2.1-fold (range, 0.8–7.0) 4 hours after the first dose of IV plerixafor. There was a positive correlation between peripheral blood CD34+ cell count 6 hr. after plerixafor SC on day -5 and both peripheral blood CD34+ cell count after G-CSF mobilization and first day stem cell collection. Human CD34+ hematopoietic stem and progenitor cells (HSPCs) were divided into four distinct subsets based on their cell surface expression of CD45RA and CD123 (IL-3Rα): (1) CD34+CD45RA-CD123+/− primitive HSPCs, (2) CD34+CD45RA+CD123+/− committed progenitors, (3) CD34dimCD45RA+CD123hi plasmacytoid dendritic cell (pDC) progenitors and (4) CD34+CD45RA-CD123hi cells of unknown function. Table 1 shows the CD34+ cell subset distribution following sequential mobilization with plerixafor, G-CSF, and plerixafor + G-CSF (PL+G). G-CSF mobilized grafts were enriched with CD45RA-CD123+/− primitive HSPCs while plerixafor preferentially mobilized the CD34dimCD45RA+CD123hi pDC precursors and CD34+CD45RA-CD123hi cells. We found no significant differences in the CD34+ subset distribution between HL and NHL pts. Flow cytometric analyses showed that the CD34+ subsets preferentially mobilized by plerixafor expressed high levels of cell surface CXCR4. Conclusion: Plerixafor IV, at doses up to 0.40 mg/kg, is well-tolerated and effective when added to G-CSF for the mobilization of stem cells from patients with lymphoma, with mobilization kinetics and stem cell collections that compare favorably with sc dosing. The Phase II study is proceeding at the 0.40 mg/kg dose. In addition, our data suggest that G-CSF and plerixafor mobilize distinct subsets of human CD34+ HSPCs. The impact of these cells on the engraftment and function of plerixafor mobilized grafts requires further study. Disclosures: Rettig: Genzyme Corp.: Consultancy, Honoraria. Vij:Genzyme: Honoraria, Speakers Bureau. Uy:Genzyme: Consultancy, Honoraria, Research Funding.

Abstract 4856 Objective: The multidrug resistance (MDR) in leukemia is a major chemotherapy obstacle, rendering many currently available chemotherapeutic drugs ineffective. The aim of this study was to explore the new strategy to early diagnose the MDR by electrochemical biosensor based on carbon nanotubes–drug supramolecular interaction. Methods: The carbon nanotubes modified glassy carbon electrodes (CNTs/GCE) as a sensor were directly immersed into the cells suspension of the sensitive leukemia cells K562 and/or its MDR cells K562/A02 to detect the response of the electrochemical probe of daunorubicin (DNR) residues after incubated with cells. Results: The fresh evidence from the electrochemical studies based on CNTs/GCE demonstrated that the homogeneous, label-free strategy could directly measure the function of cell membrane transporters in MDR cancer cells, identify the cancer cell phenotype (sensitive or MDR). The cathodic peak current showed good linear response to the changes of the fraction of MDR with a correlation coefficient of 0.995 when we further took the different ratios of the sensitive leukemia cells K562 and its MDR ones K562/A02 as the model of MDR levels to simulate the MDR occurrence in leukemia. Then, we can easily predict the MDR fraction based on the calibration curve of the cathodic peak current versus the fraction of MDR according to the obtained peak current. Conclusion: These results indicated that the biosensing assay could provide a powerful tool for assessment of MDR in leukemia. The new electrochemical biosensor based on carbon nanotubes–drug supramolecular interaction could represent promising approach in the rapid diagnosis of MDR in leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 4829 Object In many instances, Multidrug resistance (MDR) is mediated by increased expression at the cell surface of the MDR1 gene product, P-glycoprotein (P-gp), a 170-kD energy-dependent efflux pump. The aim of this study was to investigate the potential benefit of combination therapy with magnetic nanoparticle of Fe3O4 (MNP(Fe3O4)) and mdr1-shRNA Expression vetor.in K562/A02 leukemic cells. Methods To synthesis short hairpin RNA (shRNA)aiming divectly at the target sequence,we choice the 3491-3509,1539-1557and 3103-3121 nucleotide of mdr-1 mRNA as targets. Cloning in the plasmid vetor PGCSilencer-U6-neo-GFP, The recombinant plasmid vetors were called for PGY1-1,PGY1-2 and PGY1-3.The recombinant plasmid vetors were transfected into the cell 1ines K562/A02 by lipofection. After transfected 48 hours,the inhibition of mdr-1mRNA expression and the expression of P-gp was detected by realtime–PCR and Weston-blot, screening the recombinant plasmid vetor which has the most greatest mdr-1 gene inhibition ratio is PGY1-2.Analysis of the reveral ratio of multidrug resistance, the concentration of DNR and the content of mdr-1 gene and P-gp in K562/A02 cell line. Results The combination of daunorubicin (DNR) with either MNP(Fe3O4) or PGY1-2 exerted a potent cytotoxic effect on K562/A02 cells, while MNP(Fe3O4) and PGY1-2 cotreatment can synergistically down regulation the expression of mdr-1 gene and the expression of P-gp(P Conclusion MNP(Fe3O4) and PGY1-2 cotreatment can synergistically reveral multidrug resistance. Thus our in vitro data strongly suggests a potential clinical application of MNP(Fe3O4) and PGY1-2 combination on CML. Disclosures No relevant conflicts of interest to declare.

Abstract 4432 Object This study was purposed to investigate the expression of HIF-1α and VEGF in leukemia cell lines and the effect of YC-1 on the regulation of HIF-1α and VEGF and the induction of apoptosis in U937 cell, exploring the mechanism of apoptosis after treatment of YC-1. Methods RT-PCR was used to determine the levels of HIF-1α mRNA and VEGF mRNA in K562,U937 and Jurkat cells. After treatment of U937 cell with 4μmol/L YC-1 for 0 hour, 8 hours,16 hours and 24 hours respectively, the changes of morphologic features were observed by DAPI staining under fluorescent microscope and the apoptotic rates were assayed by flow cytometry with Annexin V-FITC/PI staining; the levels of HIF-1α mRNA and VEGF mRNA were measured with RT-PCR ; the protein expression of HIF-1α, VEGF, Bax,Bcl-2 and Caspase-3 were measured by Western blot. Results HIF-1α mRNA and VEGF mRNA were expressed in all three leukemia cell lines. After treatment of U937 cell with 4μmol/L YC-1 for 0 hour,8 hours,16hours and 24hours respectively, the typical apoptotic morphologic features of U937 cells were observed under fluorescent microscope and the apoptotic rates were significantly increased in a time-dependent manner, they were (4.87±0.70)%, (27.27±2.00)%, (51.53±2.81) and (60.5±3.20)% respectively, the level of VEGF mRNA reduced, while the level of HIF-1α mRNA had no obviously changes. Furthermore, the expression of HIF-1α, VEGF and Bcl-2 decreased, while the expression of Bax and Caspase-3 increased in a time-dependent manner. Conclusion HIF-1α mRNA and VEGF mRNA are expressed in leukemia, YC-1 has significant effect of down-regulation the protein expression of HIF-1α and VEGF and induction of apoptosis in U937, its mechanisms may involve in up-regulating Bax/Bcl-2 ratio and expression of Caspase-3. Disclosures: No relevant conflicts of interest to declare.

Abstract 4828 Objective This study was aimed to investigate the reversible effect of tyrosine kinase inhibitors(TKI)on Multidrug resistance cell line K562/A02. Methods The expression levels of mdr-1 mRNA and bcr-abl mRNA were assayed by RT-PCR. The protein levels of P-glycoprotein (P-gp) and P210 were detected by Western blot. The DNR accumulation of K562/A02 cells were analyzed by flow cytometry (FCM). Results Analysis of the inhibition rate showed that 0.0625μmol/L Imatinib or 5nM Nilotinib alone had no effect on the inhibition of K562/A02 cells. The fluorescence intensity of intracellular DNR of Imatinib, Nilotinib in K562/A02 cells was 7.85%, 12.02% (respectively of that in K562 cells). Imatinib or Nilotonib alone could decrease the mdr-1 mRNA and bcr-abl mRNA expression levels (Imatinib 0.65±0.02, 0.87±0.02; Nilotinib 0.48±0.04, 0.73 ±0.02) respectively, all these of which were significantly lower than the K562/A02 cells group 0.96±0.01, 1.87±0.04. The P-gp and P210 protein expression levels were also down after treated with different drugs (Imatinib 0.74±0.02, 0.68±0.01; Nilotinib 0.61±0.05, 0.60±0.01; the K562/A02 cells group 0.93±0.01, 1.25±0.03). Conclusion It is concluded that multidrug resistance (MDR) can be partially reversed by Imatinib or Nilotinib. The effect of Nilotinib was greater than Imatinib. Disclosures No relevant conflicts of interest to declare.

Abstract 4827 Purpose The present study aimed to research the machenism of magnetic nanoparticles Fe3O4 (Fe3O4-MNPs) and 5-bromotetrandrine (BrTet) on multidrug resistance cell line K562/A02 solitarily or symphysially. Mothed The proliferation of K562 and K562/A02 cells, and the cytotoxicity of PMBCs which were cultured with daunomycin (DNR) alone or in combination with Fe3O4-MNPs (0.1, V/V), BrTet (0.5μM) or both for 48 h, were evaluated by MTT assay. DNR accumulation of K562, K562/A02 cells and PMBCs were analyzed by fluorospectrophotometry after incubated with 2μM DNR in the absence or presence with Fe3O4-MNPs (0.1 V/V), BrTet (0.5μM) or both for 48 h. Real time–PCR analyses and western blotting were performed to examine the mRNA and proteins level, respectively. Result The results showed that the combination of Fe3O4-MNPs and BrTet with effective concentration could exaggerate cytotoxicity against MDR cell line K562/A02 significantly. Flow cytometry assay showed that 0.5μM BrTet in combination with Fe3O4-MNPs (0.1, V/V) significantly enhanced the intracellular accumulation of DNR in K562/A02 cells and its potency was greater than that of BrTet or Fe3O4-MNPs alone at the same concentrations. While both Fe3O4-MNPs and BrTet in company with DNR did not increase the cytotoxicity to PMBCs. Both BrTet and Fe3O4-MNPs inhibited the overexpression of MRP1, MRP2, MRP4 and MRP5, and down regulated, mrps mRNA expression in K562/A02 cells to some extent. Conclusion We propose that Fe3O4-MNPs loaded with DNR and BrTet probably have synergetic effect on reversal in multidrug resistance. However, this combination shows less cytotoxicity to normal cells and reveals better target. The results may provide evidence for clinic application of them as reversal agents of drug resistance. Disclosures No relevant conflicts of interest to declare.

Objective: To establish the xenograft leukemia model with stable multiple drug resistance in nude mice; to investigate the reversal effect of 5-Bromotetrandrine and Magnetic nanoparticle of Fe3O4 combined with DNR in vivo and to search for the possible reversal mechanisms. Methods: K562 and K562/A02 cells were respectively inoculated subcutaneously into back of athymic nude mice (1×107 cells/each) to establish the xenograft models. The tumor formation was evaluated by animal ultrasonic inspection. Tumors-bearing nude mice were assigned randomly to five groups which were treated with NS (A group); DNR 1mg/kg (B group); nanoparticle of Fe3O4 combined with DNR 0.63mg/kg(C group): 5-BrTet 2.5mg/kg combined with DNR(D group); 5-Bromotetrandrine 2.5mg/kg and Magnetic nanoparticle of Fe3O4 combined with DNR 0.63mg/kg(E group) respectively. The incidence of tumor formation, growth characteristics, weight and volume of tumor were observed. The histopathologic examination of tumors and organs were detected. For resistant tumors, the protein levels of P-glycoprotein (P-gp) were detected by Western blot. Results: The tumor incidence was 100% in the nude mice inoculated with either K562 or K562/A02 cells. In 6 to 9 days,the tumors reached a volume of more than 1 00 mm3. In vivo, MTT assay showed K562/A02 tumor maintained the drug resistance. For K562 cells xenograft tumors, there were no apparent differences in tumor suppression effect between the B AC AD AE group. For K562/A02 cells xenograft tumors, 5-BrTet and Magnetic nanoparticle of Fe3O4 combined with DNR significantly suppressed growth of tumor: the inhibition rate was 62.76% while DNR alone be used, the inhibition rate was 3.68%. Pathologic examination of resistant tumors showed the tumors necrosis obviously in E group. Application of 5-BrTet and Magnetic nanoparticle of Fe3O4 inhibited the overexpression of P-gp. Conclusion: The xenograft leukemia nude mice model was maintain the multiple drug resistance. 5-Bromotetrandrine and Magnetic nanoparticle of Fe3O4 combined with DNR had a significant tumor-suppressing effect on MDR leukemia cells xenograft model.

Object: To study the variation of p21, P-gp expression in reversion of multidrug resistance of K562/A02 leukemic line with different concentration of tetrandrine(TET), to provide new theoretic evidence for the clinical application of TET. Methods: The IC50 (the concentration causing 50% inhibition of cell growth) of DNR was analyzed by MTT method; The Expressions of p21 were assayed by westernblot; The Expressions of P-gp were assayed by FCM; The expressions of mdr1 were assayed by RT-PCR; The variation of p21 was accentuation with the accrescence concentration of TET(P Results: The variation of p21 protein in K562/A02 cells was accentuation with the accrescence concentration of TET(p Conclusion: TET may reverse multidrug resistance of K562/A02 cells by the regulation of p21,P-gp and mdr1.

Object: To the effect of Fe3O4-magnetic nanoparticle loaded with DNR on multidrugresistant K562 cells in vivo. Methods: K562-n and its MDR counterpart K562-n/VCR cell were inoculated subcutaneously into both sides of the back of nude mice (5×106 cells/each) to establish a human leukemia xenograft model. The mice were randomly divided into group A receiving normal saline every other day for 20days, group B receiving DNR every other day for 20days, group C receiving Fe3O4-magnetic nanoparticle every other day for 20days, group D receiving Fe3O4-magnetic nanoparticle loaded with DNR every other day for 20days, and group E receiving Fe3O4-magnetic nanoparticle containing DNR every other day for 20days with a magnetic field built on the surface of the tumor tissue. The tumor volume was measured on the day 1, 5, 9, 13, 17 and 21d after the first treatment. Tumor tissues were isolated for examination of the expression of mdr-1, bcl-2, bax and caspase-3 by reverse transcription polymerase chain reaction and Western blotting. Results: For K562-n/VCR tumor, the tumor volume was markedly lower in groups D and E than in groups A, B and C (group D or E vs group A, B or C, P < 0.05). The transcription of mdr-1 and Bcl-2 gene was significantly lower in groups D and E than in groups A, B and C (group D or E vs group A, B or C, P < 0.05). So did the protein expression of Bcl-2. However, there were no differences among these groups about the protein expression of P-gp. The protein and mRNA expressions of Bax and Caspase-3 in groups D and E were increased significantly compared with groups A, B and C (group D or E vs group A, B or C, P < 0.05). The tumor volume of K562-n was markedly lower in groups C, D and E than in groups A and B (group C, D or E vs group A or B, P < 0.05). Conclusion: In conclusion, DNR loaded Fe3O4-magnetic nanoparticles can suppress the growth and induce apoptosis further on the MDR K562-n/VCR tumor in vivo compared to DNR alone but not on the K562-n tumor. The external magnetic field failed to improve the antitumor effect.

Objective Previous researches confirmed that multidrugresistance(MDR) plays an important role in the failure of chemotherapy on malignant tumors. This study was to investigate the molecular biological mechanisms of cyclosporine A(CsA), tetrandrine(Tet) and their combination on multidrug resistance cell line K562/A02. Methods K562/A02 cells were treated with cyclosporine A and (or) tetrandrine. The intracellular DNR concentration and the expression of P-glyco-protein (P-gp) were observed by flow cytometry (FCM) assay. The mRNA expression of mdr-1 was measured by fluorescent semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR). Resulds CsA and Tet (alone or combination) elevated the intracellular DNR concentration in K562/A02 cells(the fluorescence intensity of intracellelar DNR in K562/A02 cells was 60%,65% and 98% respectively of that in K562 cells); The fluorescence intensity of P-gp in K562 and K562/A02 cells was 0.5% and 97.97%. The P-gp expression was down after treated with CsA,Tet and both(75.32%,76.86% and 48.61%); mdr1 mRNA was also down regulated, and the effect of their combination was greater. Conclusion Multidrug resistance (MDR) can be partially reversed by CsA or Tet, the combination of both drugs shows a great synergistic reversal effect.

Objective: To study the reversal effect of Tetrandrine(Tet) and the estrogen-receptor inhibitor,toremifene(Tor),on multidrug resistance cell line K562/A02 and to investigate the reversal mechanism of this combination. Methods: ADM accumulation and the apoptosis percentage of K562 and K562/A02 cells were analyzed by fluorospectrophotometry, respectively. The protein levels of P-glycoprotein (P-gp) were detected by fluorospectrophotometry. The mRNA levels of mdr1 and Survivin were determined by RT-PCR. Results: The IC50 of ADM for K562/A02 and K562 cells were 57.43±4.55mg/L and 1.16±0.05mg/L respectively. Pretreating K562/A02 cells with toremifene(2.5μmol/L) or Tet(1μmol/L) for 72 hours partially restored the sensitivity of K562/A02 cells to ADM (IC50 were 20.74±1.62mg/L and 14.12±1.20mg/L respectively) but had no effect on K562 cells; IC50 of combined tetrandrine and toremifene was 9.14±1.03mg/L;K562/A02 showed apoptotic characteristics after treated with tetrandrine, toremifene (alone or combination);tetrandrine and toremifene (alone or combination) elevated the intracellular ADM accumulation in K562/A02; P-gp, mdr1 and Survivin mRNA were down regulated. Conclusion: Tetrandrine, toremifene (alone or combination) showed significant MDR reversal activity in vitro The reversal activity may be related to the inhibition of P-gp overexpression and down regulation the expression of mdr1 and Survivin mRNA to increase the intracellular accumulation of anticancer drugs, which lead to more K562/A02 cells apoptosis; Multidrug resistance (MDR) can be partially reversed by Tet or Tor of which the combination shows a great synergistic reversal effect.

Objective: To investigate the effect of 6-phenylhexyl isothiocyanate (PHI) on drug resistance and sensitivity on K562/A02 cell line to ADM and elucidate the probable mechanisms. Methods: We measured growth inhibition of ADM on K562/A02 cell line by MTT assay. Apoptosis rate of K562/A02 cell line, the change of intracellular ADM and MRP1 protein level were detected by Flow Cytometry. Intracellular deoxidized GSH by spectrometric enzyme assay and MRP1 mRNA by RT-PCR semiquantitative assay were observed anteroposterior using PHI. Results: PHI can enhance the sensitivity of K562/A02 cell line to ADM, survival rate of K562/A02 cell line decreased with the increasing concentration of PHI and ADM. Apoptosis rate increased with treating by combination of two above drugs, and multiple of drug resistance had statistical significance (P0.05) no matter after or before PHI was used on different concentration. Conlusion: The depletion effect of PHI on the intracellular GSH can not only partly enhance the reverse effect of ADM, but also enhance the sensitivity of K562/A02 cell line to ADM. Such depletion may diminish side effect and treatment dosage of ADM. It provides a new view to the therapy of leukaemia.

Object: To the effect of Fe3O4-magnetic nanoparticle loaded with DNR on In order to study the multidrug-resistant reversal effect of Fe3O4-magnetic nanoparticle loaded with DNR in vivo. Methods: K562-n,a leukemia cell line with high tumorigenicity in nude mice and its multidrug-resistant counterpart K562-n/VCR cells were inoculated subcutaneously into each groins of nude mice (5×106 cells/each) to establish a human leukemia xenograft model. The mice were randomly divided into group A receiving normal saline, group B receiving anti-tumor drugs daunorubicin (DNR), group C receiving Fe3O4-magnetic nanoparticle, group D receiving Fe3O4-magnetic nanoparticle loaded with DNR, and group E receiving Fe3O4-magnetic nanoparticle containing DNR with a magnetic field built on the surface of tumor tissues. After 20 days treatments, mice were killed and tumor tissues were isolated for pathological observation. The volume and weight of tumors were measured, then tumor suppression rate was calculated. The side effects of DNR loaded Fe3O4-magnetic nanoparticle were also evaluated. Results: The results showed that DNR loaded Fe3O4-magnetic nanoparticle can significantly suppress the growth of K562-n/VCR tumor in vivo, DNR alone can greatly suppress the growth of K562-n, Fe3O4-magnetic nanoparticle loaded with DNR can not further inhibit the K562-n tumor. Conclusion: In conclusion, Fe3O4-magnetic nanoparticle loaded with DNR can reverse the leukemia multidrug resistance in vivo.

Objective: To observe the apoptosis of K562/A02 cells induced by application of Fe3O4-Magnetic Nanoparticle (Fe3O4-MNPs) loaded with Daunorubicin(DNR) and 5-Bromotetrandrine (BrTet) and to explore its possible molecular mechanism. Methods: We detected the apoptosis of K562/A02 cells after treatment with Fe3O4-MNPs and BrTet with DNR alone or their combination by flow cytometry(FCM),Wright staining and DNA ladder. The expression of Bax and Bcl-2 protein was measured by Western blot. Results: The result showed apoptotic characteristics after treated with this composite for 48 hours. Flow cytometry assay showed the percentage of apoptotic cells which were treated with Fe3O4-MNPs and BrTet with DNR alone or their combination for 48 hours increased from (7.9%±0.35)% to (15.5%±0.87)%, (19.7%±1.04)% and(40.8%±1.74)%, (P Conclusion: Our results suggest that Fe3O4-MNPs or BrTet with DNR induces apoptosis in K562/ADM cells, and when used together,they have distinct synergism. The up-regulation of Bax protein and the down-regulation of Bcl-2 protein may play important roles.

Azacitidine (AZA) has been shown to improve overall survival in patients with myelodysplastic syndrome (Silverman LR et al. J Clin Oncol2002; 20: 2429–40). AZA is postulated to work, in part, by inducing hypomethylation and thereby altering gene expression. As a correlative analysis to our phase II study of IV AZA we assessed the baseline methylome of MDS and the changes in global methylation on cycle 2 day 25 (c2d25) of treatment with AZA (75mg/m2 d1–5 of a 28 d cycle). DNA from bone marrow (BM) and peripheral blood (PB) underwent bisulfite conversion and 1500ng of DNA was hybridized to Infinium Human Methylation27 BeadArrays. Arrays were processed by the Genome Sequencing Center at Washington University. The Infinium human Methylation27 Bead Array interrogates >27,000 well annotated CpG sites analyzing 14,500 promoters with single nucleotide resolution. Data was analyzed using BeadStudio version 3 and GraphPad Prism version 5. To compare the average percentages of methylated loci, we used a mixed model for repeated measurement data which allowed us to account for potential correlations among measurements taken from the same subjects. Loci were considered methylated if their average beta value was ≥0.80, hemi-methylated if their average beta value was 0.20, and unmethylated if it was ≤0.20. Loci on the X and Y chromosomes were excluded from analyses due to gender imbalances between responders and non-responders. Fifteen patients had BM and/or PB samples at baseline and/or on c2d25 and a response assessment. Median age was 69 years (range 53–82). IPSS categories were low (7%), Int-1 (27%), Int-2 (40%) and High (7%); FAB classifications were RA (26%), CMML (7%), RAEB (47%) and RAEB-t (20%). Best responses were: 2 patients with CR, 2 with a marrow CR, 1 with a PR, 8 with SD and 2 suffered PD. Patients with CR, mCR and PR were classified as responders whereas all others were classified as non-responders. For all patients the percentage of methylated loci decreased from baseline (13.4%) to c2d25 (9.9%) (p=0.0043). This effect was driven by a decrease in methylated loci in responders (15.1% to 8.9%) (p=0.0109). The decrease in methylated loci in non-responders was not statistically significant (12.4% to 10.5%) (p=0.1083). The difference in baseline methylation between responders and non-responders was not significant (p=0.1719). Baseline methylation was not associated with age (p=0.1475), IPSS (p=0.0934) or High- vs. Low-grade MDS (p=0.3575). Interestingly, the c2d25 assessment anteceded best clinical response in 4 out of 5 responders (all that achieved a mCR or CR) (81 days v. a mean of 202 days, SD 129 days to best response). Demethylation was likewise seen in the PB (18.6% to 11.7% of loci in responders and 18.8% to 13.6% of loci in non-responders) and overall there was good correlation between the mean beta values across the loci for BM and PB (r2=0.9937). But this correlation deteriorated when only methylated loci were included (r2=0.7936), demonstrating that the correlation is driven by hemi- or unmethylated loci, which compose ≥80% of loci, and suggesting that methylation in the PB is unlikely to be a good surrogate for BM methylation. Loci specific changes were considered potentially significant if average beta scores changed by ≥0.20 from baseline to c2d25. 11 loci met this criteria in responders and the changes in several of these were confirmed by t-tests (DNTT p=0.08; APOBEC2 p=0.0015, RGPD5 p=0.0138, TMSF4 p=0.0040). These differences and others between the baseline methylation status of loci in responders and non-responders will be correlated with mRNA expression levels (such as BCL2 p=0.08, HP p=0.0057, SHANK2 p=0.003). This study represents the first methylome-wide description of MDS and the changes induced by azacitidine. If validated in larger cohorts, BM methylation changes prior to cycle 3 may be able to help discriminate responders from non-responders earlier. Further studies are needed that include a larger sample size and that correlate changes in methylation status with changes in transcription and ultimately translation.

Objective This study was to compare the reversal effect of 5-bromotetrandrine (BrTet) with Tetrandrine (Tet) when combined with ADM on multidrug resistance cell line K562/A02 and to investigate the reversal mechanism of this new derivative. Methods The protein levels of P-glycoprotein (P-gp) were detected by fluorospectrophotometry and Western blot. The mRNA levels of P-gp were determined by RT-PCR. The in vivo effect of Tet was investigated using nude mice grafted with sensitive human leukemia cell line K562 and MDR cell line K562/A02. Results Flow cytometry assay showed that 1.0 μMol/L BrTet significantly increased the apoptosis percentage. BrTet also enhanced the intracellular accumulation of ADM in K562/A02 cells and its potency was greater than that of Tet at the same concentrations. BrTet inhibited the overexpression of P-gp and down regulated MDR1 mRNA expression in K562/A02 cells in a dose-dependent manner. In nude mice bearing K562 xenografts on the left flank and K562/A02 xenografts on the right flank, i.p. injection of 10 mg/kg BrTet significantly enhanced the antitumor activity of ADM against K562/A02 xenografts with inhibitory rates of 26.1%, while ADM alone inhibited the growth of KBv200 xenografts by only 5.8%. Conclusion BrTet showed significant MDR reversal activity in vitro and in vivo. Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs, which lead to more K562/A02 cells apoptosis.

Objective: The aim of this study was to investigate the mechanism to reverse the drug resistance of leukemia cells in Cyclosporine A(CsA), tetrandrine (Tet) alone or their combination by using empirical methods, and to provide the basic evidence for further study, and lay the theoretical basis for the clinical applications. Method: By MTT assay, the IC50(the concentration causing 50% inhibitor of cell growth) of DNR was determined;By flow cytomet ry (FCM) assay, the intracellular DNR concentration and the expression of P-glyco-protein (P-gp) were examined, and the apoptotic changes were observed by Anexin-V. By fluorescent semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), the mRNA expression of multidrug resistance (MDR) was measured. Results: The IC50 of DNR for K562 and K562/A02 cells were 0.26mg/l and 21.22mg/l respectively. Treating K562/A02 cells with CsA(1mg/L), TTD(0.1mg/L)and both for 48 hours partially restored the sensitivity of K562/A02 cells to DNR(IC50 were 7.83 mg/L,5.32 mg/L,2.33 mg/L respectively.) but had not effect on K562 cells; K562/A02 cells showed apoptotic characteristics after treated with CsA, and both for 48 hours(Apoptosis rate was 10.27%,17.64%,52.79% respectively); The fluorescence intensity of intracellelar DNR in K562 and K562/A02 cells treated with DNR(1mg/L)was 522,173 respectively. CsA and TTD (alone or combination) elevated the intracellular DNR concentration in K562/A02 cells(the fluorescence intensity of intracellelar DNR in K562/A02 cells was 188,339,515 respectively); The fluorescence intensity of P-gp in K562 and K562/A02 cells was 0.5,97.97. The P-gp expression was down after treated with CsA, TTD and both(73.7,65.05,58.1); mdr1 mRNA was also down regulated, and the effect of their combination was greater. Date was analyzed by SPSS 13.0 software and expressed as mean ± SD. Conclusions: High expression of P-gp may be involved in the mechanism of multidrug resistance (MDR) of K562/A02 cells line; MDR can be partially reversed by CsA or TTD, of which the combination shows a great synergistic reversal effect.